Post-translational modification-dependent regulation of CaMKII-delta conformation in live cardiac myocytes.

Abstract

Calcium/calmodulin dependent protein kinase II (CaMKII) is a key regulator of numerous functions in cardiac myocytes including Ca regulation, contraction, and transcriptional control. Overactivation of CaMKII due to post-translational modifications like autophosphorylation at Thr287 can autonomously activate and prolong CaMKII activation, even after Ca/CaM dissociates, and thereby promote cardiac pathologies such as heart failure and arrhythmias. The specific molecular mechanisms by which CaMKII PTMs autonomously activate and prolong kinase activity are not fully understood. We found using a novel intermolecular FRET-based approach in live myocytes in a recent study that PTM-dependent alteration in CaMKII's affinity for CaM is one molecular basis by which CaMKII becomes autonomously activated. Here, we investigate how the conformational state of CaMKII in the autonomously activated state may be affected by PTMs using our FRET-based CaMKIIδ conformational state reporter, Camui (RFP-CaMKIIδ-GFP), and its phosphomimetic (T287D) and phosphoresistant (T287A) mutant variants, expressed via adenoviral infection in intact cardiac myocytes. We also conducted parallel in vitro biochemical experiments using HEK cell lysates. We found that the phosphomimetic T287D Camui exhibited prolonged durations in the active open conformational state following pacing-induced activation relative to WT or phosphoresistant T287A Camui. Moreover, the prolonged autonomous conformational state persisted even after intracellular Ca levels returned to diastolic levels at which CaM dissociates. These data suggest that PTM-dependent regulation of the conformational state of CaMKII may serve as an additional molecular mechanism for autonomous CaMKII activation that may function in tandem with PTM-dependent alterations in CaMKII's affinity for CaM.