Mechanisms Underlying Cooperativity in CaMKII Autophosphorylation and Substrate Phosphorylation

Abstract

As a member of the calmodulin-activated kinases, Ca2+/calmodulin dependent protein kinase II (CaMKII) is a serine/threonine kinase coupled to calcium signaling. Unlike other multifunctional CaMK members, CaMKII has a unique dodecameric architecture potentially permitting cooperative forms of autoregulation and substrate phosphorylation. We observed that CaMKII phosphorylation of a peptide derived from the autoregulatory domain (AC-2) displays positive cooperativity (nH=1.8). Another T-site binding peptide substrate derived from S1303 phosphorylation site on NR2B also displayed positive cooperativity (nH=2.1). Surprisingly, a truncated form of CaMKII1-317 monomer also shows this cooperativity towards NR2B and AC-2 peptides (nH=1.6 vs 1.8, respectively). Syntide-2, a traditional substrate peptide lacking T-site interactions, does not exhibit cooperativity in substrate phosphorylation for either monomeric or multimeric CaMKII (nH=1.0; nH=1.1, respectively). These data suggest that the positive cooperativity seen with substrate phosphorylation is unique to T-site site binding substrates and may involve potential allosteric substrate interactions on the catalytic surface. Cooperativity within the holoenzyme occurring between subunits has been predicted based on the fact that Ca2+/CaM binding can induce Thr286 autophosphorylation, an intersubunit intraholenzyme reaction, whereby, neighboring subunits act as both kinase and substrate following coincident Ca2+CaM binding. Using Ca2+/CaM-independent activity (i.e. autonomous) as a measure of Thr286 autophosphorylation, CaM titration experiments revealed that both Ca2+/CaM-dependent and autonomous forms of CaMKII activity were cooperative (nH=2.1 for both) for Ca2+/CaM activation and autophosphorylation. Titrating CaM levels to ratios below 1 per holoenzyme (i.e. 12 CaMKII subunits per holoenzyme) generate submaximal autonomous activity, whereas, CaM levels at a ratio of ∼2 per CaMKII holoenzyme generate maximal autonomous activity. Thus, CaM activation of CaMKII appears to follow a cooperative model whereby neighboring subunits within the holoenzyme preferentially obtain the activator to promote autophosphorylation even in the face of limiting CaM.