Identification of regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein kinase-1a/p90rsk that are inducible by MAPK

Mitogen activated protein kinase-activated protein kinase-1 (MAPKAP-K1; also known as p90(rsk)) contains two protein kinase domains in a single polypeptide, The N-terminal kinase domain is necessary for the phosphorylation of peptide substrates, whereas the C-terminal kinase domain is required for full activation of the N-terminal domain, Here we identify six sites in MAPKAP-K1a that become phosphorylated in transfected COS-1 cells, The inactive form of MAPKAP-K1a in unstimulated cells is partially phosphorylated at Ser(222) and Ser(733). Stimulation with phorbol 12-myristate 13-acetate induces the phosphorylation of Thr(360), Ser(364), Thr(574), and Ser(381) and increases the phosphorylation of Ser(222) and Ser(733). Our data indicate that mitogen-activated protein kinase activates the C-terminal kinase domain by phosphorylating Thr(574) and contributes to the activation of the N-terminal kinase domain by phosphorylating Ser(364). The activated C-terminal domain phosphorylates Ser(381), which, together with phosphorylation of Ser(364), activates the N-terminal kinase domain, The phosphorylation of Ser(222) and Ser(733), which can be catalyzed by the N-terminal domain, does not activate MAPKAP-K1a per se, but Ser(222) phosphorylation appears to be required for activation, Ser(222), Ser(364), and Ser(381) are situated in analogous positions to phosphorylation sites in protein kinase B, protein kinase C, and p70(S6K), suggesting a common mechanism of activation for these growth factor-stimulated protein kinases.