Time-dependent Autoinactivation of Phospho-Thr286-αCa2+/Calmodulin-dependent Protein Kinase II

Abstract

Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) is thought to exert its role in memory formation by autonomous Ca(2+)-independent persistent activity conferred by Thr(286) autophosphorylation, allowing the enzyme to remain active even when intracellular [Ca(2+)] has returned to resting levels. Ca(2+) sequestration-induced inhibition, caused by a burst of Thr(305/306) autophosphorylation via calmodulin (CaM) dissociation from the Thr(305/306) sites, is in conflict with this view. The processes of CaM binding, autophosphorylation, and inactivation are dissected to resolve this conflict. Upon Ca(2+) withdrawal, CaM sequential domain dissociation is observed, starting with the rapid release of the first (presumed N-terminal) CaM lobe, thought to be bound at the Thr(305/306) sites. The time courses of Thr(305/306) autophosphorylation and inactivation, however, correlate with the slow dissociation of the second (presumed C-terminal) CaM lobe. Exposure of the Thr(305/306) sites is thus not sufficient for their autophosphorylation. Moreover, Thr(305/306) autophosphorylation and autoinactivation are shown to occur in the continuous presence of Ca(2+) and bound Ca(2+)/CaM by time courses similar to those seen following Ca(2+) sequestration. Our investigation of the activity and mechanisms of phospho-Thr(286)-alphaCaMKII thus shows time-dependent autoinactivation, irrespective of the continued presence of Ca(2+) and CaM, allowing a very short, if any, time window for Ca(2+)/CaM-free phospho-Thr(286)-alphaCaMKII activity. Physiologically, the time-dependent autoinactivation mechanisms of phospho-Thr(286)-alphaCaMKII (t(1/2) of approximately 50 s at 37 degrees C) suggest a transient kinase activity of approximately 1 min duration in the induction of long term potentiation and thus memory formation.