Structural and Catalytic Mechanisms Underlying CaMKII Substrate Selection

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multimeric Ser/Thr protein kinase that orchestrates global changes in cell function via the regulation of multiple substrates. CaMKII is a dodecameric holoenzyme composed of 12 subunits that each consists of catalytic and regulatory domains that are tethered together via an association domain. The multimeric nature of CaMKII allows an intraholenzyme, intersubunit autophosphorylation reaction that is important for regulating this kinase's sensitivity to calcium-CaM. However, another possible function of this kinase's multimeric structure could be to increase the concentration of catalytic subunits to a localized environment. To address structural mechanisms underlying substrate selection, we tested soluble and immobilized substrates for differences between CaMKII and a monomeric form of CaMKII (1-316). Both kinases retain their dependence on calcium-CaM for activity and possess very little differences in their phosphorylation of high and low affinity substrates in solution (∼5 μM and ∼50 μM, respectively). Using diffusion-restricted substrates (i.e. peptide substrates immobilized synthesized on cellulose membranes), we observed that monomeric CaMKII displayed levels of phosphorylation that were proportional to the relative Km values observed in solution. Surprisingly, multimeric CaMKII displayed increased phosphorylation on substrates with a higher Km (i.e. lower affinity) and a lower level of phosphorylation on high affinity substrates (low Km). These data suggest that CaMKII diffusion-restricted substrates in specific subcellular compartments, like the post-synaptic density, may be regulated as a complex, rather than simply as individual substrates selected by their specific Km values. Thus, in addition to supporting intraholoenzyme autophosphorylation reactions, the unique multimeric architecture of the CaMKII holoenzyme may permit this multifunctional enzyme to regulate complex assemblies of substrates through dual mechanisms of restricting high affinity substrates and enhancing the phosphorylation of low affinity substrates.