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WO2001071010A1 - Proteine de levure methyltransferase hsl7p - Google Patents

Proteine de levure methyltransferase hsl7p Download PDF

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Publication number
WO2001071010A1
WO2001071010A1 PCT/US2001/009087 US0109087W WO0171010A1 WO 2001071010 A1 WO2001071010 A1 WO 2001071010A1 US 0109087 W US0109087 W US 0109087W WO 0171010 A1 WO0171010 A1 WO 0171010A1
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hsl7p
jbpl
protein
yeast
hsl7δ
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English (en)
Inventor
Sidney Pestka
Jeffry R. Cook
Terri Goss Kinzy
Jin-Hyung Lee
David Norris
Brian P. Pollack
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Rutgers State University of New Jersey
Rutgers Health
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University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to yeast protein Hsl7p, which is a homologue of Janus kinase binding protein 1, JBPl.
  • Hsl7p is a newly characterized protein methyltransferase.
  • the yeast protein Hsl7p is a sequence and functional homologue of JBPl indicating an intricate link between protein methylation and macroscopic changes in yeast morphology.
  • the Jak-Stat pathway plays a crucial role in the signal transduction of many cytokines, growth factors and hormones.
  • Central to this pathway are the Jak family of protein tyrosine kinases. This family includes the mammalian kinases Jakl, Jak2, Jak3 and Tyk2 and the Drosophila melanogaster kinase encoded by the hopscotch(hop) locus.
  • the Jaks are essential for the biological activities mediated by these ligands and defects in this family of kinases have been shown to lead to a number of disease states in both mammals and Drosophila melanogaster.
  • Jak kinases The role of the Jak kinases in cytokine signal transduction was first shown for the interferons (IFNs). Subsequently, many reports have demonstrated that Jak activation occurs rapidly after ligand stimulation. This activation initiates a cascade of events which includes receptor phosphorylation and recruitment, subsequent phosphorylation and nuclear translocation of members of the Stat (Signal Transducers and Activators of Transcription) family of proteins which then activate cytokine inducible genes. In addition to their enzymatic role, several reports have demonstrated that the Jaks play a structural role in the receptor complex and that the Jaks may have functions in addition to their kinase activity which are important for signaling.
  • IFNs interferons
  • kinase-inactive mutant of Jakl into cells that lack this kinase (and are unresponsive to interferon- ⁇ (IFN- ⁇ )) restores partial IFN- ⁇ -induced gene expression. Furthermore, the amino terminus of Tyk2 stabilizes the IFNAR1 chain of the IFN- ⁇ receptor complex.
  • Jak2 was reported to interact with SHPTP1, SHPTP2, PP2A, P13K, Yes, Fyn, She, Syp, Grb2, the angiotensin II ATI receptor and the serotonin 5-HT2A receptor (31-44).
  • Jak2 was reported to interact with SHPTP1, SHPTP2, PP2A, P13K, Yes, Fyn, She, Syp, Grb2, the angiotensin II ATI receptor and the serotonin 5-HT2A receptor (31-44).
  • Jak2 is reported to interact with SHPTP1, SHPTP2, PP2A, P13K, Yes, Fyn, She, Syp, Grb2, the angiotensin II ATI receptor and the serotonin 5-HT2A receptor (31-44).
  • JBPl Janus kinase binding protein 1
  • AdoMet S-adenosy-L-methionine
  • MBP myelin basic protein
  • HSL7 histone synthetic lethal 7 gene of Saccharomyces cerevisiae was originally defined as a gene which is lethal when mutated in combination with histone H3.
  • Hsl7p was found to be a negative regulator of Swelp and Ste20p function as well as a protein which associates with the septin ring during bud formation. Disruption of HSL7 was reported to result in cell cycle abnormalities and the production of extremely long buds
  • Figure 1 illustrates a comparison of JBPl and Hsl7p sequences.
  • Figure 2 illustrates the UV crosslinking of [ 3 H]AdoMet to Hsl7p.
  • Figure 2A shows the UN crosslinking of [ 3 H] AdoMet to Hsl7p, with details essentially the same as those described previously.
  • Figure 2B show the in vitro methylation of protein substrates by Hsl7p.
  • Figure 2C shows the inhibition of myelin basic protein in vitro methylation by homocysteine.
  • Figure 3 illustrates morphological characteristics of different yeast strains used in this study.
  • Figure 3 A shows wild type (TKY307) yeast.
  • Figure 3B shows yeast with a disrupted HSL7 gene (SPY101).
  • Figure 3C shows hsl7 ⁇ yeast transformed with pGALFLAGHSL7 (SPY103).
  • Figure 3D shows hsl7 ⁇ yeast expressing JBPl (SPY104).
  • Figure 3E shows hsl7 ⁇ yeast expressing JBPl-MT (SPY105).
  • Figure 4 is a bar graph illustrating the effect of HSL7 gene disruption and complementation on elongated bud phenotype in yeast.
  • Figure 5 illustrates the amino acid sequence of Hsl7p.
  • Figure 6 illustrates the amino acid sequence of JBPl. SUMMARY OF THE INVENTION
  • the present invention relates to yeast protein Hsl7p, which is a homologue of Janus kinase binding protein 1, JBPl.
  • Hsl7p is a newly characterized protein methyltransferase.
  • the yeast protein Hsl7p is a sequence and functional homologue of JBPl indicating an intricate link between protein methylation and macroscopic changes in yeast morphology.
  • the present invention pertains to a protein methyltransferase comprising all or a part of the sequence of Hsl7p as disclosed in Figure 5, a homologue to JBPl comprising all or a part of the sequence disclosed in Figure 6, a protein methyltransferase expressed by HSL7, a homologue to JBPl expressed by HSL7, and an hsl7 ⁇ strain of S. cerevisiae.
  • the present invention further pertains to pharmaceutical compositions for providing interferon therapy to a human comprising a therapeutically effective amount of a protein methyltransferase expressed by HSL7 admixed with a pharmaceutically acceptable vehicle or carrier.
  • the yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBPl, a newly characterized protein methyltransferase. Hsl7p also is shown to be a methyltransferase.
  • the yeast protein Hsl7p is a sequence and functional homologue of JBPl indicating an intricate link between protein methylation and macroscopic changes in yeast morphology.
  • the yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBPl, a newly characterized protein methyltransferase. As disclosed herein for the first time, Hsl7p also is shown to be a methyltransferase. It can be crosslinked to [ H]S-adenosylmethionine and exhibits in vitro protein methylation activity. Calf histones H2A and H4 and bovine myelin basic protein were methylated by Hsl7p whereas histones HI, H2B, H3, and bovine cytochrome c were not.
  • JBPl can complement Saccharomyces cerevisiae with a disrupted HSL7 gene as judged by a reduction of the elongated bud phenotype; and a point mutation in the JBPl S-adenosylmethionine consensus binding sequence eliminated all complementation by JBPl.
  • Hsl7p is also a protein methyltransferase.
  • Hsl7 ⁇ strain of S. cerevisiae.
  • the phenotype of the hsl7 ⁇ strain is characterized by elongated buds.
  • Hsl7p is a protein methyltransferase and that JBPl can complement yeast lacking the HSL7 gene. Therefore, we conclude the yeast protein Hsl7p is a sequence and functional homologue of JBPl.
  • Calf thymus histones were obtained from Roche Molecular Biochemicals (Indianapolis, IN); bovine myelin basic protein, cytochrome c, S- adenosylhomocysteine from Sigma (St. Louis, MO); [ 3 H]AdoMet (specific activity 55-85 Ci/mmol) from New England Nuclear (Boston, MA); and protein A/G PLUS-agarose beads from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
  • Plasmid constructs Yeast genomic DNA was obtained from Research Genetics, Inc. (Huntsville, AL). The HSL7 gene was amplified with 5' and 3' primers CTGCAGTACAAAGGGTTCAGTTTG and GTCGACCAGTATATAGTATACAATGC, respectively, and the amplicon digested with Pst I and Sal I, then subcloned into plasmid pTKB175 under control of the GALI promoter and containing the TRPl marker. The R368A mutant of JBPl and wild type JBPl cDNAs were constructed in plasmid pcDNA3 as reported.
  • the JBPl plasmids were digested with Barri ⁇ l and Apal and subcloned into ⁇ TKB175 producing plasmids pGALJBPl-MT and pGALJBPl-WT, respectively.
  • the Flag-HSL7 construct was produced by amplifying yeast genomic DNA with the 3' primer defined above plus a 5' primer (CGCGGATCCGCGATGGACTACAAGGACGA- CGATGACAAGATGCATAGCAACGTATTTGTTGGT) which encodes a Flag epitope.
  • the amplified DNA was digested with BamHl and Sail and subcloned into plasmid pTKB175 producing the yeast expression vector pGALFLAGHSL7.
  • hsl7 ⁇ ce ⁇ ls Production of hsl7 ⁇ ce ⁇ ls.
  • the hsl7::URA3 disruption in pBluescript construct (a gift from M. Grunstein) was transformed into haploid wild type yeast (TKY307; Table 1) and transformants were selected with SD/-Ura medium. Disruption of HSL7 was confirmed by Southern blotting.
  • the reaction contained 150 mM ⁇ aCl, 50 mM Tris HCl (pH 8.0), 1 % ⁇ P- 40, 0.8 mM PMSF, 3 ⁇ g/ml antipain, 10 ig/ml benzamidine (10 4 kallikrein- inactivating units/ml) plus 1 ⁇ g/ml each of leupeptin, chymostatin and pepstatin.
  • the 50 ⁇ l reactions were incubated at 30°C for 30 minutes.
  • Figure 1 illustrates a comparison of JBPl and Hsl7p sequences.
  • R368A indicates the location of the point mutation introduced in subdomain I of the consensus AdoMet binding site. Mutagenesis was performed as described. Similarity/identity numbers were calculated for each of the subdomains (I-IV). For domains III and IN, two percentages were calculated since the domains are of different sizes in HS 17p and JBPl. The first set of percentages indicates similarities and identities based on the length of the HSL7 seauence. The second set of sequences indicates similarities and identities based on the length of the JBPl sequence.
  • FIG. 1 illustrates the UV crosslinking of [ 3 H] AdoMet to Hsl7p.
  • Figure 2 A shows the UN crosslinking of [ 3 H] AdoMet to Hsl7p, with details essentially the same as those described previously .
  • the 7.5% gel was dried and exposed to Biomax MR film for 15 days at -70°C.
  • Figure 2B show the in vitro methylation of protein substrates by Hsl7p. Methylation reactions were done as described in "Experimental Procedures.” Each lane contained 10 ⁇ g at substrate protein. The position of the labeled bands coincided exactly with the location of the substrate proteins or the Coomassie blue-stained gel (not shown). The dried 15% gel was exposed to Biomax MR film for 21 days at - 70°C.
  • Figure 2C shows the inhibition of myelin basic protein in vitro methylation by homocysteine.
  • Protein methylation reactions were conducted as described in "Experimental Procedures," except that homocysteine was added to the other reaction components on ice. The reactions were then incubated at 30°C for 30 minutes. The 15% gel was exposed to Biomax MR film for 14 days at -70°C. Protein sizes were calculated by the migration of broad range protein standards (BioRad).
  • Figure 3 illustrates morphological characteristics of different yeast strains used in this study. Arrows indicate cells with elongated buds. All cells were grown in media containing 2% galactose to induce the gene under the control of the GALI promoter. The elongated bud phenotype was never observed in wild type yeast or in hsl7 ⁇ yeast transformed with pGALFLAGHSL7 (SPY103). In the hsl7 ⁇ strain, 15 to 20% of the yeast have elongated buds. Complementation with JBPl reduces the elongated bud phenotype significantly but not completely.
  • Figure 3A shows wild type (TKY307) yeast.
  • Figure 3B shows yeast with a disrupted HSL7 gene (SPYIOI).
  • Figure 3C shows hsl7 ⁇ yeast transformed with pGALFLAGHSL7 (SPY103).
  • Figure 3D shows hsl7 ⁇ yeast expressing JBPl (SPY104).
  • Figure 3E shows hsl7 ⁇ yeast expressing JBPl-MT (SPY105).
  • Figure 4 is a bar graph illustrating the effect of HSL7 gene disruption and complementation on elongated bud phenotype in yeast. Cells were grown in 2% galactose to induce the gene under the control of the GALI promoter. At least 3 different fields were counted for each determination.
  • JBP1-WT refers to the wild type JBPl cDNA; JBPl-MT indicates the mutated (R368A) JBPl cDNA. Values are + S.E.M.
  • Figure 5 illustrates the amino acid sequence of Hsl7p.
  • Figure 6 illustrates the amino acid sequence of JBPl.
  • JBPl The human Jak-binding protein, JBPl, was identified in a two- hybrid screen with a 3.33 kb fragment of the Jak2 cDNA as a bait.
  • JBPl is homologous to a number of other eukaryotic protein methyltransferases, including human ANMl and ANM2, rat ANMl, and S. cerevisiae Hmtlp.
  • the homology between JBPl and Hsl7p suggests that yeast Hsl7p is also a protein methyltransferase. There are four conserved subdomains in JBPl and Hsl7p.
  • the first of these regions contains a GxGRG motif which is identical between the human and yeast proteins. This motif is known to be the site at which AdoMet is bound to the protein.
  • Figure 1 shows the location of a point mutation (R368A) which was introduced in the GxGRG motif in order to produce a JBPl without methyltransferase activity when histones or myelin basic protein are used as substrates.
  • R368A point mutation
  • Hsl7p is a methyltransferase
  • Hsl7p can bind [ 3 H] AdoMet.
  • JBPl from HeLa cells was crosslinked to [ 3 H]AdoMet (Fig 2A, lane 1).
  • Hsl7p binds AdoMet, consistent with its being a methyltransferase.
  • calf histones bovine myelin basic protein and bovine cytochrome c were incubated with immunoprecipitated Hsl7p and [ 3 H] AdoMet.
  • Figure 2B shows that H2A, H4 and myelin basic protein were methylated by Hsl7p while HI, H2B, H3 and cytochrome c were not.
  • This pattern of methylation by Hsl7p is identical to that observed with human JBPl, indicating that Hsl7p and JBPl are functional as well as structural homologues.
  • the data of Figure 2C demonstrate that homocysteine, an inhibitor of methyltransferases that use AdoMet as the methyl donor, blocks the methylation of myelin basic protein by both JBPl and Hsl7p.
  • HSL7 gene was disrupted in the haploid S. cerevisiae strain TKY307 with an hsl7 ⁇ construct which has a 1.14 kb section of the gene replaced with the URA3 gene.
  • the resultant strain, SPY101 was used as a host for other constructs (Table
  • the hsl7 knock-out strain was generated by the homologous recombination as described under "Experimental Procedures.”
  • the plasmids pGALFLAGHSL7, pGALJBPl-WT and pGALJBPl-MT were constructed in the yeast expression vector pTKB175 having a TRPl marker and are shown in Table 1 illustrating the genotype of strains used in study. TABLE 1
  • Vectors pTKB175, pGALFLAGHSL7, pGAUBPl-WT and pGALJBPl-MT were transformed into hsl7 ⁇ cells (SPYIOI) to produce strains
  • SPY102, SPY103, SPY104 and SPY105 were grown in SD/-Trp with either 2% glucose (uninduced) or 2% galactose (induced).
  • the morphology of these strains was similar to that reported previously: the wild type yeast have small circular or oval buds (Figure 3A) whereas the hsl7 ⁇ cells have elongated buds ( Figure 3B).
  • Complementation of the hsl7 ⁇ cells with the HSL7 expression vector yielded cells with a normal phenotype ( Figure 3C).
  • complementation of the hsl7 ⁇ cells with the JBP1-WT expression vector produced cells which were nearly normal (Figure 3D), but complementation with the mutant
  • TKY307 were found to have no elongated buds, whereas 15% of the hsl7 ⁇ cells expressed elongated buds (SPYIOI, Figure 3B).
  • SPYIOI pGALFLAGHSL7
  • SPY102 Figure 4
  • JBPl complements its Hsl7p homologue
  • the hsl7 ⁇ cells were transformed with the cDNA for human JBPl expressed under control of the yeast GALI promoter.
  • Hsl7p and JBPl may each perform a number of cellular functions, it was important to determine whether the observed phenotypic complementation is due to protein methyltransferase activity of JBPl or to some other function.
  • JBPl was therefore mutated (R368A) at the GxGRG motif and the vector expressing the mutant JBPl was transformed into hsl7 ⁇ cells (SPYIOI).
  • hsl7 ⁇ + JBPl-MT (SPY105) cells have as many elongated buds as does the hsl7 ⁇ strain. This demonstrates that JBPl is a functional homologue of Hsl7p and that the protein methyltransferase activity is required for complementation.
  • Hsl7p is crucial for many functions in yeast. Disruption of HSL7 affects cell morphology, cell cycle progression and sensitivity to chemicals, including calcium, caffeine, calcofluor white, vanadate and verapamil.
  • Hsl7p is a protein methyltransferase and that the mutant JBPl-MT does not complement hsl7 ⁇ indicates that some of the phenotypic effects of Hsl7p and JBPl are produced by methylation of target proteins.
  • histones and myelin basic protein as methyl group acceptors however, the identity of the in vivo substrates for Hsl7p remains to be determined.
  • methyltransf erases have been identified in yeast: mRNA cap, rRNA, isoprenylcysteine and tRNA methyltransf erases; two protein methyltransferases in S. cerevisiae: Rmtlp (also referred to as Hmtlp or Odplp;) and Rmt2p.
  • Rmt2p was discovered during a search for yeast proteins containing conserved AdoMet binding motifs, it methylates the ⁇ -nitrogen atom of arginine residues, but its in vivo substrate proteins are not known.
  • Rmtlp is an arginine methyltransferase which methylates a number of yeast proteins such as Npl3p and Hrplp, which are hnRNPs and poly(A)+ RNA binding proteins.
  • Npl3p and Hrplp yeast proteins
  • Hrplp hnRNPs and poly(A)+ RNA binding proteins.
  • Rmtlp methylates mammalian hnRNP Al, cytochrome c, histones and myoglobin, but not myelin basic protein.
  • Hsl7p exhibits different substrate specificity in vitro than Rmtlp. Hsl7p methylates myelin basic protein whereas Rmtlp does not; Rmtlp methylates cytochrome c whereas Hsl7p does not.
  • JBPl does not completely rescue hsl7 ⁇ cells (SPY104).
  • Differences in the yeast lysate proteins methylated by JBPl and Hsl7p J.-H. Lee, J.R. Cook and S. Pestka, unpublished results) could account for this.
  • the R368A mutation of JBPl which did not restore normal morphology demonstrated that complementation in hsl7 ⁇ yeast absolutely requires methyltransferase activity.
  • Figure 1 JBPl and Hsl7p share extensive homology in other regions as well.
  • the S. pombe homologue of Hsl7p is skbl, a protein which is known to interact with the kinase Shkl.
  • a S. pombe skbl deletion mutant exhibits altered morphology where the wild type cells are more elongated than the mutants and over-expression of skbl results in hyper-elongated cells.
  • JBPl was shown to functionally complement skbl in terms of cell length while Hsl7p did not.
  • the roles of skbl and Hsl7p S. pombe and S. cerevisiae, respectively, are likely to be similar.
  • Skbl and Hsl7p are involved in the ras signaling pathway in S. pombe and in S. cerevisiae; and deletion of both genes produces cells with growth abnormalities.
  • Hsl7p is a functional component of the MAP kinase pathway where it was shown to compete with Cdc42p for binding to the amino-terminal half of Ste20p.
  • Ste20p is a member of the p65 PAK protein kinase family and is involved in several yeast signal transduction pathways. In the haploid mating pathway, Ste20p is a kinase downstream of the Stel2p and Ste3p receptors which bind a-factor and a-factor, respectively.
  • Ste20p, Stellp, Ste7p, and Stel2p are also required for the switch from an axial to a bipolar mode of budding which results in invasive growth.
  • Hsl7p contributes to all of these phenotypes via Ste20p. Hsl7p also inhibits the Swelp kinase that phosphorylates Cdc28, thereby producing changes in the cell cycle. In the Swelp/Cdc28p morphogenesis checkpoint pathway, Swelp and Hsllp both associate with Hsl7p.
  • Hsl7p localizes to septin rings formed at the bud necks of dividing cells where it forms a complex with Hsllp, Swelp and the septins and is involved in the Cdc28-mediated G2/M cell cycle transition.
  • McMillan et al. reported that Hsllp can phosphorylate Hsl7p. While the levels of Hsl7p appear to be relatively constant during the cell cycle, Hsllp expression is cell cycle-dependent and so is its phosphorylation of Hsl7p. Ultimately Hsl7p and Hsllp interact to promote the degradation of Swelp, possibly by polyubiquitination.
  • Hsl7p is a functional component of both the Swelp/Cdc28p morphogenesis checkpoint and MAP kinase pathways and may serve as a link between these two pathways
  • Hsl7p is intrinsically involved in at least two pathways: the Swelp/Cdc28p morphogenesis checkpoint; and Ras signaling in the MAP kinase pathway. Furthermore, because Hsl7p methylates histones, Hsl7p is likely involved in chromatin remodeling and may contribute to the "histone code" that can control downstream events. Our data presented in this report provide evidence that there is an intricate link between protein methylation and yeast morphogenesis and other pathways such as Ras signaling and histone coding; and provide a biochemical basis for understanding the mechanism by which Hsl7p modulates these many diverse actions.

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Abstract

L'invention concerne la protéine de levure Hs17p, laquelle est un homologue de la protéine de liaison à la Janus kinase1, JBP1. La protéine Hs17p est une protéine méthyltransférase nouvellement identifiée. La protéine de levure Hs17p est une séquence et un homologue fonctionnel de la protéine JBP1 indiquant une liaison complexe entre la méthylation des protéines et des modifications macroscopiques de la morphologie de la levure.
PCT/US2001/009087 2000-03-23 2001-03-22 Proteine de levure methyltransferase hsl7p Ceased WO2001071010A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6103531A (en) * 1998-02-13 2000-08-15 Ohio State Research Foundation Methods of disrupting interferon signal transduction pathways

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6103531A (en) * 1998-02-13 2000-08-15 Ohio State Research Foundation Methods of disrupting interferon signal transduction pathways

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
POLLACK ET AL.: "The human homologue of the yeast proteins Skb1 and Hs17p interacts with jak kinases and contains protein methyltransferase activity", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 44, 29 October 1999 (1999-10-29), pages 31531 - 31542, XP002941864 *

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