INTERACTION OF AUTOPHOSPHORYLATED CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE-II WITH NEURONAL CYTOSKELETAL PROTEINS - CHARACTERIZATION OF BINDING TO A 190-KDA POSTSYNAPTIC DENSITY PROTEIN

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 CaMKII alpha (mCaMKII alpha or Xenopus CaMKII beta (xCaMKII beta) P-32-auto-phosphorylated at Thr(286/287) as a probe. Numerous [P-32]CaMKII-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. [P-32]mCaMKII alpha-bound to p190 with an apparent K-d of 609 nM (subunit concentration) and a B-max of 7.0 pmol of mCaMKII alpha subunit bound per mg of P2 protein, as measured using the overlay assay. Binding of 100 nM [P-32]mCaMKII alpha to p190 was competed by nonradioactive mCaMKII alpha autophosphorylated on Thr(286) (EC(50%) = 200 nM), but to a much lesser extent by nonradioactive mCaMKII alpha autophosphorylated on Thr(306) (EC(50%) > 2000 nM). In addition, nonphosphorylated mCaMKII alpha was a poor competitor for [P-32]mCaMKII alpha binding to p190. The competition data indicate that Ca2+/CaM-dependent autophosphorylation at Thr(286) promotes binding to p190, whereas, Ca2+/CaM-independent autophosphorylation at Thr(306) does not enhance binding. Therefore, CaMKII may become localized to postsynaptic densities by p190 following its activation by an increase of dendritic Ca2+ concentration.