Mechanisms regulating Raf‐1 activity in signal transduction pathways

Abstract

Raf‐1 is a key protein involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Biochemical and genetic studies have demonstrated that Raf‐1 functions downstream of activated tyrosine kinases and Ras and upstream of mitogen‐activated protein kinase (MAPK) and MAPK kinase (MKK or MEK) in many signaling pathways. A major objective of our laboratory has been to determine how Raf‐1 becomes activated in response to signaling events. Using mammalian, baculovirus, and Xenopus systems, we have examined the roles that phosphorylation and protein‐protein interactions play in regulating the biological and biochemical activity of Raf‐1. Our studies have provided evidence that the activity of Raf‐1 can be modulated by both Ras‐dependent and Ras‐independent pathways. Recently, we reported that Arg89 of Raf‐1 is a residue required for the association of Raf‐1 and Ras. Mutation of this residue disrupted interaction with Ras and prevented Ras‐mediated, but not protein kinase C‐or tyrosine kinase‐mediated, enzymatic activation of Raf‐1 in the baculovirus expression system. Further analysis of this mutant demonstrated that kinase‐defective Raf‐1 proteins interfere with the propagation of proliferative and developmental signals by binding to Ras and blocking Ras function. Our findings have also shown that phosphorylation events play a role in regulating Raf‐1. We have identified sites of in vivo phosphorylation that positively and negatively alter the biological and enzymatic activity of Raf‐1. In addition, we have found that some of these phosphorylation sites are involved in mediating the interaction of Raf‐1 with potential activators (Fyn and Src) and with other cellular proteins (14‐3‐3). Results from our work suggest that Raf‐1 is regulated at multiple levels by several distinct mechanisms.