THE SRC FAMILY OF PROTEIN-TYROSINE KINASES - REGULATION AND FUNCTIONS

Most of the nine members of the Src family of tyrosine kinases are res tricted in their expression, often to cells of the haematopoietic line age, while some, particularly Src, Fyn and Yes, are more ubiquitously expressed. We have been studying the functions of Src, Fyn and Yes in fibroblasts. We have shown that stimulation of quiescent fibroblasts w ith platelet-derived growth factor (PDGF) causes Src, Fyn and Yes to b ecome activated, and to associate transiently with the PDGF receptor. To address the role of Src, Fyn and Yes in the response to PDGF, we ha ve used a dominant negative approach, in which cells were engineered t o express catalytically inactive forms of Src kinases. These cells wer e unable to enter S phase in response to PDGF, and we therefore conclu de that Src family tyrosine kinases are required in order for the PDGF receptor to transmit a mitogenic signal. It has previously been shown that the kinase activity of Src is negatively regulated by phosphoryl ation of tyr 527 in its carboxy-terminal tail. A kinase, Csk, that pho sphorylates tyr 527 has recently been identified. We expressed Src in yeast to test the model that phosphorylation of tyr 527 represses acti vity by promoting intramolecular association between the tail and the SH2 domain. Inducible expression of Src in S. pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated i n the SH2 domain mere as lethal as wild-type Src, but were insensitive to Csk. We interpret these results in favour of an SH2 domain : phosp horylated tail interaction repressing Src activity. However, we have a lso found that Src molecules containing mutations in the SH3 domain ar e not regulated by Csk, suggesting that the SH3 domain also functions in the intramolecular regulation of Src activity.