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) 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 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. [32P]m-CaMKII alpha-bound to p190 with an apparent Kd of 609 nM (subunit concentration) and a Bmax of 7.0 pmol of mCaMKII alpha subunit bound per mg of P2 protein, as measured using the overlay assay. Binding of 100 nM [32P]m-CaMKII alpha to p190 was competed by nonradioactive mCaMKII alpha autophosphorylated on Thr286 (EC50% = 200 nM), but to a much lesser extent by nonradioactive mCaMKII alpha autophosphorylated on Thr306 (EC50% > 2000 nM). In addition, nonphosphorylated mCaMKII alpha was a poor competitor for [32P]mCaMKII alpha binding to p190. The competition data indicate that Ca2+/CaM-dependent autophosphorylation at Thr286 promotes binding to p190, whereas, Ca2+/CaM-independent autophosphorylation at Thr306 does not enhance binding. Therefore, CaMKII may become localized to postsynaptic densities by p190 following its activation by an increase of dendritic Ca2+ concentration.
McNeill
R B
RB
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Colbran
R J
RJ
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005260:Female; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013569:Synapses / Q000378:metabolism*; D014981:Xenopus
D003598:Cytoskeletal Proteins; D009419:Nerve Tissue Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases