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Keywords:

  • Protein kinase;
  • Protein phosphatase;
  • Calmodulin;
  • Postsynaptic density;
  • Synaptic plasticity

Abstract

  1. Top of page
  2. Abstract

Abstract: Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr286 generates Ca2+-independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr286 in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A-like enzymes account for >70% of activity toward exogenous soluble Thr286-autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C 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 PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr286-autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (32P autophosphorylated in the presence of Ca2+/calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD-associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca2+-independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments.

Abbreviations used: CaMKII, calcium/calmodulin-dependent protein kinase II; mCaMKIIα, recombinant mouse α-isoform of calcium/calmodulin-dependent protein kinase II; P-I1, thiophosphorylated inhibitor 1; [32P]mCaMKIIα, Thr286-autophosphorylated recombinant mouse α-isoform of calcium/calmodulin-dependent protein kinase II; PP1, PP2A, PP2B, and PP2C, protein phosphatase 1, 2A, 2B, and 2C, respectively; PSD, postsynaptic density; PSD-CaMKII, calcium/calmodulin-dependent protein kinase II endogenous to isolated postsynaptic densities; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.