CA2261297A1

CA2261297A1 - A novel maturation-inhibited protein kinase

Info

Publication number: CA2261297A1
Application number: CA 2261297
Authority: CA
Inventors: Steven Pelech; Donna Morrison; Arthur R. Yee
Original assignee: Individual
Current assignee: University of British Columbia
Priority date: 1998-02-18
Filing date: 1999-02-18
Publication date: 1999-08-18

Abstract

The present invention provides nucleic acid and amino acid sequences for a novel MAP kinase. The present invention also provides oligomers and hybridization probes for the detection or amplification of nucleotide sequences encoding the kinase; antisense molecules to the nucleotide sequences which encode the kinase;
diagnostic tests based on sequences encoding the kinase and the activity of the kinase; genetically engineered expression vectors, host cells and organisms, antibodies capable of binding specifically to the kinase, and agonists and inhibitors with specific binding activity for the kinase.

Description

A NOVEL MATURATION-INHIBITED PROTEIN KINASE
Field of the Invention s The present invention relates to the field of molecular biology. More particularly, the present invention relates to mitogen activated protein ("MAP") kinases.
Background of the Invention to MAP kinases are a family of enzymes that regulate intracellular signalling pathways. MAP kinases are important mediators of signal transduction from cell surfaces to nuclei via protein phosphorylation cascades.
i5 Several subgroups of MAP kinases have been defined and each manifests a different substrate specificity and responds to various distinct extracellular stimuli.
Thus, the MAP kinase signalling pathways represent common 2o mechanisms for signal transduction by which different extracellular stimuli generate distinct physiological responses inside cells (Egan et al., 1993).
Various MAP kinase signalling pathways have been defined 2s in mammalian cells as well as other diverse eukaryotes such as in yeast. In mammalian cells, the extracellular stimuli activating the MAP kinase signalling pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic 30 lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1). In the yeast, Saccharomyces cerevisiae, various MAP kinase signalling pathways are activated by exposure to mating pheromone or hyperosmolar environments and 35 during cell wall construction, sporulation and mitosis.

There are at least three subgroups of MAP kinases in mammalian cells (Derijard B et al., 1995), and each subgroup is distinguished by a tripeptide sequence motif located after the conserved kinase subdomain VII region:
extracellular signal-regulated protein kinases (ERK) are characterized by Thr-Glu-Tyr; c-Jun amino-terminal kinases (JNK) or stress-activated protein kinases (SAPK) are characterized by Thr-Pro-Tyr; and p38 kinases (p38) or Hog kinases are characterized by Thr-Gly-Tyr.
io The subgroups are activated by the dual phosphorylation of the threonine and tyrosine residues in the aforementioned motifs by MAP kinase kinases (MKK or MEK) located upstream of the protein phosphorylation cascade.
Activated MAP kinases phosphorylate other effectors downstream ultimately leading to changes inside the cell.
Analysis of the cDNA sequences encoding p38 isoforms show that they are 41 kDa proteins. Their activation by dual 2o phosphorylation is catalyzed by the MAP kinase kinases, MKK3 and MKK6. The p38 signal transduction pathway is also activated by heat shock, hyperosmolar medium, IL-1 or lipopolysaccharide (LPS) endotoxin (Han et al., 1994) produced by invading gram-negative bacteria.
The function of cells and tissues rely on the fidelity of intercellular and intracellular communication via a variety of signalling pathways. Malfunction of these pathways may result in diseases including proliferative 3o maladies such as cancers and atherosclerosis, and inflammatory conditions such as psoriasis, rheumatoid arthritis, multiple sclerosis and tissue rejection (Levitski, 1994).

One of the early events in the response of cells to growth factors, hormones, immune cytokines, and neuropeptides is the stimulation of protein phosphorylation primarily due to activation of two s classes of protein kinases: those that transfer phosphate to serine or threonine and those that transfer phosphate to tyrosine (Krebs et al., l979). Phosphorylation is of particular significance in controlling mitogenesis and cellular differentiation. Receptors for a number of io polypeptide growth factors are transmembrane tyrosine protein kinases (Yarden et al., 1988), which in turn stimulate serine/threonine protein kinases such as protein kinase C, MAP kinase and Raf-1 (Hunter et al., 1984; Morrison et al., 1989) which are involved in cell i5 proliferation.
Over-expression or mutation of the genes that encode protein kinases have been found to be linked to inappropriately regulated cellular division of cells and 2o metastasis. Many such enzymes were first identified as the products of oncogenes and constitute the largest family of known oncogenes (Lindberg et al., 1990).
Mutations of genes encoding members of the protein kinase family that are involved in the regulation of 2s proliferation, differentiation and survival play a role in the etiology of human tumors. The fact that there are multiple MAP kinases indicates that individual kinases are specific in their activity.
3o Provision of nucleic acids encoding phosphatases enables screening for both activators and inhibitors. The substrates for a particular MAP kinase may be key in regulating cell proliferation and potential targets for intervention by drug inhibitors. Study of the signalling pathways and the factors that control is useful in the search for improved therapies for cancer or tissue regeneration. Furthermore, understanding the mechanism for blocking specific kinase activities may provide means s for treating inflammatory illnesses. Likewise, understanding of the various MAP kinase signalling pathways enable better understanding of other developmental and disease processes. Identification of novel MAP kinases provides the means for diagnosis and io intervention.
SUMMARY OF THE INVENTION
A MAP-like tyrosine-phosphorylated protein kinase which 15 undergoes tyrosine dephosphorylation and becomes deactivated as an oocyte matures into an ovum, has been identified. For the purposes of this specification, this new kinase (regardless of their source organism) will be called maturation inhibited protein kinase ("MIPK") and 2o the corresponding cDNA will be called "mipk". Whereas many kinases are activated during early stages of meiosis and mitosis and are required for the maturation of an oocyte to an ovum, MIPK does the opposite by becoming dephosphorylated as an oocyte matures. MIPK is activated 2s by tyrosine phosphorylation but unlike all known MAP
kinases, MIPK is reactivated following fertilization and subsequent development in cells which have begun the process of differentiation. Furthermore, MIPK is induced in terminally differentiated organs such as mammalian 3o heart and brain. The kinase falls within the p38 family, but the amino and carboxyl termini are completely different from other p38 sequences, putting it into a new category of p38 kinases.

MIPK maybe defined as a kinase activated by tyrosine phosphorylation and which is reactive with the antibody CDK5-CT. CDK5-CT is an antibody to a 31KD cylin-dependent protein kinase that is deactivated by phosphorylation rather than being activated as in the case of MIPK. MIPK has a molecular weight of about 40kD.
This invention also provides nucleic acids comprising one or more regions encoding or are complimentary to one or io more regions encoding an amino acid motif selected from the group comprising: SEQ ID NO: 3; SEQ ID NO: 4; and SEQ
ID NO: 5 (as described herein), or any motif having 50%
or more homology to any one of the aforementioned motifs.
This invention also provides a polypeptide comprising one or more of the aforementioned motifs or any motif having 50% or more homology to the aforementioned motifs.
This invention also provides nucleic acids encoding 2o peptides having at least one biological activity of MIPK.
Preferred nucleic acids are cDNAs having a nucleotide sequence shown in SEQ ID NO: 2 (mipk). The invention also provides peptides encoded by a11 or a portion of such a cDNA in SEQ ID NO: 2 and having at least one biological activity of MIPK. Also contemplated are nucleic acids which hybridize under high stringency conditions to a nucleic acid having a nucleotide sequence shown SEQ ID
N0:2 or which encode a peptide comprising all or a portion of an amino acid sequence of SEQ ID NO: 1 (MIPK).
3o Nucleic acids which encode peptides having an activity of MIPK and having at least 50% homology with a sequence shown in SEQ ID NO: 1 are also featured.

Peptides produced by recombinant expression of a nucleic acid of the invention and peptides prepared by chemical synthesis are also featured by this invention.
s Preferred peptides have the ability to treat defective cells and/or tissues in need of stimulation or regeneration (e. g. cardiac tissue, muscle and brain).
Other preferred peptides, either apart from or in to addition to the ability to treat defective cells, produce antibodies or identify inhibitors of the protein. Such peptides are useful in diagnosing defective cells and/or tissues in need of stimulation or regeneration.
15 Other preferred peptides comprise an amino acid sequence shown in SEQ ID NO: 1. In one embodiment, peptides having MIPK activity and comprising a portion of the amino acid sequence of SEQ ID NO: 1 are at least about 10-l00 amino acids in length, about 10-60 amino acids in length, 2o preferably 10-30 amino acids in length, more preferably about 10-20 amino acids in length, and most preferably about 10-16 amino acids in length.
Another aspect of the invention features antibodies 25 specifically reactive with a peptide of this invention.
Nucleic acids, peptides and antibodies of this invention may be used in compositions for pharmaceutical administration.
Brief Description of the Drawings Fig. 1. Nucleotide sequence encoding (SEQ ID NO: 2), and predicted amino acid sequence of MIPK (SEQ

ID NO: 1). The nucleotides are numbered starting at 1 for the first residue of the start codon. Oligonucleotides used to amplify mipk from P. ochraceus cDNA were based on s regions underlined, arrows indicate sense (>) and antisense (<) primers. Roman numerals indicate protein kinase subdomains (Hanks et al. l988). Residues that are identical between almost a11 protein kinases are shown in bold io type .
Fig. 2. Dephosphorylation of MIPK and phosphorylation of another MAP kinase (p44MPK) in homogenates of a P. ochraceus oocyte maturation time 15 course. Oocyte homogenates immunoprecipitated and Western blotted with antiphosphotyrosine (4G10). The numbers in the time course represent maturation in minutes from 0 (immature oocytes) to 2 h post-1-methyladenine 2o addition. On average maturation had occurred in 80% of the oocytes by 90 min. Alkaline phosphatase signals from the Western blots are quantitated by densitometric analysis and phosphotyrosine levels corrected for protein 2s levels. Values were standardized to 1 Unit = 0 min levels for MIPK (solid bars); 1 Unit - 120 min for MIPK (open bars). Data shows the mean S.E.M. of 3 independent time courses.
3o Fig. 3. Restriction map of P. ochraceus mipk in the pGEX-4T3 vector. The full length open-reading frame of mipk was amplified by PCR using primers having EcoRI and SalI tails. The fragment was then inserted into the EcoRI and - g -SalI sites of pGEX-4T3. A map of kinase-dead mipk as described herein is the same.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel protein tyrosine kinase, MIPK, and a polynucleotide sequence (mipk) encoding MIPK polypeptide. The protein is present during oocyte development and has a unique activity profile. In to a first embodiment, the invention provides a substantially pure MIPK polypeptide consisting essentially of the amino acid sequence shown in Fig. 1.
The full-length MIPK polypeptide has a mass of approximately 40 kD. Seastar MIPK has 66% identity and 74% similarity with p38 (murine), 66% identity and 74%
similarity with p38 (rat), and 65% identity and 74%
similarity with p38 (human) (percentages generated using the AGLIGN program). MIPK may be found in a variety of animlas, including but not limited to humans, mice, 2o cattle, fish, frogs and the like.
A further aspect of the invention includes mammalian MIPK. Using the anti-MIPK antibody CDK5-CT (described infra), rat tissues were screened for immunoreactive proteins. The 40 kD kinase was detected mainly in the adult rat heart and brain but not in embryonic or neonatal tissue. It is expressed to a lesser extent in other rat tissues. In another example MIPK was detected in adult bovine heart.
Another aspect of the invention pertains to mammalian MIPK present in non-dividing adult tissue in various animal species. For example, MIPK may be present in the non-dividing adult tissues of mammals including, but not limited to, cattle, humans, mice, carp and Xenopus, and the like.
Three peptide sequences within MIPK (amino acid motifs) have been identified as displaying a high degree of divergence from other MAP kinase isoforms. These motifs are preferred peptides of this invention, each having an amino acid sequence as shown in SEQ ID No.3 (100-l13), SEQ ID No: 4 (242-288) and SEQ ID No: 5 (323-345).
MIPK polypeptides of the invention and peptides of the invention include an amino acid motif the same as, or having 50% or greater homology to, one, two or three of the aforementioned amino acid motifs. Preferably, MIPK
i5 polypeptides of the invention will have three such amino acid motifs, each preferably having 60%, more preferably 75% and most preferably 90% homology to an aforementioned motif .
2o This invention pertains to isolated nucleic acids encoding a peptide or polypeptide of this invention, preferably one having at least one biological activity MIPK. The nucleic acid maybe a cDNA comprising a nucleotide sequence shown in SEQ ID NO: 2 (mipk), which 25 is a cDNA sequence which encodes seastar MIPK comprising a (1089) base pair (bp) coding region for MIPK, 17 base pairs of 5' untranslated and 15 base pairs of 3' untranslated sequence Fig. 1. The seastar cDNA includes an open reading frame of 1089 base pairs. The predicted 30 363 amino acid coding product would have a mass of about 41 kD.
Accordingly, another aspect of this invention pertains to isolated nucleic acids comprising nucleotide sequences encoding MIPK and fragments thereof, including peptides having at least one biological activity of MIPK and/or equivalents of such nucleic acids. The term Anucleic acid as used herein is intended to include such fragments and s equivalents. The term "equivalent" is intended to include nucleotide sequences encoding activity equivalent MIPK
proteins or activity equivalent peptides having an activity of MIPK. As defined herein, "a peptide having an activity of MIPK" has at least one biological activity of io the MIPK tyrosine kinase. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and will also include sequences that differ from the nucleotide sequence encoding MIPK (SEQ ID
i5 N0:2) due to the degeneracy of the genetic code.
Equivalents will also include nucleotide sequences that selectively hybridize under stringent conditions to the nucleotide sequence of MIPK (SEQ ID NO: 2).
2o Peptides or polypeptides referred to herein as having an "MIPK activity" are defined as being cross reactive to the antibody CDK5-CT as described herein. Alternatively, the peptides may have the activity of MIPK.
25 In another embodiment, the nucleic acid of the invention encodes a peptide comprising an amino acid sequence shown in SEQ ID N0: 1 or a peptide having a at least about 50%
homology, more preferably at least about 60% homology and most preferably at least about 70% homology with the 3o sequence shown in SEQ ID NO: 1. Nucleic acids which encode peptides having a MIPK activity and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence set forth in SEQ ID N0: 2 (mipk) are also within the scope 35 of the invention. The term "homology" as used herein refers to the measure of identity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. Two amino acid or nucleic acid sequences are considered substantially identical if they share at least about 75% sequence identity, preferably at least about 90% sequence identity, and more preferably at least 95% sequence identity.
Sequence identity may be determined using the BLAST
to algorithm, described in Altschul et al. (l990), J. Mol.
Biol. 215:403-10 (using the published default settings).
When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a activity of the number of matching or homologous positions shared by the sequences.
An alternate measure of homology of nucleic acid sequences is indicated when two sequences hybridize to each other 2o under low stringency, or preferably high stringency, conditions. Such sequences are substantially identical when they will hybridize under high stringency conditions.
Another aspect of the invention provides a nucleic acid which hybridizes under stringent conditions to a nucleic acid which encodes a peptide having all or a portion of an amino acid sequence shown in SEQ ID NO: 1. Hybridization to filter-bound sequences under low stringency conditions may, for example, be performed in 0.5 M NaHP04, 7% sodium 3o dodecyl sulfate (SDS), 1 mM EDTA at 65 C, and washing in 0.2 x SSC/0.1~s SDS at 42 C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under high stringency conditions may, for example, be performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65~ C, and washing in 0.1 x SSC/0.1% SDS at 68~C (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in s accordance with known methods depending on the sequence of interest (see Tijssen, l993, Laboratory Techniques in Biochemistry and Molecular Bioloay -- Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic io acid probe assays", Elsevier, New York). Generally, stringent conditions are selected to be about 5~C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. Preferably, the portion of the mipk sequence to use as such probes to i5 determine stringency of hybridization with another nucleic acid would be from nucleotide 690 to 1089, more preferably from 930 to 1089.
Isolated nucleic acids encoding peptides and polypeptides 20 of this invention, and having a sequence which differs from the nucleotide sequences shown in SEQ ID NO: 2 due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode actively equivalent peptides, but differ in sequence from the 2s sequence of SEQ ID NO: 2 due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations 3o which do not affect the amino acid sequence of the MIPK
protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequence of MIPK will exist within a particular organism.
One skilled in the art will appreciate that these 35 variations in one or more of the nucleic acids encoding peptides having an activity of MIPK may exist among a species. Any and a11 such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. Furthermore, there may be one or more isoforms or related, cross-reacting family members of MIPK. Such isoforms or family members are defined as proteins related in activity and amino acid sequence to MIPK, but encoded by genes at different loci.
io Fragments of the nucleic acid encoding MIPK are also within the scope of the invention. As used herein, a "fragment of the nucleic acid encoding MIPK" refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire amino acid i5 sequence of MIPK protein and which encodes a peptide or polypeptide of this invention, including a peptide or polypeptide having an activity of MIPK (i.e., a peptide having at least one biological activity of the MIPK
tyrosine kinase) as defined herein.
Preferred nucleic acid fragments encode peptides of at least about 10 amino acid residues in length, preferably at least about 20 amino acid residues in length, and more preferably at least about 35.
Nucleic acids encoding a peptide having an activity of MIPK will be selected from the bases encoding the mature protein, however, in some instances it may be desirable to select all or part of a peptide from the leader 3o sequence portion of the nucleic acids of the invention.
Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant peptides having an activity of MIPK.

A nucleic acid encoding a peptide having an activity of MIPK may be obtained from mRNA present in P. ochraceus or from other animals' nucleic acids encoding MIPK from seastar or other animals' genomic DNA. For example, the s gene encoding MIPK can be cloned from either a cDNA or a genomic library in accordance with protocols herein described. A cDNA encoding MIPK can be obtained by isolating total mRNA from seastar. Double stranded cDNAs can then be prepared from the total mRNA. Subsequently, to the cDNAs can be inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques. Genes encoding MIPK can also be cloned using established polymerase chain reaction (PCR) techniques in accordance with the nucleotide sequence information i5 provided by the invention. The nucleic acids of the invention can be DNA or RNA. A preferred nucleic acid is a cDNA encoding MIPK having the sequence depicted in SEQ
ID NO: 2 (mipk) .
zo This invention also provides expression vectors containing a nucleic acid encoding a peptide or polypeptide of the invention, operably linked to at least one regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a 2s regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences known in the art and are selected from the peptide or polypeptide of this invention. Accordingly, the term regulatory sequence includes promoters, enhancers and other 3o expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. In one embodiment, the expression vector includes a DNA encoding a peptide having an activity of MIPK. Such expression vectors can be used to transfect cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein.
This invention further provides to a host cell io transfected to express a peptide or polypeptide of this invention having an activity of MIPK. The host cell may be any prokaryotic or eukaryotic cell. For example, a peptide having an activity of MIPK may be expressed in bacterial cells such as E. coli, insect cells i5 (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO). Other suitable host cells can be found in Goeddel, (1990) supra or known to those skilled in the art.
2o DNA sequences of the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to 25 detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA
fragments with shared structural features; and 3) PCR
amplification of a desired nucleotide sequence using oligonucleotide primers. Laboratories which devote a 3o portion of their time to DNA sequencing services for contracting parties include Automated DNA Sequencing in Utah State University, Core Facility for Protein/DNA
Chemistry at Queen's University in Canada, and the DNA
Core Facility at San Diego University.

A preferred method for obtaining genomic DNA is the Polymerase Chain Reaction (PCR), which relies on an in vitro method of nucleic acid synthesis by which a particular segment of DNA is specifically replicated.
s Two oligonucleotide primers that flank the DNA fragment to be amplified are utilized in repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase. These primers io hybridize to opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the region between the primers. Since the extension products themselves are also complementary to and capable of binding primers, successive cycles of 15 amplification essentially double the amount of the target DNA synthesized in the previous cycle. The result is an exponential accumulation of the specific target fragment, approximately 2n, where n is the number of cycles of amplification performed (see PCR Protocols, 2o Eds. Innis, et al., Academic Press, Inc., 1990).
A cDNA expression library, such as lambda gtll, can be screened indirectly for MIPK peptides having at least one epitope, using antibodies specific for MIPK. Such 2s antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of MIPK cDNA.
The polynucleotide sequence of this invention also 3o include sequences complementary to the polynucleotide encoding MIPK including antisense sequences. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA
molecule (Weintraub, 1990). The invention embraces a11 antisense polynucleotides capable of inhibiting production of MIPK polypeptide. In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense s nucleic acids may interfere with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded, or alternatively, the double-stranded mRNA is degraded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily io synthesized, small enough to enter the cell, and are less likely to cause problems than larger molecules when introduced into the target MIPK-producing cell. The use of antisense methods to inhibit the translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem., 15 172:289, 1988) .
In addition, ribozyme nucleotide sequences for MIPK are included in the invention. Ribozymes are RNA molecules possessing the ability to specifically cleave other 2o single-stranded RNA in a manner analogous to DNA
restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it 25 (Cech, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
In the present invention, the MIPK polynucleotide 3o sequences may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the MIPK genetic sequences. Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific s genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., 1987), the pMSXND expression vector io for expression in mammalian cells (Lee and Nathans, 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or i5 polyhedrin promoters).
Polynucleotide sequences encoding MIPK can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms.
2o Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
Biologically active viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate 2s DNA sequences of the invention. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the MIPK coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro 3o recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic techniques. See, for example, the techniques described in Maniatis, et al., 1989.

A variety of host-expression vector systems may be utilized to express the MIPK coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the MIPK coding sequence; yeast transformed with recombinant yeast expression vectors containing the MIPK
coding sequence; plant cell systems infected with recombinant virus expression vectors (e. g., cauliflower 1o mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e. g., Ti plasmid) containing the MIPK coding sequence;
insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the MIPK coding sequence; or animal cell systems infected with recombinant virus expression vectors (e. g., retroviruses, adenovirus, vaccinia virus) containing the MIPK coding sequence, or transformed animal cell systems engineered for stable expression.
In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the expressed. For example, when large quantities of MIPK are to be produced, vectors which 2s direct the expression of high levels of fusion protein products that are readily purified may be desirable.
Those which are engineered to contain a cleavage site to aid in recovering are preferred. Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther, 1983), in which the MIPK coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid lac Z protein is produced; pIN
vectors (Inouye and Inouye, 1985; Van Heeke and Schuster, J., 1989) and the like. In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et s al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu and Grossman, 1987, Acad. Press, N.Y., Vol. l53, pp.516-544; Glover, l986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, l987, Heterologous Gene Expression in Yeast, io Methods in Enzymology, Eds. Berger and Kimmel, Acad.
Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds.
Strathern, et al., Cold Spring Harbor Press, Vols. I and II. A constitutive yeast promoter such as ADH or LEU2 or i5 an inducible promoter such as GAL may be used (Rothstein R., 1986.). Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
2o In cases where plant expression vectors are used, the expression of the MIPK coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson, et al., 1984), or the coat protein 2s promoter to TMV (Takamatsu, et al., 1987) may be used;
alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi, et al., 1984; Broglie, et al., 1984); or heat shock promoters, e.g., soybean hsp17.5-E
or hsp17.3-B (Gurley, et al., 1986) may be used.
Means of production of MIPK may be done using yeast, bacterial, or insect cell-based expression. Mammalian cells available in the art for expression of a heterologous protein include Chinese hamster ovary cells, HeLa cells, baby hamster kidney ells, COS cells and many others. A common, preferred bacterial host is E. coli.
Suitable vectors can be chosen or constructed, which s contain regulatory sequences appropriate to the goal including promoters, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details, see, for example, Molecular Cloning: a 1o Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Transformation procedures depend on the host used, but are well known.
One strategy to maximize recombinant MIPK expression in 15 E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, Calif. (1990) 1l9-128). Another strategy would 2o be to alter the nucleic acid encoding the MIPK protein to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al., (l992) Nuc. Acids Res., 20: 2111-25 21l8). Such alteration of nucleic acids of the invention can be carried out by standard DNA synthesis techniques.
The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of 3o chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al., U.S. Pat. No. 4,598,049; Caruthers et al., U.S. Pat. No.

4,458,066; and Itakura, U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
The present invention further pertains to methods of s producing peptides and polypeptides of this invention.
For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding a peptide having an activity of MIPK can be cultured under appropriate conditions to allow expression to of the peptide to occur. The peptide may be secreted and isolated from a mixture of cells and medium containing the peptide. Alternatively, the peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, 15 media and other byproducts. Suitable media for cell culture are well known in the art. The desired peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel 2o filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for a peptide having an activity of MIPK.
2s Another aspect of the invention pertains to isolated peptides having an activity of MIPK. A peptide having an activity of MIPK has at least one biological activity of MIPK. A peptide having an activity of MIPK may differ in amino acid sequence from the MIPK sequence depicted in 3o SEQ ID NO: 1 but such differences result in a modified protein with activities in the same or similar manner as a native MIPK protein or which has the same or similar characteristics of a native MIPK protein. Various modifications of the MIPK protein to produce these and other active equivalent peptides are described in detail herein.
A peptide can be produced by modification of the amino acid sequence of the MIPK protein shown in SEQ ID N0: 1 (MIPK), such as a substitution, addition, or deletion of an amino acid residue which is not directly involved in the activity of the protein. Peptides of the invention can be at least about 10 amino acid residues in length, io preferably about greater than 20 amino acid residues in length, and more preferably greater than about 30 amino acid residues in length. Peptides having an activity of MIPK and which are at least about 30 amino acid residues in length, at least about 40 amino acid residues in i5 length, at least about 60 amino acid residues in length, at least about 80 amino acid residues in length, and at least about 100 amino acid residues in length are also included within the scope of this invention.
2o The biological activity, for example, can vary from a polypeptide fragment (e.g. an epitope to which an antibody can bind (10 to 20 amino acids, such as MIPK-CT
peptide KELTFQLIQAVRHQSRR residues 347-363 (SEQ ID N0:6) and MIPK-NT peptide TGSGETLSDDGYHRYE residues 6-21 (SEQ
2s ID N0:7) to a large polypeptide which is capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An enzymatically functional MIPK polypeptide or fragment thereof possesses MIPK tyrosine kinase activity.
The terms peptide, protein and polypeptide are used interchangebly herein.

An "active polynucleotide" denotes a polynucleotide which encodes a active polypeptide as described herein.
Minor modifications of the MIPK primary amino acid s sequence may result in proteins which have substantially equivalent activity as compared to the MIPK polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous.
All of the polypeptides produced by these modifications io are included herein as long as the tyrosine kinase activity of MIPK is present. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its kinase activity. This can lead to the i5 development of a smaller active molecule which may have broader utility. For example, it is possible to remove amino or carboxyl terminal amino acids which may not be required for MIPK kinase activity.
2o The MIPK polypeptide of the invention also includes conservative variations of the polypeptide sequence. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative 2s variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, 3o and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

Another embodiment of the invention provides a substantially pure preparation of a peptide having an activity of MIPK. Such a preparation is substantially free of proteins and peptides with which the peptide naturally occurs (i.e., other MAP kinase proteins), either in a cell or when secreted by a cell.
The term "isolated" or "substantially pure" as used herein refers to a nucleic acid or peptide that is io substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Such proteins or peptides are also characterized as being free of a11 other MAP kinase proteins. Accordingly, an isolated peptide having an activity of MIPK is produced recombinantly or synthetically and is substantially free of cellular material and culture medium or substantially free of chemical precursors or other chemicals and is 2o substantially free of all other MAP kinase proteins. An isolated nucleic acid is also free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the organism from which the nucleic acid is derived.
Peptides of this invention can be obtained, for example, by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid of MIPK
encoding such peptides. In addition, fragments can be 3o chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, the MIPK protein may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptides having a MIPK
activity.
It is possible to modify the structure of a peptide of this invention for such purposes as increasing solubility, enhancing therapeutic or prophylactic efficacy, or stability (e.g., shelf life ex vivo and to resistance to proteolytic degradation in vivo). Such modified peptides are considered activityal equivalents of peptides having an activity of MIPK as defined herein.
A modified peptide can be produced in which the amino acid sequence has been altered, such as by amino acid i5 substitution, deletion, or addition, to modify activity, or to which a component has been added for the same purpose. Preferred amino acid substitutions for non-essential amino acids include, but are not limited to substitutions with alanine, glutamic acid, or a methyl 2o amino acid.
Another example of modification of a peptide having an activity of MIPK is substitution of cysteine residues preferably with alanine, serine, threonine, leucine or 25 glutamic acid residues to minimize dimerization via disulfide linkages. In addition, amino acid side chains of fragments of the protein of the invention can be chemically modified. Another modification is cyclization of the peptide or removal of the amino or carboxyl 3o terminal amino acids residues. Additionally, a peptide having an activity of MIPK can be modified by replacing an amino acid shown to be essential for MIPK kinase activity.

The MIPK polypeptide of the invention also include conservative variations of the polypeptide sequence. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, to glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also i5 immunoreact with the unsubstituted polypeptide.
In order to enhance stability and/or reactivity, a peptide having an activity MIPK can be modified to incorporate one or more polymorphisms in the amino acid 2o sequence of the MIPK protein resulting from any natural allelic variation. Additionally, D-amino acids, non-natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention.
Site-directed mutagenesis of a nucleic acid encoding a peptide having an activity of MIPK can be used to modify the structure of the peptide by methods known in the art.
Such methods may, among others, include polymerase chain 3o reaction (PCR) with oligonucleotide primers bearing one or more mutations (Ho et al., (1989) Gene, 77: 51-59) or total synthesis of mutated genes (Hostomsky, Z. et al., (1989) Biochem. Biophys. Res. Comm. l61: 1056-1063). To enhance recombinant protein expression, the aforementioned methods can be applied to change the codons present in the cDNA sequence of the invention to those preferentially utilized by the host cell in which the recombinant protein is being expressed (Wada et al., s supra ) .
Another aspect of the invention pertains to an antibody specifically reactive with a peptide or polypeptides of this invention. The antibodies of this invention can be to used to standardize MAP kinase activity or to isolate the naturally-occurring or native form of MIPK. For example, by using peptides having an activity of MIPK based on the cDNA sequence of MIPK, anti-protein/anti-peptide antisera or monoclonal antibodies can be made using standard i5 methods. A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., MIPK protein or an antigenic fragment which is capable of eliciting an antibody response). Techniques for conferring immunogenicity on a protein or peptide 2o include conjugation to carriers or other techniques well known in the art. A peptide having an activity of MIPK
can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or 2s other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.
Following immunization, anti-MIPK antisera can be obtained and, if desired, polyclonal anti-MIPK antibodies 3o isolated from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, for example the hybridoma technique originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497) as well as other techniques such as the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of to antibodies specifically reactive with a peptide having an activity of MIPK and the monoclonal antibodies isolated.
The term "antibody" as used herein is intended to include fragments thereof which are also specifically reactive with a peptide or polypeptide of this invention.
Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating 2o antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention is further intended to include bispecific and chimeric molecules having an anti-MIPK portion.
The invention also provides a method for detecting a cell proliferative disorder associated with MIPK in a subject, comprising contacting a target cellular component containing MIPK, with a reagent which detects 3o MIPK. The target cell component can be nucleic acid, such as DNA or RNA, or it can be protein. When the component is nucleic acid, the reagent is a nucleic acid probe or PCR primer. When the cell component is protein, the reagent is preferably an antibody probe. The probes can be detestably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.
Preferred antibodies according to the invention are isolated, in the sense of being free form contaminants such as antibodies able to bind other polypeptides and or free of serum components. Monoclonal antibodies are io preferred for some purposes, although polyclonal antibodies may be capable of distinguishing the polypeptides. Antibodies are tested for affinity to immunizing peptide by ELISA, and tested for cross-reactivity to Seastar MIPK purified extract as well i5 as various rat tissue extracts using Western blot technique.
Antibodies that may be used in the identification and characterization of MIPK such as: CDK5-CT, ERKl-CT, 2o Cdc2-CT, PSTAIRE, Cdc2-IX, and CdCK-2B may be prepared as described by Sanghera et al., 1992 or obtained commercially from Upstate Biotechnology Ins. (Lake Placid). Immunizing peptides may be produced using an ABI automated peptide synthesizer. The peptide is 2s cleaved from synthesis resin, and purified by reverse-phase HPLC. Purity may be assessed by analytical RP-HPLC and the sequence confirmed by amino acid analysis. The peptide may be coupled to KLH prior to immunization into rabbits. New Zealand white rabbits may 3o be subcutaneously injected with KLH-coupled immunizing peptide in PBS with Freund's incomplete adjuvant every 4 weeks. The sera from these animals may be applied onto an agarose column with 0.1 M glycine, pH 2.5.
Subsequently the antibody solution may be neutralized to pH 7.0 with saturated Tris. Antibody affinities may be assessed by ELISA.
Such peptides can also be used to study the mechanism of MAP kinase activity and to design modified derivatives or analogs useful in therapeutic treatment of proliferative diseases, such as cancer and atherosclerois, and inflammatory diseases such as psoriasis, rhematoid arthritis, multiple sclerosis and tissue rejection. It is to now also possible to design an agent or a drug capable of blocking or inhibiting the ability of MIPK to act as a protein kinase. Such agents could be designed, for example, in such a manner that they would bind to MIPK, thus preventing malfunction.
Diagnostic Screens The invention includes a method for determining if a subject is at risk of a disorder characterized by MAP
2o kinase mafunction. In preferred embodiments, the subject method can be generally characterized as comprising detecting, in a tissue of the subject (e. g. a human patient), the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding one of the subject MIPKs or (ii) the misexpression of an MIPK gene. To illustrate, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a subject MIPK gene, (ii) an 3o addition of one or more nucleotides to such an MIPK gene, (iii) a substitution of one or more nucleotides of an MIPK gene, (iv) a gross chromosomal rearrangement of one of the subject MIPK genes, (v) a gross alteration in the level of a messenger RNA transcript of an MIPK gene, (vi) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an MIPK gene, and (vii) a non-wild type level of an MIPK protein. One aspect of the invention may provide a nucleic acid probe or a PCR
primer comprising an oligonucleotide containing a region of the nucleotide sequence (SEQ ID N0:2) which is capable of hybridizing to a sense or antisense, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the io subject MIPK genes. The probe is exposed to nucleic acid of a tissue sample; and the hybridization of the probe to the sample nucleic acid is detected. In certain embodiments, detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,l95 and 4,683,202) or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science, 241:1077-l080; and Nakazawa et al. (1944) PNAS 91:360-364) the later of which can be particularly useful for detecting point 2o mutations in the MIPK gene. Alternatively, the level of MIPK protein can detected in an immunoassay.
Anti-sense techniques (e. g. microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to an MIPK mRNA or gene sequence) can be used to inhibit endogenous production of MIPK as well as to investigate the role of and the normal cellular activity of each MIPK. Such techniques can be utilized in cell culture, but can also 3o be used in the creation of transgenic animals.

Drug Screening Assays The invention includes assays which can be used to screen for drugs which are either agonists or antagonists of the s normal cellular activity of the subject MIPK proteins or their role in cellular activity. In one embodiment, the assay evaluates the ability of a compound to modulate protein kinase activity of MIPK. A variety of assay formats will suffice and, in light of the present io inventions, will be comprehended by skilled artisan.
Transgenic Animals The invention includes transgenic animals which include 15 cells (of that animal) which contain an MIPK transgene and which preferably (though optionally) express (or misexpress) an endogenous or exogenous MIPK protein in one or more cells in the animal. The MIPK transgene can encode the wild-type form of the protein, or can encode 2o homologs thereof, including both agonists and antagonists, as well as antisense constructs. Tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns. Temporal patterns 2s of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
Genetic techniques which allow for the expression of 3o transgenes, that are regulated in vivo via site-specific genetic manipulation, are known to those skilled in the art. For example, genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination a target sequence. As used herein, the phrase "target sequence" refers to a nucleotide sequence that is genetically recombined by a recombinase. The target sequence is flanked by recombinase recognition sequences and is generally either s excised or inverted in cells expressing recombinase activity. Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of the subject MIPK polypeptide. For io example, excision of a target sequence which interferes with the expression of a recombinant MIPK gene, such as one which encodes an antagonistic homolog, can be designed to activate expression of that gene. This interference with expression of the protein can result i5 from a variety of mechanisms, such as spatial separation of the MIPK gene from the promoter element or an internal stop codon. Moreover, the transgene can be made wherein the coding sequence of the gene is flanked recombinase recognition sequences and is initially transfected into 2o cells in a 3' to 5' orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter 2s driven transcriptional activation.
In an illustrative embodiment, either the cre-loxP
recombinase system of bacteriophage P1 (Lakso et al.
(l992) PNAS 89:6232-6236; Orban et al. (1992) PNAS
30 89:686l-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 25l:1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences. loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination. The orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:l509-5l4);
catalyzing the excision of the target sequence when the to loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP
sequences are oriented as inverted repeats.
Accordingly, genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added 2o agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element. Thus, the activation expression of the recombinant MIPK protein can be regulated via control of recombinase expression.
Use of the cre-loxP recombinase system to regulate expression of a recombinant MIPK protein requires the construction of a transgenic animal containing transgenes 3o encoding both the Cre recombinase and the subject MIPK
protein. Animals containing both the Cre recombinase and a recombinant MIPK gene can be provided through the construction of "double" transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., an MIPK gene and recombinase gene.
One advantage derived from initially constructing transgenic animals containing a MIPK transgene in a recombinase-mediated expressible format, particularly derives from the likelihood that the subject protein will be deleterious upon expression in the transgenic animal.
In such an instance, a founder population, in which the io subject transgene is silent in a11 tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues. Thus, the creation of a founder population in which, for example, an antagonistic MIPK transgene is silent will allow the study of progeny from that founder in which disruption of MIPK mediated induction in a particular tissue or at developmental stages would result in, for example, a lethal phenotype.
Similar conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene. Exemplary 2s promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Pat. No.
4,833,080. Moreover, expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, 3o e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner. By this method, the MIPK
transgene could remain silent into adulthood until "turned on" by the introduction of the trans-activator.

In an exemplary embodiment, the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
Embryonic target cells at various developmental stages s can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonic target cell. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in io diameter which allows reproducible injection of 1-2 pl of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS
1s 82:4438-4442). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the 2o transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.
Retroviral infection can also be used to introduce 2s transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS
73:1260-1264). Efficient infection of the blastomeres is 30 obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (l985) PNAS
82:6927-6931; Van der Putten et al. (1985) PNAS 82:6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al.
(1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al.
(1982) Nature 298:623-628). Most of the founders will be io mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al . ( 1982 ) supra) .
2o A third type of target cell for transgene introduction is the embryonic stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (l986) Nature 322:445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human 3o animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240:l468-1474.

Methods of making knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
s Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert recombinase target sequences flanking portions of an endogenous MIPK
gene, such that tissue specific and/or temporal control of inactivation of an MIPK allele can be controlled as i o above .
Gene Therapy The gene constructs of the invention can also be used as i5 a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic agent.
One application is to inhibit MIPK in terminally differentiated tissue, for example to contribute to a release of neurons or cardiomyocytes from cell cycle 2o block. Thus, another aspect of the invention features expression vectors for in vivo transfection and expression of an MIPK protein in particular cell types so as to reconstitute the activity of, or to deliver a form of the protein which inhibits MIPK malfunction by 2s interfering with the biological activity of MIPK.
Expression constructs of the subject MIPK proteins, and mutants thereof, may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the MIPK gene to cells 3o in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids.
Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e. g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular s carriers, as well as direct injection of the gene construct or CaP04 precipitation carried out in vivo.
Because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend io on such factors as the phenotype of the intended target and the route of administration, e.g., locally or systemically. Furthermore, it will be recognized that the particular gene construct provided for in vivo transduction of MIPK expression may also be useful for in i5 vitro transduction of cells, such as for use in the diagnostic assays described above.
A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing 2o nucleic acid, e.g., a cDNA, encoding the MIPK protein.
Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral 2s vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the 3o transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only s replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:27l). Thus, recombinant to retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the subject receptors rendering the retrovirus replication defective.
The replication defective retrovirus is then packaged i5 into virions which can be used to infect a target cell through the use of a helper virus by standard techniques.
Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such-viruses can be found in Current Protocols in Molecular Biology, 2o Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (l989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus 2s lines for preparing both ecotropic and amphotropic retroviral systems include psi Crip, psi Cre, psi 2 and psi Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see 3o for example Eglitis, et al. (1985) Science 230:1395-1398;
Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA
85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.
USA 85:30l4-3018; Armentano et al. (1990) Proc. Natl.
Acad. Sci. USA 87:6141-6l45; Huber et al. (1991) Proc.

Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (199l) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al.
(l991) Science 254:l802-l805; van Beusechem et al. (l992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al.
(l992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al.
(1993) J. Immunol. 150:4l04-41l5; U.S. Pat. No.
4,868,1l6; U.S. Pat. No. 4,980,286; PCT Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO
89/05345; and PCT Application WO 92/07573).
In choosing retroviral vectors as a gene delivery system for the subject MIPK gene, successful infection of target cells by most retroviruses, and therefore stable introduction of the recombinant MIPK gene, may require that the target cells be dividing. With certain exceptions, such as lymphatic cancers, such a requirement will not be a hindrance to use of retroviral vectors. In fact, where gene therapy constructs of the present 2o invention, such as antagonistic forms of MIPK, are intended to be delivered to cells containing malafunctioning MIPK, such limitation on infection can be beneficial in that the tissue (e. g., nontransformed cells) surrounding the target cells do not likely undergo 2s as extensive cell division and is therefore somewhat refractory to infection with retroviral vectors.
It is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based 3o vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT
publications W093/25234 and W094/06920). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (l989) PNAS 86:9079-9083; Julan et al. (l992) J. Gen Virol 73:325l-3255; and Goud et al.
(1983) Virology 163:251-254); or coupling cell surface receptor ligands to the viral env proteins (Neda et al.
(l991) J Biol Chem 266:14143-14146). Coupling can be in the form of the chemical cross-linking with a protein or other variety (e. g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating to fusion proteins (e. g., single-chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, can also be used to convert an ecotropic vector in to an amphotropic vector.
The use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the MIPK gene of the retroviral vector.
Another viral gene delivery system useful in the present invention used adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (199l) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
Suitable adenoviral vectors derived from the adenovirus 3o strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al. (l992) cited supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be s modified so as to affect the spectrum of infectivity.
Additionally, introduced adenoviral DNA (and foreign DNA
contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of to insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e. g., retroviral DNA). The carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al.
i5 cited supra; Haj-Ahmand and Graham (1986) J. virol.
57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral 2o genetic material (see, e.g., Jones et al. (1979) Cell l6:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp, l09-l27). Expression of the inserted MIPK gene can be under control of, for 2s example, the ElA promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
Another viral vector system useful for delivery of the 3o subject MIPK gene is the adeno-associated virus (AAV).
Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol.
(l992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (l992) Am. J Respir. Cell. Mol.
Biol. 7:349-356; Samulski et al. (1989) J. Virol.
63:3822-3828; and McLaughlin et al. (1989) J. Virol.
62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for 1o exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol.
Cell Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl Acad. Sci. USA
81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol.
4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol.
2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619;
and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).
In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of an MIPK protein in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject MIPK gene by the targeted cell.
3o Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.

In a representative embodiment, a gene encoding one of the subject MIPK proteins can be entrapped in liposomes bearing positive charges on their surface (e. g., lipofectins) and (optionally) which are tagged with s antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551;
PCT publication W091/06309; Japanese patent application l047381; and European patent publication EP-A-43075). For example, lipofection of cells containing malfunctioning io MIPK can be carried out using liposomes tagged with monoclonal antibodies against, for example, squamous cells.
In clinical settings, the gene delivery systems for the 15 therapeutic MIPK gene can be introduced into a patient by methods, known to those skilled in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in 2o the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other 2s embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No.
5,328,470) or by stereotactic injection (e.g., Chen et 3o al. (1994) PNAS 91: 3054-3057).
The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced in tact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation s can comprise one or more cells which produce the gene delivery system.
Antisense Therapy io Another aspect of the invention relates to the use of the isolated nucleic acid in "antisense" therapy. As used herein, "antisense therapy" refers to administration or in situ generation of oligonucleotides or their derivatives which specifically hybridizes (e. g. binds) 15 under cellular conditions, with the cellular mRNA and/or genomic DNA encoding an MIPK protein so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation. One application would be to inhibit expression of MIPK in terminally zo differentiated tissue in order to contribute to regeneration of that tissue. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In 2s general, antisense therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
3o An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an MIPK protein.

Alternatively, the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an MIPK gene. Such oligonucleotide probes are preferably modified oligonucleotide which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo.
Exemplary nucleic acid molecules for use as antisense io oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (l988) Cancer Res 48:2659-2668.
Accordingly, the modified oligomers of the invention are useful in therapeutic, diagnostic, and research contexts.
2o In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general.
For such therapy, the oligomers of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous for 3o injection, the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included in the invention.
The compounds can be administered orally, or by s transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration bile salts and io fusidic acid derivatives, and detergents. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical i5 administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as known in the art.
In addition to use in therapy, the oligomers of the 2o invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA
sequences to which they specifically bind. Such diagnostic tests are described in further detail herein.
2s The antisense constructs of the present invention, by antagonizing the normal biological activity of MIPK, can be used in the manipulation of tissue, both in vivo and in ex vivo tissue cultures.
3o EXAMPLES
Degenerate sense and antisense oligonucleotides were designed based on protein microsequence data of tryptic fragments of purified MIPK from P. ochraceus oocytes compared to p38 sequences, and were used to amplify portions of the MIPK cDNA by PCR. Similarly, primers were derived for use in RACE, cloning and sequencing the cDNA, and for PCR site directed mutagenesis.
The following antibodies were used:
Anti-Cdc2 Kinase-C-Terminus recognizes the 34 kDa protein encoded by the cdc2 gene in various mammalian io species. In rat tissues, it cross reacts with 46 kDa and 70 kDa proteins. It recognizes p34cdc2 from mouse, rat, sheep and human. The immunogen was a 36 residue synthetic peptide, based upon the C-terminal residues 263-297 of the mouse 34 kDa cdc2-encoded protein kinase (Cisek et al. l989):
(Cdc2-IX): Anti-Cdc2 Kinase-IX recognizes the 34 kDa protein encoded by the cdc2 gene in diverse species. It recognizes p34cdc2 from sea star, mouse, rat, sheep and zo human. The immunogen was a 25 residue synthetic peptide based upon residues 202-223 of the mouse 34 kDa cdc2-encoded protein kinase (Cisek et al., 1989) in catalytic subdomain IX region (Hanks et al., 1988).
(CDK5-CT): Anti-Cyclin-Dependent Kinase 5-C-Terminus recognizes the 32 kDa protein purified from bovine brain (Lew et al., 1992), which corresponds to cyclin-dependent protein kinase 5. It recognizes CDK5 from cow and human.
The immunogen was a 16 residue synthetic peptide based 3o upon the C-terminal residues 268-283 of the human 31 kDa CDK5- (or PSSALRE kinase) encoded protein kinase (Hanks, l987; Meyerson et al., 1992). This antibody is available from Upstate Biotechnology Inc. (Lake Placid, N.Y., USA) under catalogue no. 06-258.

(ERK1-CT): Anti-Erkl-C-Terminus recognizes the 43 kDa MAP
kinase encoded by the Erkl gene, 42 kDa MAP kinase encoded by the ERK2 gene and the 44 kDa MAP kinase encoded by the seastar Mpk gene. It also appears to recognize other putative MAP kinases in the range of 40 to 70 kDa. Recognizes the various MAP kinases in cytosolic and nuclear extracts from sea star, clam, frog, chicken, mouse, rat, sheep and human. The immunogen was a 38 residue peptide based upon residues 333-367 of the to rat 43 kDa Erkl MAP kinase (Boulton et al., 1990), which corresponds to the C-terminus of the protein. This antibody is available from Upstate Biotechemisty Inc.
(Lake Pacid, N.Y., USA) under catalogue no. 06-194.
Anti-Cdc2 Kinase-Subdomain III-PSTAIRE region (PSTAIRE) recognizes p34cdc2 from fission yeast, Paramecium, Dictyostelium, sea star, frog, chicken, mouse, rat, sheep and human. The immunogen was a 19 residue synthetic peptide based upon residues 42-57 of the human 34 kDa 2o Cdc2-encoded protein kinase (Lee et al., 1987) in kinase subdomain III region according to the classification of Hanks et al. (1988).
(Mipk-CT): Anti-Maturation-inhibited Protein Kinase-C
Terminus recognizes recombinant expressed MIPK and purified seastar Mipk. The immunogen was a 19 residue synthetic peptide based upon residues 347-363 of seastar MIPK.
3o Anti-Cyclin Dependent Kinase 6 (Santa Cruz) Cdk6 (C-21):is an affinity purified rabbit polyclonal antibody raised against a peptide corresponding to as 306-326 of human Cdk6.

4G10 Antiphosphotyrosine monoclonal (Upstate Biotechnology Inc.) recognizes phosphotyrosine containing proteins by immunoprecipitation, Western blotting and cell staining.
(A) Extraction of Proteins from P. ochraceus oocyte P. ochraceus oocyte isolation 1o Ovaries were surgically removed from the arms of the P.ochraceus harvested from the beaches around Vancouver, Canada, and kept on ice in sea water (sea water obtained from the Department of Fisheries & Oceans, West Vancouver Laboratory, 4160 Marine Drive, West Vancouver, Canada, which acquired it 50 feet below the surface of the ocean and 360 feet off shore; this is relatively normal seawater composition). Ovaries were first gently teased apart with forceps and razor blades to release the oocytes, then strained through a large mesh filter ("Hiwei" brand miniature food strainer or similar model) to remove connective tissue and residue. Oocytes thus extracted were then washed three times with cold sea water, and pelleted by centrifugation at 400 x g for 5 min.

Induction of maturation Four hundred mL of the resultant packed oocytes were suspended in 4 L of sea water containing 4 ~.M
1-methyladenine (Sigma Chemical Co.) at 14EC for 90-120 min. Maturation was defined by the onset of germinal vesicle breakdown ("GVBD") as determined by the disappearance of the nucleus within the oocyte when viewed under 100X magnification. Mature oocytes were harvested io when GVBD occurred in over 80% of the oocytes, about 2 hours after initiation of maturation.
Measured volumes of oocyte suspension were removed at discrete time points after the addition of 1-methyladenine for time related measurements.
P. ochraceus oocyte homogenization Oocytes were pelleted by centrifugation at 400 x g for 5 2o min. To each 200 mL of packed cells were added 400 mL of homogenization buffer (50 mM (-glycerophosphate, 20 mM
3-[N-morpholino]propanesulfonic acid ("MOPS"), 5 mM
ethylenebis(oxyethylenenitrilo)tetraaceticacid ("EGTA"), 2 mM ethylenediaminetetraaceticacid ("EDTA"), 1 mM Na3V04, 0.25 ~M dithiothreitol ("DTT"), 5.0 uM -methylaspartic acid, 1.0 mM phenylmethylsulphonylfluoride("PMSF"), 1.0 mM benzamidine, pH 7.2). The oocytes were homogenized in a blaring blender in two 15 s bursts, and centrifuged at 9000 rpm for 15 min in a Beckman J2-HS centrifuge to 3o remove particulate matter and organelles. The post-mitochondrial supernatant was centrifuged in a Sorval Combi Ultracentrifuge (Dupont, Canada) at 250,000 x g for min and the supernatant immediately aliquoted and frozen at -70~C until required.

Analysis of P. ochraceus oocyte extracts for tyrosine phosphorylated proteins Differentiation or identification of kinases is performed s by testing for kinase activity (tyrosine, serine, or threonine) and immune reactivity with various established kinase reactive antibodies. Molecular weight is another characteristic used to separate out different types of kinases. Once such an identification is made, the DNA and io amino acid sequences of the new kinase maybe compared with those of other kinases using PCR.
To identify proteins undergoing changes in tyrosine phosphorylation with oocyte maturation, cytosolic extracts i5 from immature and mature stage oocytes were fractionated on a 1 mL ResourceQ (PHARMACIA) anion exchange column.
Columns were equilibrated before and after use with 2 mL
of 2.0 M NaCl, and a11 buffers were filtered (0.22 ~,), and samples filtered (0.45 ~,) before application to the 2o column. Five mg of protein were diluted to 2.1 mL with buffer A (10 mM MOPS, pH 7.2, 25 mM (glycerophosphate, 2 mM EDTA, 5 mM EGTA, 2 mM Na3V04). Two mL of sample were applied to the column and a standard elution program including a 10 mL 0-0.8 M NaCl linear gradient was 25 performed. Fractions of 500 ~L were collected, further separated by SDS-PAGE (Laemmli, l970), and subjected to western blotting using the anti-phosphotyrosine antibody 4G10. Proteins were boiled for 5 min with 1 volume of 4x sample loading buffer (125 mM Tris-HC1 (pH 6.8), 4% SDS
30 (w/v), 20% glycerol (v/v), 0.3 M (mercaptoethanol, 0.01%
bromophenol blue (w/v)). Proteins were subjected to electrophoresis on 1.5 mm thick polyacrylamide gels with 4% stacking gels and loo separating gels. The gels were electrophoresed in running buffer (25 mM Tris, 192 mM
35 glycine, 3.5 mM SDS) at 10 mA overnight, until the bromophenol blue reached the bottom of the gel. The gel was then equilibrated in transfer buffer (20 mM Tris, 120 mM glycine, 20% methanol (v/v), pH 8.6) to remove SDS.
Nitrocellulose membrane was hydrated in transfer buffer s for at least 1 min before transfer. Proteins were electrophoretically transferred in a Hoeffer transfer cell at 4~C for 3 h at 300 mA. For 4G10 blotting, membranes were blocked overnight at room temperature using low-salt TBS (20 mM Tris, pH 7.5, 50 mM NaCl) containing 3% bovine to serum albumin ("BSA") (w/v). Primary antibody was incubated for 4 h at room temperature, and alkaline phosphatase conjugated secondary antibody incubated for 2 h. A11 washes were performed using low-salt TBS
containing 0.05% Nonidet-P40 (NP-40). A11 other Western i5 blots were blocked in 5% skim milk (w/v) in TBS (50 mM
Tris base, 150 mM NaCl, pH 7.5) for 2 h. Primary antibody was diluted to the optimum concentration (usually 1/500-1/1000), in TTBS (0.05% Tween-20 in TBS) with 0.1%
azide (w/v) and incubated with the membrane overnight at 2o room temperature with agitation. The blots were washed extensively before incubation with the alkaline phosphatase conjugated secondary antibody, diluted to the optimum concentration in TTBS (1/2000) for 2 h at room temperature. Excess secondary antibody was removed by 2s thoroughly washing the membranes with TTBS, and the Tween-20 rinsed away with TBS. Western blots were developed in 5-bromo-4-chloro-3-indolyl phosphate ("BCIP")/nitro blue tetrazolium ("NBT") colour development solution (mixture of 3% NBT in 1 mL 70% dimethylformamide 30 ("DMF") and 1.5% BLIP in 1 mL 100% DMF before addition of 100 mL of AP buffer (0.1 M Tris, pH 9.5, 0.1 M NaCl, 5 mM
MgCl2) ) .
Several major bands of tyrosine phosphorylation were 35 identified using this procedure, some of which were undergoing significant changes in response to 1-methyladenine (Sigma Chemical Co.) stimulation. To characterize these 4G10 immunoreactive proteins, antibodies were used to assess elution profiles of various known tyrosine phosphorylated proteins.
The predominant protein showing an increase in phosphotyrosine signal was 44 kDa in size. Western blotting with the ERK1-CT antibody indicated that this to protein belonged to the MAP kinase family and further characterization confirmed the identification of this protein as the previously characterized p44MPK referenced in Sanghera et al. (1990).
i5 A major band shown to decrease in tyrosine phosphorylation was identified as a member of the cyclin dependent kinase ("CDK") family through cross reactivity with a polyclonal PSTAIRE antibody known to bind to CDK1 and CDK2 in the sea star oocyte system. Western blotting with a specific CDK1 2o antibody confirmed that the tyrosine phosphorylation occurred on CDK1 in the immature oocyte cytosol, and that it was activated in response to 1-methyladenine. CDK1 was first identified in the oocyte system as a histone H1 kinase that underwent activation with oocyte maturation 25 (Pelech et al., 1987; Meijer et al., 1987).
One protein showed a decrease in tyrosine phosphorylation with oocyte maturation. It was a 40 kDa protein that was found to cross react with a polyclonal CDK5-CT antibody, 3o but not ERK1-CT. Cyclin-dependent kinase 5 (Cdk5, also called PSSALRE, or "nclk" for neuronal cdc2-like kinase) expression has been noted as being high in adult mouse brain and in human heart, placenta, kidney, lung, liver, pancreas, and skeletal muscle (Meyerson et al., 1992).
35 The 40 kDa protein did not exhibit the expected molecular mass of known Cdk5-like proteins (Meyerson et al., 1992) and required further characterization to identify it.
CDK5-CT has been known to recognize Cdk5 in other systems and close analysis of the immunizing peptide used to generate the antibody revealed the potential for crossreactivity within the CDK and MAP kinase families.
This and the molecular weight indicate a close relationship to the MAP kinase family. The protein was named Maturation-Inhibited Protein Kinase ("MIPK") since to tyrosine dephosphorylation of MAP kinases is correlated with their activation.
(B) Initial Characterization of MIPK
i5 To confirm that MIPK was the protein initially identified through western blotting with 4G10, immunoprecipitation experiments were performed. Cytosolic extracts were brought to 1% SDS (w/v) with the addition of 20% SDS. The extracts were then diluted with and equal volume of 6%
2o NETF (6% Nonidet P-40 ("NP-40") (v/v) in NETF buffer (100 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl (pH 7.4), 50 mM NaF)).
Ten ~g of antibody were incubated with the denatured cytosolic extracts for 1 h at 4~C with rotation. To the mixture were added 20 ~L of Protein A-Sepharose CL4B
25 (Pharmacia) 1:1 slurry in 3%NETF (3% NP-40 (v/v) in NETF
buffer) and the antibody was allowed to complex for 45 min at 4~C with rotation. The beads were washed 2 x with 6%NETF and once with NETF. Immunoprecipitates were boiled for 5 min in 4x SDS sample buffer and subjected to 3o SDS-PAGE and Western blotting as described. Under denaturing conditions (1% SDS), MIPK could be immunoprecipitated with the CDK5-CT antibody coincident with a depletion of phosphotyrosine signal in the ResourceQ (Pharmacia) fractions . Immunoprecipitated MIPK
35 was also subjected to Western blotting with a panel of antibodies from the MAP kinase and cyclin-dependent kinase superfamilies of protein kinases. Only the CDK5-CT
antibody crossreacted with MIPK while two other Cdk5 antibodies failed to crossreact. MIPK also did not react s with Cdc2-NT, an antibody known to immune react with Cdk5 (Meyerson et al., 1992). This data, along with the different molecular weight, strongly indicates that MIPK
is not the sea star cognate of Cdk5.
to The 44 kDa protein identified in the immunoprecipitation Western blotted effectively with Erkl-CT, but not with CDK5-CT. The crossreactivity with Erkl-CT indicated that this protein was p44MPK and this was confirmed through Western blotting with a polyclonal anti-p44MPK antibody.
Dephosphorylation of MIPK Durin~~ Oocyte Maturation The cross-reactivity of CDK5-CT with MIPK indicated that MIPK could be a member of the MAP kinase superfamily of 2o protein kinases. This family is activated via tyrosine and threonine phosphorylation in a position equivalent to the Thr-Glu-Tyr site in the activation loop of ERK1. It is therefore possible to predict MAP kinase activity based on phosphotyrosine levels. The effects over time of 2s dephosphorylation and potential inhibition of MIPK was assessed during 1-methyladenine-induced maturation of the oocyte. At discreet time points following 1-methyladenine addition, aliquots of oocytes were removed and processed to yield cytosolic extracts. Immunoprecipitation with 3o CDK5-CT was used to isolate MIPK. Eighty percent of the immunoprecipitate was analyzed for phosphotyrosine levels, while the remainder was used to assess MIPK protein levels through Western blotting with CDK5-CT.

Densitometric analysis was used to quantify the phosphotyrosine signals, which could then be plotted (Figure 2). Although there was some variability during the first 15 min after 1-methyladenine stimulation, a s consistent decrease in phosphotyrosine levels was evident through the maturation process. A minimum signal was observed at the 50 min time point, coincident with the appearance of tyrosine phosphorylation of MIPK. This activation of MIPK continued through the maturation to process to full activity at the end of maturation, which occurred at time point 90-120 min post-1-methyladenine addition. The inactivation of MIPK was complete in a period prior to the activation of known maturation activated protein kinases including p44MPK, CDK1, and 15 ribosomal S6 kinase (Pelech et al., 1988).
(C) Purification of MIPK from P. ochraceus oocytes MIPK was purified from P. ochraceus oocyte cytosol using 2o sequential column chromatography steps, leading to the isolation of the protein for micropeptide sequencing.
Hydroxylapatite columns were made (the sample was split among 24 such columns) by resuspending 10 g of dry hydroxylapatite resin (BioRad) in 50 mL KII buffer to 25 obtain a smooth slurry. This slurry was diluted to 500 mL
with KII and allowed to settle (2-3 min). KII containing fines was poured off and the resin was resuspended in 500 mL KII buffer, allowed to settle, and supernatant was poured off. The resin was then packed in a 1" diameter 3o Flex column (Konte brand, distributed by VWR in Mississauga, Canada).
Approximately 16 g of cytosolic extract was thawed, diluted to 1:l0 with KII buffer (5 mM MOPS, pH 7.2, 5 mM
35 EGTA, 5 mM NaF, 1 mM Na2V03, and 0.25 mM DTT) and applied to previously described 24 (10 g) hydroxyapatite columns.
MIPK was eluted with 50-100 mL per each column of 40 mM
potassium phosphate buffer, pH 7.2. A total of 1 L of eluate was diluted to 6 L with KII and applied to six 25 mL Q Sepharose columns. Proteins were eluted with a 280 mL, 0-0.8 M NaCl gradient in KII and through immunoblotting MIPK was found to elute between concentration of 0.23 to 0.33 M NaCl. The pooled peak fractions, 240 mL in total, were brought up to a final io concentration of 1 M NaCl, with 300 mL 4 M NaCl in KII and 660 mL KII. This was applied to three 25 mL
phenyl-Sepharose columns and eluted with a 300 mL linear gradient, from 1 to 0.67 M NaCl and from 0 to 0.5% Brij 35 in KII. MIPK was found to elute between 0.73 M NaCl, 0.41% Brij 35 and 0.67 M NaCl, 0.5% Brij 35. The pooled peak fractions were 150 mL in volume and were diluted to 1.2 L adsorbed to 3 - 25 mL polylysine-agarose columns.
The columns were developed with a 280 mL linear gradient of 0-0.8 M NaCl, with the MIPK eluting between 0.24 and 0.32 M NaCl.
The final step in the purification involved fractionation of the pooled peak fractions on a 1 mL MonoQ column. The polylysine fractions were diluted 1:1 with KII to be applied onto the MonoQ column, and eluted with a 10 mL
linear gradient of 0 to 0.8M NaCl. MIPK eluted with 0.42 to 0.44 M NaCl. The two peak fractions were pooled and prepared for sequencing as described below.
3o Identification of Purified MIPK Protein Band Peak fractions containing MIPK from the MonoQ column were further separated by SDS-PAGE and silver stained. In preparation for silver staining (Merril et al., 1981), the gel was first soaked in fixative 1 (40% methanol/10%

acetic acid (v/v/v)) for 30 min and fixative 2 (10%
ethanol/5% acetic acid (v/v/v)) for 2 x 15 min. The gels were oxidized for 5 min in oxidizer (3.4 mM KZCr07, 3.2 mM
nitric acid) followed by three washes with dH20. The gels were then stained with 0.204% AgN03 (w/v) for 20 min. Gels were washed briefly in dH20 and developed with 0.28 M
Na2C03 in 0.166% formaldehyde solution (v/v) . Development was stopped by soaking the gel in 5% acetic acid (v/v) and the gel then stored in dH20. To identify the specific io protein band, an identical gel was western blotted with CDKS-CT. Following the Western blot procedure, the membrane was Ponceau S (Sigma) stained and the profile compared with the silver stained gel. A common band appeared in both, representing MIPK migration. The protein i5 band for MIPK appeared to be quite well resolved.
Sample Preparation for Protein Sequencing Peak MonoQ fractions containing the 40 kDa protein were 2o determined by Western blotting with CDK5-CT. The 2 peak fractions (500 ~,L in volume) were loaded 250 ~.L at a time into one lane of an 11% SDS-PAGE. The resulting gel was transferred onto a nitrocellulose membrane with a second nitrocellulose membrane backer. Both membranes were 25 stained with Amido Black 10B (ICN) revealing an abundance of protein on both membranes. The edge of the lane on the first membrane was cut off to allow Western blotting with CDK5-CT. The pieces of the lane were realigned and the MIPK band identified. This band of interest was excised 3o and placed in dH20 to keep it from drying out.
Micropeptide Sequencing of P. ochraceus MIPK
The protein sample was subjected to trypsin digestion 35 followed by peptide separation by reverse phase-high pressure liquid chromatography(RP-HPLC)-electrospray ionizing mass spectroscopy. Approximately 900 of the sample was collected following RP-HPLC, and approximately 10% of this sample was diverted into the mass spectrometer. The masses of the peptides were determined and, for a few peptides, collision-induced fragmentation data aided in the interpretation of the Edman degradation peptide sequencing data (Edman, P. 1950). By this mode six peptides were sequenced and database searches were to performed on the sequences. Results indicated that MIPK
is related to the MAP kinase superfamily of protein kinases, with closest homology to the p38 family of stress activated kinases.
In summary, MIPK was shown to co-elute over 5 chromatography steps, with a major tyrosine phosphorylated protein in the immature oocyte cytosol.
The protein was purified to a state where it could be resolved by SDS-PAGE from all contaminating proteins in 2o the preparation. This MIPK was identified by a combination of staining and Western blotting techniques, and the protein band was subjected to micropeptide sequencing. This resulted in the peptide sequencing of six discrete peptides indicating homology between MIPK and the p38 family of protein kinases. These peptide sequences may be used for cloning MIPK.
(D) Cloning and Sequencing of P. ochraceus MIPK
3o Good tools for characterization of MIPK are the cDNA clone and predicted amino acid sequence of the protein. The closest homology appears to be with the p38 family of stress activated protein kinases. From this information it is possible to clone MIPK utilizing the polymerase chain reaction (PCR). PCR is a method for rapidly amplifying a specific DNA sequence from a sample of total DNA or cDNA (White et al., 1989). From the information obtained by protein sequencing, degenerate primers may be designed to allow direct amplification of MIPK from seastar cDNA.
Degenerate PCR of MIPK from P. ochraceus MIPK peptide sequences were aligned with the protein to sequence for human p38. The first step in designing a PCR
strategy for MIPK cloning was the alignment of the sequenced peptides with the intact p38 sequence. This allows for the creation of PCR primers in the correct orientation for amplification.
RNA isolation from P. ochraceus was performed using the RNeasy isolation kit (Qiagen). Immature oocytes were extracted from the arm of one starfish for RNA extraction.
Three hundred ~.L of packed cells were diluted in 3.5 mL of lysis buffer with (mercaptoethanol and lysed using a polytron homogenizer (Brinkman) 2 x 10 s. The lysate was centrifuged for 3 min at maximum speed, and 350 ~,L of supernatant was used for each purification column. The sample was purified with a phenol/chloroform extraction, and centrifuged for 10 min at 10,000 x g. The supernatant was extracted with 1 volume of chloroform, mixed and centrifuged again for 10 min at 10,000 x g.
One volume of 70% ethanol was added to the sample before 3o application to an Rneasy column. Columns were washed with the appropriate buffers before elution with 50 ~,L of sdH20. The quantity of RNA was assessed on a formaldehyde gel.

RNA aaarose One percent (w/v) agarose was dissolved in lx MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0).
After cooling to 55~C, 5% deionized formaldehyde was added and the gel poured. Two volumes of RNA loading buffer (600 ~,L 10x MOPS buffer, 2.1 mL deionized formaldehyde, 6.0 mL deionized formamide, 4.0 mL glycerol, bromophenol blue) were added to each RNA sample, and the samples were to heated at 60~C for 15 min. One ~,L of 1 mg/mL ethidium bromide was added to each sample before loading on the gel. The gel was electrophoresed at 75 V until the bromophenol blue was 75% through the gel.
mRNA purification mRNA preparations were made using the Qiagen Oligotex mRNA
kit. Approximately 250 ~,L of total seastar RNA (about 440 ~,g) were diluted to 500 ~,L with sdH20 and 500 ~,L binding 2o buffer were added. Thirty ~.L of 37~C preheated Oligotex resin were added and the sample incubated for 3 min at 65~C. The sample was further incubated at room temperature for 10 min prior to centrifugation for 2 min at maximum speed. The supernatant was aspirated off and the resin resuspended in 400 ~L of wash buffer. This slurry was applied to a spin column and washed a second time. The mRNA was eluted with 2 x 30 ~,L of 70~C elution buffer. The final concentration of mRNA was estimated at 72-360 ng/~,L.
3o Quantitation of oligonucleotides Oligonucleotides were synthesized in the 40 nmole scale.
Primer pellets were dissolved in 100 ~.L of sdH20 and vortexed to resuspend. Five ~,L of primer was diluted to 1000 ~L sdH20 and an absorbance reading measured at 260 nm in a spectrophotometer. The molarity of the solution was calculated according to the following equation:
Conc (M) - A26~ x 33 mg/L x dilution factor s # base pairs x 330 g/mole x 1000 mg/g Reverse transcriptase reaction The Perkin-Elmer GeneAmp RNA PCR kit was used in the 1o synthesis of P. ochraceus cDNA. To 10 ng of mRNA were added 5 mM dNTPs, 1 U RNasin (1 U is the amount of RNasin required to inhibit by 50% the activity of 5 ng of ribonuclease A), 2.5 ~M random hexamer primers, and 50 U
Moloney murine leukemia virus (MMLV) reverse transcriptase 15 (1 U of MMLV RT incorporates 1 nmol of dTTP into acid precipitable material in 10 min at 37~C using poly(A) (oligo (dT) 1z_18 as template-primer) in a 20 ~L reaction.
The mixture was incubated for 10 min at room temperature, 60 min at 42~C, 99~C for 5 min, and held at 4~C until ready 2o to proceed.
Specific PCR conditions for MIPK
To the 20 ~L reverse transcriptase reaction mix were added 2s 3 ~L 10x PCR Buffer II, 24.5 ~,L sdH20, 1.5 ~,L of each primer, and 0.5 ~,L Taq polymerase. The PCR reaction conditions were an initial 1 min at 99~C, 45 cycles of 99~C
for 30 s, 35~C for 60 s, 72~C for 90 s, followed by a 10 min incubation at 72~C to fill all ends. The PCR reaction 3o was either frozen immediately if not used immediately in ligation reactions.
To clone the unpurified PCR products, ligations were set up in a total volume of 20 ~L, using 200 ng of vector per 35 ligation and a vector to insert ratio of from 1:1 to 1:3.

The vector agarose was melted at 65~C for 5 min and pipetted into a microfuge tube containing the prescribed amount of deionized H20. Up to 10 ~L of PCR product were added along with 2 ~.L 10x ligation buffer (Sambrook et al., 1989), 0.5 mM ATP, pH 7, 1 unit T4 Lipase (BRL) and incubated at 14-15~C overnight. The ligation mix was warmed at 65~C for about 1 min and 5 ~,L added to a microfuge tube containing 20 ~,L prewarmed sterile deionized H20. Transformation into DHa (competent E. coli to cells was performed as described above.
The best PCR results came from the following pairs of primers:
S4A CCI GTI CA(A/G) TA(T/C) CA(A/G) AAA (SEQ ID N0:8) S2C AA(T/C) TGI AC(A/G) TG(T/C) TC(A/G) TC (SEQ ID N0:9) and S2B GA(T/C) GA(A/G) CA(T/C) GTI CA(A/G) TTC (SEQ ID
NO:10) 2o S5A TTI GCI AC(A/G) TAI GGA TG (SEQ ID NO:11) Using very low stringency conditions, PCR products of the expected size could be generated. The products are preferentially amplified over background, by loading lesser amounts of the reaction on a gel.
The products were cloned into the pBluescript vector (Stratagene Cloning Systems, La Jolla, California) without the need of a further purification step.
The PCR products were cloned by exploiting the properties of the Taq polymerase used in the PCR reaction. Taq polymerase has been observed to add a single non-template-directed deoxyadenosine (A) residue to the 3' end of duplex PCR products. Use of terminal transferase to add a single T residue to a blunt ended vector created a sticky end which was specific for the Taq polymerase generated PCR product. This allowed for direct and efficient cloning of the PCR product without the need for s enzymatic modification (Holton and Graham., 1991).
This method of cloning is specific for PCR reactions involving Taq polymerase and is based on the procedure by Holton and Graham (l991). Five ~.g of pBluescript KS+
io vector were digested with 20 U of EcoRV for about 2 h in a 20 ~L volume with BRL React two buffer. While the vector is being digested, the PCR reaction was carried out to amplify the fragment to be cloned, including a final 10 min 72~C step to finish a11 the ends of the PCR products.
i5 The PCR products should be used immediately after preparation or stored at -20~C after preparation.
After EcoRV digestion, the vector was incubated at 65~C for min to kill the enzyme. The vector was then 2o precipitated in 1/10 volume (2 ~,L) 3M NaOAc, pH 5.2 and 2 volumes (45 ~.L) 100~s ethanol for 20 min at -20~C. The mixture was centrifuged for 5 min at 15,000 rpm, the supernatant aspirated off, the pellet washed with 70%
ethanol, and centrifuged again. The supernatant was again 25 aspirated off and the pellet dried.
The Boehringer terminal deoxytransferase kit was used to T-Tail the vector in a 50 ~,L total volume. Five ~,g or vector in 28 ~.L sdH20, 10 ~,L 5X TdT buffer, 3 ~L 25 mM
3o CoCl2, 5 ~L 100 ~M ddTTP (Pharmacia) and 4 ~,L of TdTransferase (Boehringer 25 U/~L with 1 U equal to the amount of enzyme that incorporates 1 nmol dAMP into acid-insoluble products within 60 min at 37~C using d(pT)6 as primer) were incubated at 37~C for 1 h. The above 35 reaction conditions are 25 mM CoCl2, 10 ~,M ddTTP and 100 U

of terminal transferase. The T-tailed vector was gel purified in 0.9 0 low melting point agarose, the band excised and weighed to determine the DNA concentration.
Legations For the majority of legations, the vector, the insert, or both, were present in low melting point (LMP) agarsoe and the following legation protocol was used (adapted from io Kalvakolanu and Livingston (1991)). The amount of agarose in the mixture was never more than 25 per 50 ~L reaction.
To 18.5 ~L of sdH20 were added 5 JCL 5x legation buffer (Sambrook et al., 1989), and 0.5 ~,L 100 mM ATP (pH 7), and the mixture heated to 65-70~C. Vector agarose was also heated to melting for about 2-5 minutes at 65-70~C.
Approximately 250 ng of vector was added to the preheated buffer mixture and the mixture cooled to room temperature on ice. To each tube was then added lU (lU = amount of enzyme which will catalyze the exchange of 1 nmol 32PPi 2o into [ (a - 32P] ATP in 20 min at 37~C) T4 ligase. The insert agarose was then melted at 65~C for 2-5 min and 20 ~.L added to the legation mixture and mixed gently. The legation was incubated at 16~C overnight.
Products of legation reactions were heated to 65~C to melt.
Ten ~,L of the legation mixture were added to 50 ~.L
prewarmed sdH20 and placed on ice. Two hundred ~,L of competent DH5(E. coli cells were added and incubated on ice for 30 min, 42~C for 90 sec, and on ice for 2 min. One 3o mL of LB media (10 g bactotryptone, 5 g bactoyeast extract, 10 g NaCl, pH 7, Sambrook et al., 1989) was added and incubated at 37~C, 225 rpm for 1 h. The cells were plated on LB/ampicillin/X-gal(for pBluescript vectors) agar.

For pure vector, a quick transformation was performed.
Fifty ~L of competed DHSa or UT 5600 E. coli cells were combined with a few ng of DNA (1 ~L) and incubated at 37~C
for 1 min and chilled on ice for 1 min. Two hundred ~.L of LB media were added and the mixture plated on LB/ampicillin agar.
To clone the unpurified PCR products, ligations were set up in a total volume of 20 ~,L, using 200 ng of vector per io ligation and a vector to insert ratio of 1:1 to 1:3. The vector agarose was melted at 65~C for 5 min and pipetted into a microfuge tube containing the correct amount of deionized H20. Up to 10 ~.L of PCR product were added along with 2 ~,L 10x ligation buffer (Sambrook et al., 1989), 0.5 mM ATP, pH 7, 1 U T4 ligase (BRL) and the mixture was incubated at 14-15~C overnight. The ligation mix was warmed at 65~C for about 1 min and 5 ~.L were added to microfuge tube containing 20 ~,L prewarmed sdH20.
Transformation into DHSa competent E. coli cells was zo performed as described above.
Duplicate PCR reactions were performed for the two working primers pairs. Six clones from each PCR reaction were tested for correctly sized inserts by direct PCR. In total, four of twelve clones showed appropriate insert size from the S4A-S2C reactions, while five of twelve clones indicated appropriate insert size from the S2B-S5A
reactions. These clones were further analyzed by restriction mapping. Three of the four clones from the 3o S4A-S2C reactions gave identical restriction maps and two were sent for sequencing. Three of the five clones from the S2B-S5A reactions gave appropriate sized inserts upon restriction digest, although they did not appear to be identical in size. As a result, three of the clones were sequenced.

Confirmation of the identity of the PCR clones This protocol was adapted from Sathe et al. (1991) and Barnes (1994). Positive colonies from transformation plates were picked with sterile tooth picks and swirled into 20 ~,L of sdH20 in microamp PCR tubes. The toothpicks were then stabbed into numbered spots on a LB/ampicillin plate to be grown overnight as stock cultures. To the PCR
tube were added 13 ~,L sdH20, 5 ~L 1 mM dNTPs (final 100 to (M) , 5 ~L of lox Vent Pol. Buffer (final 2 mM MgS04) , 5 ~.L
mg/mL BSA ( f final 100 ~,g/mL) , 1 ~,L 1M Tris base ( f final 2 mM
Tris pH 9.1), 1 ~,L each 20 ~.M M13 Forward and Reverse primers, and 0.5 ~,L Taq:Vent mix (100:1, 2.5 U). The PCR
reaction conditions were 5 min at 94~C for 1 min, 1 min i5 annealing at 55~C, 2 min elongation at 72~C, followed by a min final extension at 72~C. Five hundred ~,g of RNAse were added per mL of 6x gel loading buffer (0.25% w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, 30% (v/v) glycerol in water) and 10 ~L were added to each reaction 2o tube. Twenty ~,L of the reaction were electrophoresed on an agarose gel to assess the insert size.
DNA sequence information confirmed that the PCR products amplified encode a protein that is related to the p38 25 family of protein kinases. The S2B-S5A fragment peptide sequence matched the information obtained from the MIPK
protein. The 5' and 3' regions were cloned to ensure that one gene was giving rise to the PCR products.
3o Cloning and sequencing of MIPK from immature seastar oocytes A series of sequence specific primers were designed based on DNA fragments generated by degenerate PCR. The primers 35 were tested for their ability to amplify internal sequences and all were found to work under these conditions. The primers would give large products in the RACE reactions, with the large areas of overlap.
Rapid amplification of cDNA ends (RACE) 5' and 3' RACE reactions were performed using the CLONTECH
Marathon cDNA Amplification Kit (Clontech Laboratories Inc., Cambridge, United Kingdom). This kit involves the io production of an adaptor ligated cDNA library to be used for PCR amplification. Five ~,l (~l ~,g) mRNA from sea star oocytes was used for first strand synthesis and mRNA was combined with 1 ~L 10 mM (1 mM final) cDNA synthesis primer and heated to 70~C for 2 min followed by 2 min on ice. Two ~,L 5x 1st strand buffer, 10 ~M dNTPs, 100 Units Murine Leukemia Virus Reverse Transcriptase ("MMLV-RT"), 100 U Superscript II (BRL) (1 U incorporates 1 nmol dTTP
in acid precipitate material in 10 min at 37~C using polyA(oligo(dT)25 as template primer) were added, and the 2o reaction incubated at 42~C for 1 hour. The tube was placed on ice to terminate the reaction.
For second-strand synthesis, the 10 ~,L first-strand reaction was combined with 16 ~L 5x concentrated second-strand buffer, 0.5 mM dNTPs, 4 ~,L 20x second-strand enzyme cocktail, and sterile deionized HZO to a final volume of 80 ~,L. The reaction was incubated at 16~C for 1.5 hours. Ten units T4 DNA Polymerase (1 U incorporates 10 nmol of deoxyribonucleotide into acid-precipitable 3o material in 30 min at 37~C) were added, and the reaction continued for 45 min at 16~C whereupon it was terminated with EDTA/glycogen mixture. The DNA was purified by extraction and precipitated with 95% ethanol. The pellet was resuspended in 10 ~L sterile deionized H20.

Adaptor ligation was performed on the entire 10 ~L of ds cDNA. Two ~,M Marathon cDNA adaptor, 4 ~L 5x DNA ligation buffer, 2 units T4 ligase (Clonetech), 2 units T4 ligase (Boehringer), and 250 ~M ATP were incubated with the cDNA
at 16~C overnight. The reaction was terminated by heating at 70~C for 5 min The adaptor ligated (Ad)-cDNA was diluted with sterile deionized H20 (1/250, v/v) for use in RACE reactions.
1o Amplifying full length MIPK cDNA clone using PCR
Specific primers were designed containing the start and stop codons of MIPK as described above. Approximately 0.5 ~,L of Ad-cDNA were used to amplify the full length cDNA.
To the reaction mixture were added 2 ~L 25 mM dNTPs, 10 ~.L
10x Vent buffer (NEB) , 1 ~L 100 mM MgS04, 5 ~,L 20 ~,M ATGE
primer and STOPS primer, and 0.5 U Vent polymerase (1 U is the amount of enzyme that will incorporate 10 nmol of dNTP
into acid-insoluble material at 75~C in 30 min in lx 2o buffer). The mixture was preheated at 94~C for 3 min followed by 30 cycles of 1 min at 94~C, 1 min at 65~C, and 2 min at 72~C. The product ends were filled using an additional incubation of 10 min at 72~C.
The PCR product was purified using the Qiaquick PCR
purification protocol (Qiagen). The fragment and the pGEX-4T3 vector were digested with EcoRI and SalI to prepare for ligation. The PCR product was again purified using the Qiaquick procedure while the vector was gel 3o purified on a 0.9% low melting point-agarose gel. The vector band was excised from the gel and used directly in the ligation as described. Ligations were transformed into DH5 cells as described above and colonies screened by direct PCR. Positive clones were tested for expression levels and an appropriate clone selected and sequenced.

Analysis of Sequence Data The open reading frame of MIPK was 1089 base pairs (bp), which predicted a 363 amino acid protein (Fig. 1). The protein sequence contained a11 of the conserved domains (I-XI) characteristics of a protein-serine/threonine kinase. The Thr189 and Tyr191 residues in subdomain VIII
are in an equivalent position to the TEY, TPY or TGY
sequences in known MAP kinases and stress-activated io kinases. The sequence shares a TGY sequence with the p38 kinase family. As in p38, kinase subdomain VII is separated by only 6 amino acids from the activation region in subdomain VIII, whereas the gap is 8 residues in the JNK family and over 12 amino acids in known MAP kinases i5 such as Erkl and Erk2. The predicted amino acid sequence and overall structure identify this protein as a member of the p38 family. All of the peptides sequenced in the purified MIPK protein were identified in the predicted amino acid sequence.
A search of various nucleotide and protein databases revealed that the deduced amino acid sequence of MIPK was a novel sequence most closely related to p38 from Xenopus.
Protein sequence alignment of MIPK with p38 from various species indicated a high homology in the kinase domains.
However, there were significant differences with MIPK.
The N-terminus of MIPK contains an extra 8 amino acids which are not found in any of the known p38 kinases, and there is a stretch of 10 amino acids between subdomains IV
3o and V which was 80% different than p38. The largest region of sequence variation between MIPK and p38 begins in subdomain X and continues to the C-terminus of the protein. In this 124 amino acid region there was 46%
identity between MIPK and human p38 compared to a 65%
identity in the entire protein. In the final 36 amino acids, identity dropped to 28%, with the last 6 residues in p38 missing from MIPK. While it is clear that MIPK was a member of the p38 family of serine/threonine kinases, MIPK was not the sea star homolog of p38 kinase.
Tissue Distribution of MIPK
Using the anti-MIPK antibody CDK5-CT, rat tissues were screened by Western Blotting for immunoreactive proteins.
to The 40 KDa kinase was detected mainly in the hearts and brains and to a lesser extent in kidney, liver, lung, spleen, testis and thymus of 50 day old rats. It was present at much lower levels in the hearts of 1 day old rats and minimally in embryonic and neonatal tissue. The kinase was also present in adult bovine heart. Kinase activity of the 40 KDa protein was confirmed by binding to ATP-agarose beads.
(E) Comparison of seastar MIPK with other proteins MIPK was clearly a member of the p38 family of protein kinases based on the TGY sequence in the activation loop.
To more closely investigate the relationship between MIPK
and p38, sequence homologies were compared between p38 homologs from a number of different species. p38 homologs are distinct in their degree of identity, 84%-99%. MIPK
was found to be 65%-66% identical, 74-75% conserved, with the p38 homologs. This is a significantly lesser degree of conservation than expected for a p38 homolog.
3o Comparisons were therefore expanded to the other p38-like proteins that have been identified. Four p38-related proteins in the human system are p38a, p38,, p38~y and p38b. Although some sequence discrepancies have been noted between different groups, these variations account for an insignificant portion of the proteins. Human homologs within the p38 family vary from 57%-71%
identical, and from 72%-83% conserved.
MIPK was found to be 54%-65% identical to, 68%-74%
conserved with the human p38 family, but is no more or less related to any of the family members. Another source of p38 homologs is the yeast system, with Hogl from S. cerevisiae and Styl from S. pombe both containing the characteristic TGY sequence in subdomain VIII. Even the to yeast homologs are 43%-53% identical to, 61%-70% conserved with the human p38 family, and 48% identical to, 61%-66%
conserved with MIPK. MIPK is not the clear homolog to any of the currently identified p38 family proteins.
Amino acid motifs of MIPK:
1. as 100-113 CRGDTLSSFRDVYM (SEQ ID N0:3) 2. as 242-288 SRIMDLTGTPDDEILAKIQSEDARNFVKSQPKTKKKDFRGYFAGANE
(SEQ ID N0:4) 3. as 323-345 ESDEPIGKQFDDSFEQQDLTVQQ (SEQ ID N0:5) The sequences of the motifs are well conserved within the p38-alpha isoform of MAP kinase in diverse species (see below).
Region l00-l13 242-288 323-345 Sea star Mipk 100% 100% 100%

Human p38-alpha 36% 38% 39%

Rat p38-alpha 36% 34% 35%

Mouse p38-alpha 36% 34% 39%

Carp p38-alpha 36% 21% 35%

Xenopus p38-alpha 29% 36% 39%

Human p38-beta-1 21% 47% 26%

Human p38-beta-2 21% 47% 26%

Human p38-gamma 42% 30% 30%

To further investigate the relationship between MIPK and the MAP
kinase superfamily, MIPK homology trees were constructed using PHYLIP
(Felsenstein, 1993). Amino acid sequences and nucleotide sequences were aligned using Clustal. To achieve more reliable comparisons, N-and C-terminal overhangs were removed. PHYLIP was used to build a tree from the species alignments.
Western blotting comparisons of MIPK and p38 in P. ochraceus Assessments were made for evidence of other p38-like species. The p38 antibodies available are not able to immunoprecipitate from the seastar system. Cytosolic extracts from P. ochraceus oocytes were therefore fractionated on a Resource column before being subjected to SDS-PAGE. Duplicate gels were Western-blotted with CDKS-CT, p38-CT, or both antibodies.
Clean bands were found in both individual Western blots, although both proteins appeared in the same fraction from i5 the ResourceQ column. To clarify the results, one of the membranes was probed with both antibodies. Two proteins could be resolved using this technique, although differing by only a few kDa, with MIPK representing the larger of the two proteins. The result clearly indicates that two 2o p38-like proteins are expressed in the seastar oocyte system. One of these proteins, MIPK, is recognized only by the CDK5-CT antibody, while p38-CT could only detect the lower molecular weight protein. Phosphotyrosine Western blots indicate that only MIPK was activated in the 25 immature oocyte blocked at prophase, based on the detection of a single band.
Design of Probes for MIPK
3o To determine whether MIPK homologs exist in other species.
MIPK-specific probes were made. Through analysis of the MIPK sequence, it is clear that the regions of highest diversity between MIPK and p38 family members involved the N- and C- termini. Peptides from these regions may be 35 used for immunization and antibody generation. A
C-terminal peptide corresponding to amino acids 348-363 _ 77 _ was synthesized. This peptide is novel for MIPK versus the p38 isoforms, and also shows no homology with any other proteins in the available databases.
(F) Post Fertilization Activation of MIPK
In the sea star oocyte system, the majority of available information is limited to the events surrounding meiotic maturation, with very little study having been io made on the time following fertilization.
Sea stars were induced to spawn by injection into the body cavity of a minimum of 1 mL per arm of 0.14 mM
1-methyladenine in Millipore filtered seawater (Eraser et al., 1981). Shedding of mature oocytes typically commenced between 60-90 min following primary injection.
During spawning, sea stars were inverted over 400 ml beakers containing Millipore filtered seawater. After approximately 60 min of shedding, the oocytes were allowed 2o to settle and were washed with three changes of filtered seawater. The oocytes were resuspended at a concentration of 1% (v/v) in filtered seawater. The container was placed in a refrigerator equilibrated to 12~0.5~C and the seawater was gently aerated and oscillated at 40 rpm. The oocyte suspension was allowed to equilibrate at this temperature for at least 1 h prior to addition of sperm.
Sperm was collected from male sea stars and diluted 1:200 (v/v) with filtered seawater. Sperm viability and motility were verified by phase microscopy. Equilibrated 3o mature oocytes were fertilized by addition of 1:100 (v/v) of the sperm dilution (effective dilution 1:20000 (v/v)).
Fertilization of oocytes was confirmed in an aliquot of oocytes at approximately 1 h following sperm addition by observing the elevation of fertilization membranes.

_ 78 _ Embryo cultures with less than 70% fertilization of oocytes were discarded.
At specific time points during development, the embryos were pelleted at 4~C in a Beckman J2-HS centrifuge (1500 rpm for 5 min). A 33% (v/v) suspension was prepared in chilled homogenization buffer and the embryos disrupted with 2 x 30 sec bursts at 19,000 rpm of a Polytron (PT3000, Brinkman, USA). Homogenates were immediately 1o centrifuged in a Sorval Combi ultracentrifuge at l0,000 x g for 10 min. The supernatant was then decanted and centrifuged at 250,000 x g for 30 min. Supernatants were quickly aliquotted and stored at -70~C.
Extracts from various post-fertilization time points were assessed for MIPK tyrosine phosphorylation levels, and the levels compared with those found in immature and mature oocytes. Results showed that the very low level of phosphorylation that exists at the time of fertilization 2o decreases to nothing within the first 6 hours post-fertilization. From 12-20 hours, the tyrosine phosphorylation of MIPK increased dramatically. This level dropped by 24 hours and appeared to stay low through 48 hours.
The early development of P. ochraceus embryos is characterized by an initial rapid increase in cell number to the 256-cell stage. At 12~C, this period of synchronous cleavage lasts approximately 14 h. After that, the 3o individual cells within the embryo assume independent division rates, and there is a flattening of the developmental curve (Eraser et al., 1981). It appears that the activation of MIPK is coincident with the transition from synchronous cell cleavages to differential cleavage. Maturation and stress time courses in which _ 79 _ phosphorylation of MIPK is measured following osmotic or heat shock indicate that MIPK is activated in cells which are arrested in the cell cycle. Thus, MIPK appears to act as a cytostatic factor. The clear decrease in the rate of cell division would require activation of enzymes which promote quiescence and cell cycle blockages, in preparation for differentiation. It therefore follows that the role of MIPK post-fertilization involves cells exiting from the cell cycle.
io (G) Activation of MIPK by Osmotic Shock Treatment Seastar oocytes were subjected to high osmolarity conditions to assess the activity of MIPK during osmotic shock. Native seawater has a concentration of salt of approximately 0.5 M. Oocytes from individual P. ochraceus were resuspended in natural seawater containing 1.0 M, 1.5 M, 2.0 M, or 3.0 M NaCl based on the initial concentration of NaCl in sea water of 0.5 M. A first volume of oocyte zo suspension was removed immediately, pelleted, and the sea water removed. The oocytes were quick frozen in a dry ice/ethanol bath. The remaining suspensions were incubated at 14~C, with aliquots removed at discrete time points after the addition of the high salt sea water.
Frozen, packed cells were stored at minus 70~C.
One M NaCl proved to be the only concentration at which the oocytes could survive. Results showed a dramatic activation of MIPK in response to osmotic shock. This is 3o despite an extreme decrease in the amount of MIPK protein found in the oocyte. Results indicate a relatively constant level of MIPK tyrosine phosphorylation in the cell, despite this decrease in protein. This indicates that the activation of MIPK is essential for the osmotic shock response and that it is required at a minimum threshold level. To ensure that tyrosine phosphorylation was due to MIPK and not p38 isoforms in the oocyte, immunoprecipitations from various times were Western blotted with a p38-CT antibody. Results confirmed that there was no known p38 isoforms present.
(H) Activation of MIPK by Heat Shock Treatment Heat shock effects were also tested for MIPK activation.
io Normal conditions for oocyte maturation ranges from 10~C-14~C. Heat shock temperatures of 25~C, 35~C and 45~C
were tested. Oocytes from individual P. ochraceus were resuspended in a measured volume of natural sea water preheated to 25~C, 35~C, or 45~C. The zero time point of i5 oocyte suspension was removed, pelleted, the sea water removed, and the oocytes quick frozen in a dry ice/ethanol bath. Suspensions were incubated at the given temperatures, with aliquots removed at discrete time points after the addition of the preheated sea water.
2o Frozen, packed cells were stored at minus70~C.
At 45~C, the oocytes appeared to melt and the effects were found to be unreliable. At 25~C, a small activation of MIPK was apparent after 60 min incubation. At 35~C, MIPK
z5 activation was visible within 20 min and continued increasing through 30 min After 30 min the total tyrosine phosphorylation remained constant, while the amount of MIPK protein began to drop. As with the osmotic shock time course, it appeared that the MIPK was activated to a 3o threshold level, and that this level was maintained despite a net decrease in MIPK protein.
A11 publications and patents cited in this specification are incorporated herein by reference. Although the 35 foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that changes and modification may be made thereto without departing from the spirit or scope of the appended claims.
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25 Boulton TG, Yancopoulos GD, Gregory JS, Slaughter C, Moomaw C, Hsu J, Cobb MH. (1990) An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249:4964 64-7 3o Boulton TG, Gregory JS, Jong SM, Wang LH, Ellis L, Cobb MH
(1990) Evidence for insulin-dependent activation of S6 and microtubule-associated protein-2 kinases via a human insulin receptor/v-ros hybrid. J Biol Chem 265:5 2713-9 Cisek, L. J., and Corden, J. L. (1989) Phosphorylation of RNA polymerase by the murine homolog of the cell cycle control protein cdc2. Nature 339:679-684.
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Coruzzi, et a1.(1984) EMBO J., 3:1671-1680.
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Derijard, B., Raingeaud, J., Barret, T., Wu, I.-H., Han, J., Ulevitch, R.J., and Davis, R.J. (1995) Independent 3o human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267:682-685.
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Egan, S.E. and Weinberg, R.A. (1993) The pathway to signal achievement. Nature 365:781-783 .
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Patent References MAP Kinase Phosphatase Gene And Uses Thereof; WO 9706245 WO 952l923 88a SEQUENCE LISTING
(1) GENERAL INFORMAT:LON
(i) APPLICANT: Pelec~h, Steven (ii) TITLE OF INVENT:LON: A Novel Maturation-Inhibited Protein Kinase ( i i i ) NUMBER OF SEQUENCES : 11 (iv) CORRESPONDENCE i~DDRESS:
(A) ADDRESSEE: Smart & Biggai:
(B) STREET: 650 West Georgia Street, Suite 2200 (C) CITY: Vancouver (D) STATE: British Columbia (E) COUNTRY: Canada (F) ZIP: V6B 4N8 (v) COMPUTER READABL1~ FORM
(A) MEDIUM TYPE: Dis)~ette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: Patent:In Ver. 2.0 (vi) CURRENT APPLICATION DATA
(A) APPLICATION NUf~fBER: 2, 2 61, 297 (B) FILING DATE: 1u99-02-18 (vii) PRIOR APPLICAT:CON DATA:
(A) APPLICATION NUMBER: 2,224,112 (B) FILING DATE: 1998-02-18 (viii) ATTORNEY/AGEN~C INFORMATION:
(A) NAME: Smart & Bi<_~gar (B) REFERENCE/DOCKET NUMBER: 80021-90 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (604)--682-7295 (B) TELEFAX: (604)-6E32-0274 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARAC~'ERISTICS
(A) LENGTH: 363 am=_no acids (B) TYPE: amino ac=_d (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide 88b (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pisaster ochraceus (xi) SEQUENCE DESCR:CPTION: SEQ ID NO.: 1:
Met Asn Asn Pro Val '.Chr Gly Ser Gly Glu Thr Leu Ser Asp Asp Gly Tyr His Arg Tyr Glu heu Asn Lys Thr Thr Trp Glu Val Pro Val Gln Tyr Gln Lys Leu Ser Ala Val Gly Ala Gly Ala Tyr Gly Ser Val Cys Ser Ser Leu Asn Thr hys Thr C~ly Ile Lys Ile Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Ser Ala Ile His Ala Lys Arg Thr Tyr Arg Glu Leu Arg Leu Leu Gln Isis Met Asp His Glu Asn Ile Ile Ser Leu Leu Asp Val Phe Cys Arg Gly Asp Thr Leu Ser Ser Phe Arg Asp Val Tyr l00 105 110 Met Val Thr His Leu Met Gly Ala Asp Leu Asn Ser Ile Thr Lys Thr l15 7.20 125 Gln Lys Leu Ser Asp Glu His Val Gln Phe Leu Val Tyr Gln Ile Leu Arg Gly Leu Lys Tyr ..le His Ser Val Gly Val Ile His Arg Asp Leu 145 ._50 155 160 Lys Pro Ser Asn Leu Ala Val Asn Glu Asp Cys Glu Leu Arg Ile Leu Asp Phe Gly Leu Ala Arg Gln Ala Asp Asp Glu Met Thr Gly Tyr Val 180 185 l90 Ala Thr Arg Trp Tyr Arg Ala Pro Glu Ile Met Leu Asn Trp Met His Tyr Thr Asn Thr Val ~~sp Met Trp Ser Val Gly Cys Ile Met Ala Glu 2l0 215 220 Leu Leu Thr Gly Lys ~'hr Leu Phe Pro Gly Ser Asp His Ile Asp Gln 88c Leu Ser Arg Ile Met Asp Leu Thr Gly Thr Pro Asp Asp Glu Ile Leu Ala Lys Ile Gln Ser G1u Asp Ala Arg Asn Phe Val Lys Ser Gln Pro Lys Thr Lys Lys Lys Asp Phe Arg Gly Tyr Phe Ala Gly Ala Asn Glu Ile Ala Val Asp Leu 7~eu Glu Lys Met Leu Leu Leu Asp Val Asp Lys Arg Ile Thr Ala Glu (slu Ala Leu Ser His Pro Tyr Val Ala Lys Tyr His Asp Glu Ser Asp G1u Pro Ile Gly Lys Gln Phe Asp Asp Ser Phe Glu Gln Gln Asp Leu ~Chr Val Gln Gln Trp Lys Glu Leu Thr Phe Gln Leu Ile Gln Ala Val ~~rg His Gln Ser Arg Arg (2) INFORMATION FOR SEQ ID NO: 2:
( i ) SEQUENCE CHARAC':'ERISTICS
(A) LENGTH: 1344 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Pisast:er ochraceus 88d (xi) SEQUENCE DESCR:CPTION: SEQ ID NO.: 2:
GCCGGACACA TCCGTACAT'C CAGCCTGGGA TTATAAGAAA ACTNATTTAG TCAAAGTAAA 60 TTAGAATTAG TCATTCGAT'.C TTGATTTGGT AGAGCACTAA AAAATACTCT TAGTCTTAGA 120 GTTAGTGTTA CCATACCAA'.C TAACTTAATT AAATCATGAA CAACCCAGTA ACAGGATCAG 180 GAGAAACGTT ATCTGATGA(: GGGTATCATC GATATGAACT GAATAAAACT ACATGGGAGG 240 TGCCGGTTCA GTACCAAAA~~ CTCTCCGCAG TGGGAGCTGG TGCATATGGA TCCGTGTGCT 300 CATCCTTAAA CACAA.AAAC'.C GGCATAAAGA TTGCTATCAA GAAGCTTTCT CGACCATTTC 360 AGAAACTCTC TGATGAACA'.C GTGCAGTTCC TTGTGTATCA AATACTTCGT GGGCTCAAGT 600 AAGACTGCGA ATTGAGGAT~~ CTAGATTTTG GTCTTGCTCG TCAAGCTGAT GATGAGATGA 720 CAGGTTACGT AGCTACACG~~ TGGTATAGAG CACCAGAAAT CATGCTGAAT TGGATGCATT 780 ACACCAATAC TGTC~GATAT(~ TGGTCTGTTG GATGTATAAT GGCAGAACTT CTCACAGGTA 840 AAACGCTATT TCCTGGATCCz GATCACATTG ATCAGTTGAG TCGCATCATG GATCTAACTG 900 GTACACCTGA TGATGAAAT(~ CTTGCCAAAA TCCAGAGTGA AGATGCACGG AACTTTGTTA 960 TTGCTGTTGA CCTTCTGGAG AAAATGC:TTC TGTTGGATGT AGACAAGCGT ATCACTGCTG 1080 AAGAGGCACT GAGTCATCC~C TATGTTGCCA AATATCATGA TGAAAGTGAT GAGCCTATTG 1140 GTAAGCAGTT TGATGATTC(~ TTTGAACAGC AAGACTTGAC TGTGCAGCAG TGGAAAGAGC 1200 TTACTTTTCA GCTGATTCA~~ GCAGTAAGAC ATCAAAGCAG AAGGTAAATA GCTACAACAT l260 AAAAAAAAAA P,AAAAAAAA~~ AAAA 13 4 4 (2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARAC~'ERISTICS
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (vi ) ORIGINAL SOURCF's (A) ORGANISM: Pisaster ochraceus (xi) SEQUENCE DESCR=PTION: SEQ ID NO.: 3:
Cys Arg Gly Asp Thr heu Ser Ser Phe Arg Asp Val Tyr Met 88e (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARAC'.CERISTIC S
(A) LENGTH: 47 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pisasi=er ochraceus (xi) SEQUENCE DESCR:LPTION: SEQ ID NO: 4:
Ser Arg Ile Met Asp heu Thr Gly Thr Pro Asp Asp Glu Ile Leu Ala Lys Ile Gln Ser Glu ~~sp Ala Arg Asn Phe Val Lys Ser Gln Pro Lys Thr Lys Lys Lys Asp I?he Arg Gly Tyr Phe Ala Gly Ala Asn Glu (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARAC':'ERISTICS
(A) LENGTH: 23 amino acids (B) TYPE: amino ac:_d (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pisast:er ochraceus (xi) SEQUENCE DESCR::PTION: SEQ ID NO: 5:
Glu Ser Asp Glu Pro ::le Gly Lys Gln Phe Asp Asp Ser Phe Glu Gln Gln Asp Leu Thr Val C~ln Gln 88f (2) INFORMATION FOR SEQ ID NO.: 6:
(i) SEQUENCE CHARAC'.C'ERISTICS
(A) LENGTH: 17 amino acids (B) TYPE: amino ac_Ld (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pisast~er ochraceus (xi) SEQUENCE DESCR:CPTION: SEQ ID NO: 6:
Lys Glu Leu Thr Phe C~ln Leu Ile Gln Ala Val Arg His Gln Ser Arg Arg (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARAC'.CERISTICS
(A) LENGTH: 16 amino acids (B) TYPE: amino ac=_d (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pisast:er ochraceus (xi) SEQUENCE DESCR::PTION: SEQ ID NO: 7:
Thr Gly Ser Gly Glu ~_'hr Leu Ser Asp Asp Gly Tyr His Arg Tyr Glu (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARAC~""ERISTICS
(A) LENGTH: 16 nuc:_eic acids (B) TYPE: nucleotide (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

88g (xi) SEQUENCE DESCR:CPTION: SEQ ID NO: 8:

( 2 ) INFORMATION FOR :iEQ ID NO : 9 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 16 nuc:Leic acids (B) TYPE: nucleotide (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRI1?TION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARAC~CERISTICS
(A) LENGTH: 17 nuc:Leic acids (B) TYPE: nucleotide (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRII?TION: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARAC'.CERISTICS
(A) LENGTH: 14 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRI1?TION: SEQ ID NO: 11:

Claims

1. A protein kinase, wherein the kinase is activated by tyrosine phosphorylation, has a mass of about 40 kD, is cross-reactive with CDK5-CT antibody, not cross-reactive with ERK1-CT antibody, and is present as a composition having at least 50% weight percent of the kinase.

2. A polypeptide comprising one or more amino acid motifs selected from a group of amino acid sequences which are at least 50% homologous to SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

3. The polypeptide of claim 2 wherein said amino acid sequences are at least 75% homologous to SEQ ID NO:3, SEQ
ID NO:4 and SEQ ID NO:5.

4. The polypeptide of claim 2 which is reactive on an immunoblot with CDK5-CT antibody.

5. The polypeptide of claim 2 which has the activity of a protein kinase and is activated by tyrosine phosphorylation.

6. The polypeptide of claim 2 which comprises an amino acid sequence having at least 50% homology to SEQ ID NO:1.

7. The polypeptide of claim 2 which comprised an amino acid sequence having at least 75% homology to SEQ ID NO:1.

8. The polypeptide of claim 2 which comprises an amino acid sequence having at least 90% homology to SEQ ID NO:1.

9. The polypeptide of claim 2 comprising an amino acid sequence substantially the same as SEQ ID NO:1.

10. An antibody which is not CDK5-CT and is specific for the protein kinase of the composition of claim 1.

11. A peptide having at least one T cell epitope recognized by a T cell receptor, said peptide consisting of a portion of the polypeptide of claim 9.

12. A method of detecting a protein kinase of claim 1 or a fragment thereof, comprising the steps of contacting a biological sample with an antibody specific for the protein kinase and determining whether said antibody binds to a protein in said sample.

13. An isolated nucleic acid comprising a nucleotide sequence encoding or complementary to a nucleotide sequence encoding all or a fragment of at least 6 amino acids of the protein kinase of claim 1.

14. The nucleic acid of claim 13 comprising a nucleotide sequence which is at least 75% homologous to SEQ ID NO:2.

15. The nucleic acid of claim 14 wherein said nucleotide sequence is at least 90% homologous to SEQ ID NO:2.

16. The nucleic acid of claim 13 comprising a nucleotide sequence the same as or complementary in a sense or antisense orientation to a segment of from 18 to 1088 contiguous nucleotides of SEQ ID NO:2.

17. An expression vector comprising the nucleic acid of claim 13.

18. A cell or an organism comprising the cell, wherein the cell is transformed with the expression vector of claim 17.

19. A method of affecting protein kinase expression in an animal, comprising the steps of contacting cells in the animal with a antisense oligonucleotide comprising a nucleic acid of claim 14.

20. A method for detecting a nucleic acid encoding a protein kinase in a biological sample comprising the steps of contacting said sample with a first nucleic acid comprising a nucleic acid of claim 14 and detecting hybridization of said first nucleic acid to a nucleic acid in said sample.