Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions

Raf-1 activation is a complex process which involves plasma membrane recrui tment, phosphorylation, protein-protein and lipid-protein interactions, We now show that PP1 and PP2A serine-threonine phosphatases also have a positi ve role in Ras dependent Raf-1 activation, General serine-threonine phospha tase inhibitors such sodium fluoride, or beta-glycerophosphate and sodium p yrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I-1 or protein phosphatase inhibitor 2 al l abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitor s block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to s erine phosphatase inhibitors. Sucrose gradient analysis demonstrates that s erine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribut ion of 14-3-3 and Raf-1 across different plasma membrane microdomains, Thes e observations suggest that dephosphorylation of S259 is a critical early s tep in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement . Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.