Phosphorylation of the kinase suppressor of Ras by associated kinases

The kinase suppressor of Ras (KSR) is a loss-of-function allele that suppre sses the rough eye phenotype of activated Ras in Drosophila and the multivu lval phenotype of activated Ras in Caenorhabditis elegans. The physiologica l role of mammalian KSR is nor known. We examined the mechanisms regulating the phosphorylation of this putative kinase in mammalian cells. Wild-type mouse KSR and a mutated KSR protein predicted to create a kinase-dead prote in are phosphorylated identically in intact cells and in the immune complex . Phosphopeptide sequencing identified 10 in vivo phosphorylation sites in KSR, all of which reside in the 539 noncatalytic amino terminal amino acids . Expression of the amino terminal portion of KSR alone demonstrated that i t was phosphorylated in the intact cell and in an immune complex in a manne r indistinguishable from that of intact KSR. These data demonstrate that th e kinase domain of KSR is irrelevant to its phosphorylation state and sugge st that the phosphorylation of KSR and its association with a distinct set of kinases may affect intracellular signaling.