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

Abstract

Subcellular localization of Ca2+/calmoduli-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 CaMKIIa (mCaMKIIa) or Xenapus CaMKIIB (xCaMKIIB) 32P-autophosphorylated at Thr286/287 as a probe. Numerous [32P]CaMKII-binding proteins were identified in various whole rat tissue extracts, but binding was most prominent to forebrain proteins of 190kDa (p190) and 140kDa (p140). Fractionation of forebrain extracts localized to p190 and p140 to a crude particulate/cytoskeletal fraction anbd isolated postsynaptic densities. [33P]mCaMKIIa-bound to p190 with an apparent Kd of 609 nM (subunit concentration) and a B-max of 7.0 pmol of mCaMKIIa subunit bound per mg of P2 protein, as measured using hte overlay assay. Binding of 100nM [32P]mCaMKIIa to p190 was competed by nonradioactive mCaMKIIa autophosphorylated on Thr286 (EC-50% = 200nM) but to a much lesser extent by nonradioactive mCaMKIIa autophosphorylated on Thr306 (EC-50%>2000nM). In addition, non-phosphorylated mCaMKIIa was a poor competitor for [32P]mCaMKIIa binding to p190. The competition data indicate that Ca2+/CaM-dependent autophosphorylation at Thr286 promotes binding to p190, whereas, Ca2+/CaM-independent autophosphorlyation at Thr306 does not enhance binding. Therefore, CaMKII may become localized to postsynaptic dendritic Ca2+ concentration.