Generation of a temperature-sensitive cSRC.

Abstract

We have created retroviruses encoding a temperature-sensitive variant of the cSrc protein, and of its activated allele cSrc(Y527F). Cells expressing the activated allele harboring this mutation displayed a transformed phenotype when grown at 34 degrees, but not at 39 degrees, as quantitated by measuring the susceptibility of the cells to contact inhibition and their ability to grow anchorage-independently at the two temperatures. The level of the mutant protein in cells grown at the high temperature was slightly reduced, and the in vitro kinase activity of the protein was hypersensitive to inactivation by heat treatment: the combination of these two defects probably explains the temperature-sensitive nature of the transformed phenotype. Surprisingly, the pattern of tyrosine phosphorylated proteins differed very little between the two temperatures, suggesting that critical substrates are few and of low abundance. The introduction of the same mutation into the wild-type allele of cSrc, Y527, also rendered the protein thermolabile. These ecotropic retroviruses should prove useful in assessing the function of cSrc in mammalian cells.