The Multimeric Architecture of CaMKII Alters Substrate Regulation of Diffusion-Restricted Environments

Abstract

Calcium/calmodulin dependent protein kinase II (CaMKII) is a family of multifunctional Ser/Thr kinases that play a key role in calcium signaling in many cell types, including neurons, where it performs both structural and signaling roles in learning and memory. CaMKII exists as a dodecameric holoenzyme. Whether the multimeric nature of the CaMKII holoenzyme also produces unique regulation and substrate interactions is unknown; however, having multiple catalytic subunits in each holoenzyme could afford enhanced substrate and binding partner interactions leading to altered rules for substrate selection and phosphorylation compared to monomeric kinases. Using a peptide-based model of CaMKII substrates, we measured substrate phosphorylation in soluble versus immobilized assays designed to represent the compartmentalized, diffusion-restricted environments in which CaMKII is known to function (such as the post-synaptic density [PSD] in the dendritic boutons of neurons) using both CaMKII and a monomeric form of CaMKII (1-316). Solution kinetics revealed very minimal differences in substrate phosphorylation of various substrates, yet significant changes were observed in phosphorylation profiles of immobilized peptide substrates. Strikingly, whereas monomeric CaMKII phosphorylation correlated proportionally to relative Km values from solution assays, multimeric CaMKII displayed preferential phosphorylation of low-affinity substrates (higher Km) and diminished phosphorylation of high-affinity substrates (lower Km). Subsequent experiments involving the phosphorylation of purified PSDs produced >4-fold global enhancement of phosphorylation by multimeric CaMKII compared to the addition of equal catalytic units of monomer, consistent with findings that many of the CaMKII substrates in the PSD lack a canonical phosphorylation motif and could therefore, as low-affinity substrates, take full advantage of CaMKII's multimeric structure. Thus, the multimeric architecture of CaMKII may confer a unique and novel mechanism for regulating substrate phosphorylation within diffusion-restricted subcellular compartments, which may underscore CaMKII's dominant role in synaptic plasticity and learning.