Interaction of Autophosphorylated Ca2+/Calmodulin-dependent Protein Kinase II with Neuronal Cytoskeletal Proteins

Subcellular localization of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by interaction with specific anchoring proteins may be an important mechanism contributing to the regulation of CaMKII. Proteins capable of binding CaMKII were identified by the use of a gel overlay assay with recombinant mouse CaMKIIα (mCaMKIIα) or Xenopus CaMKIIβ (xCaMKIIβ) 32P-autophosphorylated at Thr286/287as a probe. Numerous 32PCaMKII-binding proteins were identified in various whole rat tissue extracts, but binding was most prominent to forebrain proteins of 190 kDa (p190) and 140 kDa (p140). Fractionation of forebrain extracts localized p190 and p140 to a crude particulate/cytoskeletal fraction and isolated postsynaptic densities. 32PmCaMKIIα-bound to p190 with an apparent Kdof 609 nM (subunit concentration) and a Bmax of 7.0 pmol of mCaMKIIα subunit bound per mg of P2 protein, as measured using the overlay assay. Binding of 100 nM 32PmCaMKIIα to p190 was competed by nonradioactive mCaMKIIα autophosphorylated on Thr286 (EC50= 200 nM), but to a much lesser extent by nonradioactive mCaMKIIα autophosphorylated on Thr306(EC50> 2000 nM). In addition, nonphosphorylated mCaMKIIα was a poor competitor for 32PmCaMKIIα binding to p190. The competition data indicate that Ca2+/CaM-dependent autophosphorylation at Thr286promotes binding to p190, whereas, Ca2+/CaM-independent autophosphorylation at Thr306does not enhance binding. Therefore, CaMKII may become localized to postsynaptic densities by p190 following its activation by an increase of dendritic Ca2+concentration. Subcellular localization of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by interaction with specific anchoring proteins may be an important mechanism contributing to the regulation of CaMKII. Proteins capable of binding CaMKII were identified by the use of a gel overlay assay with recombinant mouse CaMKIIα (mCaMKIIα) or Xenopus CaMKIIβ (xCaMKIIβ) 32P-autophosphorylated at Thr286/287as a probe. Numerous 32PCaMKII-binding proteins were identified in various whole rat tissue extracts, but binding was most prominent to forebrain proteins of 190 kDa (p190) and 140 kDa (p140). Fractionation of forebrain extracts localized p190 and p140 to a crude particulate/cytoskeletal fraction and isolated postsynaptic densities. 32PmCaMKIIα-bound to p190 with an apparent Kdof 609 nM (subunit concentration) and a Bmax of 7.0 pmol of mCaMKIIα subunit bound per mg of P2 protein, as measured using the overlay assay. Binding of 100 nM 32PmCaMKIIα to p190 was competed by nonradioactive mCaMKIIα autophosphorylated on Thr286 (EC50= 200 nM), but to a much lesser extent by nonradioactive mCaMKIIα autophosphorylated on Thr306(EC50> 2000 nM). In addition, nonphosphorylated mCaMKIIα was a poor competitor for 32PmCaMKIIα binding to p190. The competition data indicate that Ca2+/CaM-dependent autophosphorylation at Thr286promotes binding to p190, whereas, Ca2+/CaM-independent autophosphorylation at Thr306does not enhance binding. Therefore, CaMKII may become localized to postsynaptic densities by p190 following its activation by an increase of dendritic Ca2+concentration.