ABERRANT PROTEIN-PHOSPHORYLATION AT TYROSINE IS RESPONSIBLE FOR THE GROWTH-INHIBITORY ACTION OF PP60(V-SRC) EXPRESSED IN THE YEAST SACCHAROMYCES-CEREVISIAE

Expression of pp60(v-src), the transforming protein of Rous sarcoma vi rus, arrests the growth of the yeast Saccharomyces cerevisiae. To dete rmine the basis of this growth arrest, yeast strains were constructed that expressed either wild-type v-src or various mutant v-src genes un der the control of the galactose-inducible, glucose repressible GAL1 p romoter. When shifted to galactose medium, cells expressing wild-type v-src ceased growth immediately and lost viability, whereas cells expr essing a catalytically inactive mutant (K295M) continued to grow norma lly, indicating that the kinase activity of pp60(v-src) is required fo r its growth inhibitory effect. Mutants of v-src altered in the SH2/SH 3 domain (XD4, XD6, SPX1, and SHX13) and a mutant lacking a functional N-terminal myristoylation signal (MM4) caused only a partial inhibiti on of growth, indicating that complete growth inhibition requires eith er targeting of the active kinase or binding of the kinase to phosphor ylated substrates, or both. Cells arrested by v-src expression display ed aberrant microtubule structures, alterations in DNA content and ele vated p34(CDC28) kinase activity. Immunoblotting antiphosphotyrosine a ntibody showed that many yeast proteins, including the p34(CDC28) kina se, became phosphorylated at tyrosine in cells expressing v-src. Both the growth inhibition and the tyrosine-specific protein phosphorylatio n observed following v-src expression were reversed by co-expression o f a mammalian phosphotyrosine-specific phosphoprotein phosphatase (PTP 1B). However a v-src mutant with a small insertion in the catalytic do main (SRX5) had the same lethal effect as wild-type v-src, yet induced only very low levels of protein-tyrosine phosphorylation. These resul ts indicate that inappropriate phosphorylation at tyrosine is the prim ary cause of the lethal effect of pp60(v-src) expression but suggest t hat only a limited subset of the phosphorylated proteins are involved in this effect.