Aberrant protein phosphorylation at tyrosine is responsible for the growth-inhibitory action of pp60v-src expressed in the yeast Saccharomyces cerevisiae.
Expression of pp60v-src, the transforming protein of Rous sarcoma virus, arrests the growth of the yeast Saccharomyces cerevisiae. To determine the basis of this growth arrest, yeast strains were constructed that expressed either wild-type v-src or various mutant v-src genes under the control of the galactose-inducible, glucose repressible GAL1 promoter. When shifted to galactose medium, cells expressing wild-type v-src ceased growth immediately and lost viability, whereas cells expressing a catalytically inactive mutant (K295M) continued to grow normally, indicating that the kinase activity of pp60v-src is required for its growth inhibitory effect. Mutants of v-src altered in the SH2/SH3 domain (XD4, XD6, SPX1, and SHX13) and a mutant lacking a functional N-terminal myristoylation signal (MM4) caused only a partial inhibition of growth, indicating that complete growth inhibition requires either targeting of the active kinase or binding of the kinase to phosphorylated substrates, or both. Cells arrested by v-src expression displayed aberrant microtubule structures, alterations in DNA content and elevated p34CDC28 kinase activity. Immunoblotting with antiphosphotyrosine antibody showed that many yeast proteins, including the p34CDC28 kinase, became phosphorylated at tyrosine in cells expressing v-src. Both the growth inhibition and the tyrosine-specific protein phosphorylation observed following v-src expression were reversed by co-expression of a mammalian phosphotyrosine-specific phosphoprotein phosphatase (PTP1B). However a v-src mutant with a small insertion in the catalytic domain (SRX5) had the same lethal effect as wild-type v-src, yet induced only very low levels of protein-tyrosine phosphorylation. These results indicate that inappropriate phosphorylation at tyrosine is the primary cause of the lethal effect of pp60v-src expression but suggest that only a limited subset of the phosphorylated proteins are involved in this effect.
Florio
M
M
Department of Molecular and Cell Biology, University of California at Berkeley 94720.
Wilson
L K
LK
Trager
J B
JB
Thorner
J
J
Martin
G S
GS
D001358:Avian Sarcoma Viruses / Q000235:genetics; D002453:Cell Cycle; D005656:Fungal Proteins / Q000378:metabolism; D015870:Gene Expression; D006131:Growth Inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014170:Transformation, Genetic; D014443:Tyrosine / Q000378:metabolism
D005656:Fungal Proteins; D006131:Growth Inhibitors; D014443:Tyrosine; D015688:Oncogene Protein pp60(v-src); D017027:Protein Tyrosine Phosphatases