DIFFERENTIAL INACTIVATION OF POSTSYNAPTIC DENSITY-ASSOCIATED AND SOLUBLE CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE-II BY PROTEIN-PHOSPHATASE-1 AND PROTEIN-PHOSPHATASE-2A/

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (Ca MKII) at Thr(286) generates Ca2+-independent activity. As an initial s tep toward understanding CaMKII inactivation, protein phosphatase clas ses (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Th r(286) in rat forebrain subcellular fractions were identified using ph osphatase inhibitors/activators, by fractionation using ion exchange c hromatography and by immunoblotting. PP2A-like enzymes account for >70 % of activity toward exogenous soluble Thr(286)-autophosphorylated CaM KII in crude cytosol, membrane, and cytoskeletal extracts; PPI and PP2 C account for the remaining activity, CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs) ; each protein phosphatase is also present in isolated PSDs, but only PPI is enriched during PSD isolation, When isolated PSDs dephosphoryla ted exogenous soluble Thr(286)-autophosphorylated CaMKII, PP2A again m ade the major contribution. However, CaMKII endogenous to PSDs (P-32 a utophosphorylated in the presence of Ca2+/calmodulin) was predominantl y dephosphorylated by PP1. In addition, dephosphorylation of soluble a nd PSD-associated CaMKII in whole forebrain extracts was catalyzed pre dominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associ ated forms of CaMKII appear to be dephosphorylated by distinct enzymes , suggesting that Ca2+-independent activity of CaMKII is differentiall y regulated by protein phosphatases in distinct subcellular compartmen ts.