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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of California Berkeley
Schools of Arts and Sciences
United States
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