PROTEIN-KINASE-C IS REGULATED IN-VIVO BY 3 FUNCTIONALLY DISTINCT PHOSPHORYLATIONS

Background: Protein kinase Cs are a family of enzymes that transduce t he plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphoryla tions before it is competent to respond to second messengers. Results: We have identified the positions and functions of the in vivo phospho rylation sites of protein kinase C by mass spectrometry and peptide se quencing of native and phosphatase-treated kinase from the detergent-s oluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl termi nus of protein kinase C beta II are phosphorylated in vivo. T500 and S 660 are selectively dephosphorylated in vitro by protein phosphatase 2 A to yield an enzyme that is still capable of lipid-dependent activati on, whereas all three residues are dephosphorylated by protein phospha tase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytoso l, and that T500 is not all autophosphorylation site. Conclusions: Str uctural and biochemical analyses of native and phosphatase-treated pro tein kinase C indicate that protein kinase C is processed by three pho sphorylations. Firstly, trans-phosphorylation on the activation loop ( T500) renders it catalytically competent to autophosphorylate. Secondl y, a subsequent autophosphorylation on the carboxyl terminus (T641) ma intains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular local ization. The conservation of each of these residues (or an acidic resi due) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family .