Ca2؉ -calmodulin-dependent protein kinase II (CaMkinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (␣, ␤, ␥, and ␦) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 Å revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their K m for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca 2؉ -calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (␥ > ␤ > ␦ > ␣) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (␤ > ␥ > ␦ > ␣). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform.1 is a major downstream effector of Ca 2ϩ signaling in eukaryotic cells. A rise in intracellular Ca 2ϩ concentration leads to binding of Ca 2ϩ ions to calmodulin (CaM), which binds to and activates CaM-kinase II. Upon activation, this enzyme has the ability to autophosphorylate, a process that confers Ca 2ϩ -independent activity upon the kinase (1) and greatly increases its affinity for CaM (2). Once activated, CaM-kinase II phosphorylates numerous target proteins and is involved in many cellular functions, including synaptic plasticity, synaptic vesicle mobilization, regulation of gene expression, regulation of smooth muscle contractility, and modulation of ion channel function (3-7). The fact that CaM-kinase II has so many potential substrates raises the question of the relationship between its activation and a specific response to a particular Ca 2ϩ signal. Possibly, the regulated expression of the multiple isoforms of CaM-kinase II confers these unique properties.CaM-kinase II is expressed from a family of four closely related genes, ␣, ␤, ␥, and ␦, each of which produces mRNA that can be alternatively spliced, giving rise to at least 30 different proteins (8, 9). The overall organization of each of the four kinase isoforms is similar: an N-terminal catalytic domain is followed by a regulatory domain that contains an autoinhibitory region and a CaM-binding site, and a C-terminal association domain, through which the subunits interact to assemble into holoenzymes (10). Between the CaM-binding domain and the association domain is a region termed the...

Excitotoxicity mediated by glutamate receptors plays crucial roles in ischemia and other neurodegenerative diseases. Whereas overactivation of ionotropic glutamate receptors is neurotoxic, the role of metabotropic glutamate receptors (mGluRs), and especially mGluR1, remains equivocal. Here we report that activation of NMDA receptors results in calpain-mediated truncation of the C-terminal domain of mGluR1alpha at Ser(936). The truncated mGluR1alpha maintains its ability to increase cytosolic calcium while it no longer activates the neuroprotective PI(3)K-Akt signaling pathways. Full-length and truncated forms of mGluR1alpha play distinct roles in excitotoxic neuronal degeneration in cultured neurons. A fusion peptide derived from the calpain cleavage site of mGluR1alpha efficiently blocks NMDA-induced truncation of mGluR1alpha in primary neuronal cultures and exhibits neuroprotection against excitotoxicity both in vitro and in vivo. These findings shed light on the relationship between NMDA and mGluR1alpha and indicate the existence of a positive feedback regulation in excitotoxicity involving calpain and mGluR1alpha.

No abstract

No abstract

binding to the C-domain of CaM. Specifically, PEP-19 accelerates the rates of both association and dissociation of Ca 2ϩ without greatly affecting the overall K Ca of the C-domain (5). RC3 accelerates the rate of Ca 2ϩ dissociation from CaM, but has a lesser effect on the association rate, thereby decreasing the affinity of binding Ca 2ϩ to the C-domain of CaM (6). Importantly, both PEP-19 and RC3 exert these effects even when CaM is bound to CaM-dependent protein kinase II (CKII␣) (5, 6).These results suggest that PEP-19 and RC3 could have broad extrinsic effects on CaM-related signaling pathways by modulating the Ca 2ϩ binding properties of free or enzyme-bound CaM. This is consistent with the phenotype of RC3 knock-out mice, which show defects in synaptic plasticity (7), attenuated phosphorylation of hippocampal protein kinase A and C substrates (8), and altered Ca 2ϩ dynamics in cortical neurons (9). Both PEP-19 and RC3 contain an IQ motif. This rather loose consensus sequence (IQXXXRGXXXR) was first identified as the light chain binding site in conventional myosins, but was subsequently recognized as a CaM binding sequence in numerous other proteins (10). IQ motif proteins exhibit diverse modes of interaction with CaM that include Ca 2ϩ -dependent or independent binding (10), binding to both or only one domain of CaM (5, 11-13), binding multiple CaMs to multiple IQ motifs (14), and exchange of CaM between the IQ motif and other sites in the same protein (15, 16).These intriguing structure-function relationships of IQ motifs led us to identify amino acids in PEP-19 that are required to modulate Ca 2ϩ binding to CaM. We show here that the consensus IQ CaM binding motif is necessary, but not sufficient to mimic the effect of intact PEP-19 on CaM. An adjacent highly acidic amino acid sequence acts in synergy with the IQ motif to modulate Ca 2ϩ binding to the C-domain of CaM. We propose that this acidic/IQ sequence constitutes a new CaM regulatory motif. EXPERIMENTAL PROCEDURESRecombinant Proteins and Peptides-Recombinant CaM, CaM(K75C), CaM(T110C), CaM(T34C), CaM(T34C,T110C), PEP19, and RC3 were cloned, expressed, and purified as described previously (5, 6, 16 -18). The expression plasmid for the C-domain of CaM (residues 78 -148) was a generous gift * This work was supported in part by National Institutes of Health Grants GM069611 and NS038310 and Robert A. Welch Foundation Grant AU1144. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. -bound calmodulin; CaM ACR , acrylodan labeled CaM(K75C); CaM DANS , IAEDANS labeled CaM(K75C); CKII, CaM-dependent protein kinase II; RC3, neurogranin; FRET, fluorescence resonance energy transfer; MOPS, 4-morpholinepropanesulfonic acid; acrylodan, 6-acryloyl-2-dimethylaminonaphthalene; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; DDPM,maleimide; HPLC, high performance liq...