Lobe-specific functions of Ca2+·calmodulin in alphaCa2+·calmodulin-dependent protein kinase II activation.

N-methyl-D-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.

Time-dependent autoinactivation of phospho-Thr286-alphaCa2+/calmodulin-dependent protein kinase II.

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.

A two-state model for Ca2+/CaM-dependent protein kinase II (alphaCaMKII) in response to persistent Ca2+ stimulation in hippocampal neurons.

Persistent elevation of the intracellular free Ca(2+) concentration [Ca(2+)](i) is neurotoxic and therefore it is important to understand how it affects downstream components of the Ca(2+) signaling pathway. The response of calmodulin (CaM) and alphaCa(2+)/CaM-dependent protein kinase II (alphaCaMKII), to intracellular Ca(2+) overload in hippocampal neurons is studied by confocal imaging of fluorescently tagged proteins. Transient and persistent redistribution of CaM and alphaCaMKII together is seen from the cytosol to dendritic and somatic punctae. Typical persistent redistribution occurs following a lag of 138+/-(S.E.M.) 12 s and is complete at 460+/-(S.E.M.) 34 s (n=18), lack of Thr(286)-autophosphorylation of alphaCaMKII however promotes the formation of early transient punctae (peak at 40 s). In contrast, the T286D-mimick of phospho-Thr(286)-alphaCaMKII forms punctae with a delay >10 min, indicating that Thr(286)-autophosphorylation is antagonistic to CaMKII clustering. A two-state model is proposed in which phospho-Thr(286)-alphaCaMKII, formed immediately upon Ca(2+) stimulation, is primarily responsible for target interactions and memory functions of alphaCaMKII. However, a distinct clustering form denoted alphaCaMKII(c), generated upon persistent intracellular free Ca(2+) elevation, is deposited in the punctae which are made of self-interacting CaM/CaMKII complexes. Punctate deposition disables both the interactions and the activity of CaMKII.