Dynamic Substrate Gating in CaMKII by Autophosphorylation

Abstract

Calcium/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulates diverse substrates in cellular processes ranging from metabolism and cell cycle control to calcium homoeostasis, excitable cell activity and plasticity. CaMKII is a multimeric holoenzyme composed of 8–14 subunits from four closely related isoforms (α, β, γ, δ). The calcium sensor CaM binds to a target sequence within the autoregulatory domain (ARD) of each CaMKII subunit. Coincident CaM binding within the holoenzyme supports intersubunit autophosphorylation (T286 α-isoform and T287 β-, γ-, and δ-isoforms) within the ARD; a process enabling calcium-spike frequency encoding in excitable cells. We now show that T287 autophosphorylation tunes substrate phosphorylation to couple the activation state of CaMKII to substrate selectivity. Specifically, unlike high-affinity substrates, weak substrates require T286 autophosphorylation. Substrate switching may allow many of CaMKII substrates with weak non-consensus sites to be coupled to T287 autophosphorylation. The mechanism of this substrate filtering appears linked to the ARD as a catalytic fragment devoid of the ARD (and T286) exhibits broad substrate specificity as seen with T287 autophosphorylation (and phosphomimetic substitutions). Consistently, we find that substrates differential compete for access to the catalytic domain as indicated by differential ARD displacement as measured by associative changes in CaM binding. Graded CaMKII activity/autophosphorylation within the holoenzyme produces differential substrate phosphorylation profiles consistent with calcium-spike frequency encoding. We provide a structural model to explain T287 autophosphorylation-associated regulation of substrate selectivity and hypothesize that calcium-spike frequency encoding into T286 autophosphorylation tunes CaMKII's substrate selectivity to yield diverse cellular responses.