Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites.
BACKGROUND
Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated.


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.
Ory
Stéphane
S
Regulation of Cell Growth Laboratory, NCI-Frederick, Frederick, MD 21702, USA.
Zhou
Ming
M
Conrads
Thomas P
TP
Veenstra
Timothy D
TD
Morrison
Deborah K
DK
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D004789:Enzyme Activation; D066298:In Vitro Techniques; D020935:MAP Kinase Signaling System; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054648:Protein Phosphatase 2; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D048948:14-3-3 Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D019908:Proto-Oncogene Proteins c-raf; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D018631:ras Proteins