Results: Using mass spectrometry to analyze the KSR1-scaffolding complex, we identify the serine/threonine protein phosphatase PP2A as a KSR1-associated protein and show that PP2A is a critical regulator of KSR1 activity. We find that the enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, we find that PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation.
Conclusions: These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites.