Several retrovirus transforming proteins possess a protein-tyrosine kinase activity which is believed to mediate transformation by these viruses. In animal cells certain growth factor and peptide hormone receptors also display a similar protein tyrosine kinase activity. The mechanism by which the expression of these enzymes leads to growth activation remains obscure. Several protein-tyrosine kinases have been detected in the yeast Saccharomyces cerevisiae and one of them has been purified. The protein- tyrosine kinase activity purified from yeast is associated with a 40kDa protein which undergoes autophosphorylation at tyrosine in vitro and which is phosphorylated at tyrosine in vivo. Although yeast cells are eukaryotic, their growth and division do not involve the complex morphogenetic and tissue-specific interactions characteristic of metazoa; furthermore, yeast is amenable to both classical genetic analysis and molecular genetic techniques. For these reasons it may be possible to use this organism to define the role of protein phosphorylation at tyrosine in the cell division cycle. The gene encoding the 40 kDa enzyme will be cloned by sequencing tryptic peptides and synthesizing mixed oligonucleotide probes, and the cloned gene will be used to isolate related genes from genomic or cDNA libraries. The function of the genes will be determined by gene disruption analysis and site-directed mutagenesis. To facilitate the identification of tyrosine kinase substrates, the kinases will be expressed at elevated levels using vectors which contain an inducible promoter. Proteins phosphorylated at tyrosine in vivo will be identified using an antiphosphotyrosine antibody or radiolabeling techniques. The physiological functions of tyrosine phosphorylation will be examined by determining the effects of peptide mating hormones, nutritional status and mutations in cell cycle genes on protein kinase activity in vitro and protein tyrosine phosphorylation in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM044173-01
Application #
3303349
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Trager, J B; Martin, G S (1997) The role of the Src homology-2 domain in the lethal effect of Src expression in the yeast Saccharomyces cerevisiae. Int J Biochem Cell Biol 29:635-48
Brazill, D T; Thorner, J; Martin, G S (1997) Mck1, a member of the glycogen synthase kinase 3 family of protein kinases, is a negative regulator of pyruvate kinase in the yeast Saccharomyces cerevisiae. J Bacteriol 179:4415-8
Wilson, L K; Dhillon, N; Thorner, J et al. (1997) Casein kinase II catalyzes tyrosine phosphorylation of the yeast nucleolar immunophilin Fpr3. J Biol Chem 272:12961-7
Jiang, W; Lim, M Y; Yoon, H J et al. (1995) Overexpression of the yeast MCK1 protein kinase suppresses conditional mutations in centromere-binding protein genes CBF2 and CBF5. Mol Gen Genet 246:360-6
Ma, D; Cook, J G; Thorner, J (1995) Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway. Mol Biol Cell 6:889-909
Benton, B M; Zang, J H; Thorner, J (1994) A novel FK506- and rapamycin-binding protein (FPR3 gene product) in the yeast Saccharomyces cerevisiae is a proline rotamase localized to the nucleolus. J Cell Biol 127:623-39
Kelly, T J; Martin, G S; Forsburg, S L et al. (1993) The fission yeast cdc18+ gene product couples S phase to START and mitosis. Cell 74:371-82
Lim, M Y; Dailey, D; Martin, G S et al. (1993) Yeast MCK1 protein kinase autophosphorylates at tyrosine and serine but phosphorylates exogenous substrates at serine and threonine. J Biol Chem 268:21155-64
Dailey, D; Schieven, G L; Lim, M Y et al. (1990) Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity. Mol Cell Biol 10:6244-56