Search tips
Search criteria 


Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1991 February; 11(2): 987–1001.
PMCID: PMC359764

Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.


A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (4.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Celenza JL, Carlson M. A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science. 1986 Sep 12;233(4769):1175–1180. [PubMed]
  • Comoglio PM, Di Renzo MF, Tarone G, Giancotti FG, Naldini L, Marchisio PC. Detection of phosphotyrosine-containing proteins in the detergent-insoluble fraction of RSV-transformed fibroblasts by azobenzene phosphonate antibodies. EMBO J. 1984 Mar;3(3):483–489. [PubMed]
  • Cooper JA, Sefton BM, Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. [PubMed]
  • Draetta G, Piwnica-Worms H, Morrison D, Druker B, Roberts T, Beach D. Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate. Nature. 1988 Dec 22;336(6201):738–744. [PubMed]
  • Foster R, Thorner J, Martin GS. Nucleotidylation, not phosphorylation, is the major source of the phosphotyrosine detected in enteric bacteria. J Bacteriol. 1989 Jan;171(1):272–279. [PMC free article] [PubMed]
  • Gorelick FS, Wang JK, Lai Y, Nairn AC, Greengard P. Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals. J Biol Chem. 1988 Nov 25;263(33):17209–17212. [PubMed]
  • Gould KL, Nurse P. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature. 1989 Nov 2;342(6245):39–45. [PubMed]
  • Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. [PubMed]
  • Hanley RM, Means AR, Ono T, Kemp BE, Burgin KE, Waxham N, Kelly PT. Functional analysis of a complementary DNA for the 50-kilodalton subunit of calmodulin kinase II. Science. 1987 Jul 17;237(4812):293–297. [PubMed]
  • Johnston LH, Eberly SL, Chapman JW, Araki H, Sugino A. The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle. Mol Cell Biol. 1990 Apr;10(4):1358–1366. [PMC free article] [PubMed]
  • Kamps MP, Sefton BM. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [PubMed]
  • Kornbluth S, Paulson KE, Hanafusa H. Novel tyrosine kinase identified by phosphotyrosine antibody screening of cDNA libraries. Mol Cell Biol. 1988 Dec;8(12):5541–5544. [PMC free article] [PubMed]
  • Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. [PMC free article] [PubMed]
  • Lee MG, Nurse P. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature. 1987 May 7;327(6117):31–35. [PubMed]
  • Leto TL, Pleasic S, Forget BG, Benz EJ, Jr, Marchesi VT. Characterization of the calmodulin-binding site of nonerythroid alpha-spectrin. Recombinant protein and model peptide studies. J Biol Chem. 1989 Apr 5;264(10):5826–5830. [PubMed]
  • Levin DE, Hammond CI, Ralston RO, Bishop JM. Two yeast genes that encode unusual protein kinases. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6035–6039. [PubMed]
  • Lewin B. Driving the cell cycle: M phase kinase, its partners, and substrates. Cell. 1990 Jun 1;61(5):743–752. [PubMed]
  • Lin CR, Kapiloff MS, Durgerian S, Tatemoto K, Russo AF, Hanson P, Schulman H, Rosenfeld MG. Molecular cloning of a brain-specific calcium/calmodulin-dependent protein kinase. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5962–5966. [PubMed]
  • McGuinness TL, Lai Y, Greengard P. Ca2+/calmodulin-dependent protein kinase II. Isozymic forms from rat forebrain and cerebellum. J Biol Chem. 1985 Feb 10;260(3):1696–1704. [PubMed]
  • McLeod M, Beach D. Homology between the ran1+ gene of fission yeast and protein kinases. EMBO J. 1986 Dec 20;5(13):3665–3671. [PubMed]
  • Nadin-Davis SA, Nasim A. A gene which encodes a predicted protein kinase can restore some functions of the ras gene in fission yeast. EMBO J. 1988 Apr;7(4):985–993. [PubMed]
  • Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. [PubMed]
  • Riabowol K, Draetta G, Brizuela L, Vandre D, Beach D. The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell. 1989 May 5;57(3):393–401. [PubMed]
  • Roussou I, Thireos G, Hauge BM. Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase. Mol Cell Biol. 1988 May;8(5):2132–2139. [PMC free article] [PubMed]
  • Schieven G, Thorner J, Martin GS. Protein-tyrosine kinase activity in Saccharomyces cerevisiae. Science. 1986 Jan 24;231(4736):390–393. [PubMed]
  • Sefton BM, Beemon K, Hunter T. Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo. J Virol. 1978 Dec;28(3):957–971. [PMC free article] [PubMed]
  • Slice LW, Taylor SS. Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli. J Biol Chem. 1989 Dec 15;264(35):20940–20946. [PubMed]
  • Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. [PubMed]
  • Snyder M, Elledge S, Sweetser D, Young RA, Davis RW. Lambda gt 11: gene isolation with antibody probes and other applications. Methods Enzymol. 1987;154:107–128. [PubMed]
  • Stern DF, Heffernan PA, Weinberg RA. p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity. Mol Cell Biol. 1986 May;6(5):1729–1740. [PMC free article] [PubMed]
  • Stern DF, Kamps MP. EGF-stimulated tyrosine phosphorylation of p185neu: a potential model for receptor interactions. EMBO J. 1988 Apr;7(4):995–1001. [PubMed]
  • Stern DF, Kamps MP, Cao H. Oncogenic activation of p185neu stimulates tyrosine phosphorylation in vivo. Mol Cell Biol. 1988 Sep;8(9):3969–3973. [PMC free article] [PubMed]
  • Takio K, Wade RD, Smith SB, Krebs EG, Walsh KA, Titani K. Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families. Biochemistry. 1984 Aug 28;23(18):4207–4218. [PubMed]
  • Tan JL, Spudich JA. Developmentally regulated protein-tyrosine kinase genes in Dictyostelium discoideum. Mol Cell Biol. 1990 Jul;10(7):3578–3583. [PMC free article] [PubMed]
  • Toda T, Cameron S, Sass P, Wigler M. SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits. Genes Dev. 1988 May;2(5):517–527. [PubMed]
  • Wang JY, Queen C, Baltimore D. Expression of an Abelson murine leukemia virus-encoded protein in Escherichia coli causes extensive phosphorylation of tyrosine residues. J Biol Chem. 1982 Nov 25;257(22):13181–13184. [PubMed]
  • Weber IT, Shabb JB, Corbin JD. Predicted structures of the cGMP binding domains of the cGMP-dependent protein kinase: a key alanine/threonine difference in evolutionary divergence of cAMP and cGMP binding sites. Biochemistry. 1989 Jul 11;28(14):6122–6127. [PubMed]
  • Weber W, Bertics PJ, Gill GN. Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation. J Biol Chem. 1984 Dec 10;259(23):14631–14636. [PubMed]
  • Wittenberg C, Reed SI. Conservation of function and regulation within the Cdc28/cdc2 protein kinase family: characterization of the human Cdc2Hs protein kinase in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Sep;9(9):4064–4068. [PMC free article] [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)