Carboxyl-terminal phosphorylation regulates the function and subcellular localization of protein kinase C beta II

Protein kinase C is processed by three phosphorylation events before it is competent to respond to second messengers. Specifically, the enzyme is firs t phosphorylated at the activation loop by another kinase, followed by two ordered autophosphorylations at the carboxyl terminus (Keranen, L, M,, Duti l, E, M,, and Newton, A. C, (1995) Curr, Biol, 5, 1394-1403). This study ex amines the role of negative charge at the first conserved carboxyl-terminal phosphorylation position, Thr-641, in regulating the function and subcellu lar localization of protein kinase C beta II Mutation of this residue to Al a results in compensating phosphorylations at adjacent sites, so that a tri ple Ala mutant was required to address the function of phosphate at Thr-641 , Biochemical and immunolocalization analyses of phosphorylation site mutan ts reveal that negative charge at this position is required for the followi ng: 1) to process catalytically competent protein kinase C; 2) to allow aut ophosphorylation of Ser-660; 3) for cytosolic localization of protein kinas e C; and 4) to permit phorbol ester-dependent membrane translocation. Thus, phosphorylation of Thr-641 in protein kinase C beta II is essential for bo th the catalytic function and correct subcellular localization of protein k inase C, The conservation of this residue in every protein kinase C isozyme , as well as other members of the kinase superfamily such as protein kinase A, suggests that carboxyl-terminal phosphorylation serves as a key molecul ar switch for defining kinase function.