Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes

Abstract

Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged Balpha/delta regulatory subunits by approximately 3-fold or knock-down of Balpha by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-Balpha/delta subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in Balpha knock-down cells but elevated in Balpha- and Bdelta-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing Balpha or Bdelta. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABalphaC and ABdeltaC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABalphaC and ABdeltaC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.