Mechanism of reversion of Rous sarcoma virus transformation by herbimycin A: reduction of total phosphotyrosine levels due to reduced kinase activity and increased turnover of p60v-src1.

We studied the mechanism of reversion of Rous sarcoma virus (RSV)-transformation by herbimycin A in a temperature-sensitive RSV-infected NRK cell line. Herbimycin reduced the total cellular phosphotyrosine level to 10% of the control value. Intracellular p60src kinase activity was reduced significantly within 3 h of herbimycin treatment, but recovered to approximately 50% of the control within 24 h, and the kinetics paralleled the decrease of the phosphorylation of a cellular target p36. Partial proteolytic phosphopeptide analysis of p60src revealed decreased phosphorylation of tyrosine 416 in the C-terminal half. Analysis of the steady state level of p60src indicated 30% decrease in src protein. Measurement of the rates of p60src synthesis and degradation showed that the decrease in the level was due to the accelerated degradation of src protein. Cycloheximide blocked this enhanced turnover of p60src, but did not block the herbimycin-induced inactivation of p60src kinase. Subcellular distribution and myristoylation of p60src protein were not altered. These results support the idea that RSV transformation is associated with elevated phosphotyrosine levels and indicate that inhibition of src kinase activity can reverse RSV-transformation by reducing cellular phosphotyrosine content.