Identification of constitutive and Ras-inducible phosphorylation sites of KSR: Implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression

Genetic and biochemical studies have identified kinase suppressor of Ras (K SR) to be a conserved component of Ras-dependent signaling pathways. To bet ter understand the role of KSR in signal transduction, we have initiated st udies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phospho rylation sites of KSR In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KS R contains three additional sites of phosphorylation (Thr260, Thr274, and S er443), all of which match the consensus motif (Px[S/T]P) for phosphorylati on by mitogen-activated protein kinase (MAPK). Further, we find that treatm ent of cells with the MEK inhibitor PD98059 blocks phosphorylation of the R as-inducible sites and that activated MAPK associates with KSR in a Ras-dep endent manner. Together, these findings indicate that KSR is an in vivo sub strate of MAPK. Mutation of the identified phosphorylation sites did not al ter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, sugg esting that phosphorylation at these sites may serve other functional roles , such as regulating catalytic activity. Interestingly, during the course o f this study, we found that the biological effect of KSR varied dramaticall y with the level of KSR protein expressed. In Xenopus oocytes, KSR function ed as a positive regulator of Ras signaling when expressed at low levels, w hereas at high levels of expression, KSR blocked Ras-dependent signal trans duction. Likewise, overexpression of Drosophila KSR blocked R7 photorecepto r formation in the Drosophila eye. Therefore, the biological function of KS R as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.