Identification of regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein kinase-1a/p90(rsk) 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 Ser²²² and Ser⁷³³. Stimulation with phorbol 12- myristate 13-acetate induces the phosphorylation of Thr³⁶⁰, Ser³⁶⁴, Thr⁵⁷⁴, and Ser³⁸¹ and increases the phosphorylation of Ser²²² and Ser⁷³³. Our data indicate that mitogen-activated protein kinase activates the C-terminal kinase domain by phosphorylating thr⁵⁷⁴ and contributes to the activation of the N-terminal kinase domain by phosphorylating Ser³⁶⁴. The activated C-terminal domain phosphorylates Ser³⁸¹, which, together with phosphorylation of Ser³⁶⁴, activates the N-terminal kinase domain. The phosphorylation of Ser²²² and Ser⁷³³, which can be catalyzed by the N-terminal domain, does not activate MAPKAP-K1a per se, but Ser²²² phosphorylation appears to be required for activation. Ser²²², Ser³⁶⁴, and Ser³⁸¹ 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.