Lobe-specific Functions of Ca2+·Calmodulin in αCa2+·Calmodulin-dependent Protein Kinase II Activation

Homan Soleymani,Katalin Török,Philip Beesley,Abdirahman M. Jama,Ahmed J. Al-Nagar,Zawahir Chowdhury,Stephen Martin,Sana Z. Baig,Jonathan Gabriel

doi:10.1074/jbc.m110.157057

Abstract

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.

Highlights

␣Ca2ϩ1⁄7calmodulin-dependent protein kinase II (␣CaMKII)3 [1] function is essential for memory formation, as demonstrated by studies of spatial learning [2] and of the electrophysiologically testable memory model, N-methyl-D-aspartic acid receptor (NMDAR)-dependent long term potentiation (LTP) [3, 4]
Frequency NMDAR-mediated Ca2ϩ signals [5,6,7,8]. ␣CaMKII is activated by Ca2ϩ stimulation in a frequency-dependent manner [9], and here we propose that Ca2ϩ signal transduction is determined by the activation mechanism of ␣CaMKII by Ca2ϩ1⁄7CaM
Characterization of EF-Hand CaM Mutants—The role of each Ca2ϩ binding site of CaM in the activation mechanism of ␣CaMKII was investigated to understand how signaling by Ca2ϩ influx is decoded by CaM in neurons

Summary

Introduction

␣Ca2ϩ1⁄7calmodulin-dependent protein kinase II (␣CaMKII)3 [1] function is essential for memory formation, as demonstrated by studies of spatial learning [2] and of the electrophysiologically testable memory model, N-methyl-D-aspartic acid receptor (NMDAR)-dependent long term potentiation (LTP) [3, 4]. The first active state is formed by the binding of Ca2ϩ1⁄7CaM and ATP This complex is ready to phosphorylate protein or peptide substrates. ␣CaMKII is a substrate for itself, and the ␣CaMKII active complex is converted to its second active state, Ca2ϩ1⁄7CaM1⁄7phospho-Thr286-␣CaMKII, by rapid autophosphorylation. This latter process is essential for NMDAR-dependent. Ca2ϩ1⁄7CaM-bound phospho-Thr286-␣CaMKII exists at activating (Ͼ500 nM) and at resting (Ͻ100 nM) free Ca2ϩ concentrations [14]. At free Ca2ϩ concentrations of Ͻ20 nM, when Ca2ϩ and CaM dissociate, activity is reduced to Յ5% of that of fully Ca2ϩ1⁄7CaM-stimulated phospho-Thr286-␣CaMKII [14]. The activity of phospho-Thr286-␣CaMKII is transient in LTP and is time-limited by inhibitory Thr305/306 autophosphorylation [8, 15]

Methods

Results

Conclusion