Calmodulin-dependent protein kinase II

Three multifunctional protein kinases, cyclic AMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II, are involved in signal transduction in response to their respective second messengers, cyclic AMP, diacylglycerol, and Ca2+. This review will summarize the key findings on calmodulin-dependent protein kinase II.     

Calmodulin-dependent protein kinase II

One of the most important mechanisms for regulating neuronal functions is through second messenger cascades that control protein kinases and the subsequent phosphorylation of substrate proteins. Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is the most abundant protein kinase in mammalian brain tissues, and the alpha-subunit of this kinase is the major protein and enzymatic molecule of synaptic junctions in many brain regions. CaM-kinase II regulates itself through a complex autophosphorylation mechanism whereby it becomes calcium-independent following its initial activation. This property has implicated CaM-kinase II as a potential molecular switch at central nervous system (CNS) synapses. Recent studies have suggested that CaM-kinase II is involved in many diverse phenomena such as epilepsy, sensory deprivation, ischemia, synapse formation, synaptic transmission, long-term potentiation, learning, and memory. During brain development, the expression of CaM-kinase II at both protein and mRNA levels coincides with the active periods of synapse formation and, therefore, factors regulating the genes encoding kinase subunits may play a role in the cell-to-cell recognition events that underlie neuronal differentiation and the establishment of mature synaptic functions. Recent findings have demonstrated that the mRNA encoding the alpha-subunit of CaM-kinase II is localized in neuronal dendrites. Current speculation suggests that the localized translation of dendritic mRNAs encoding specific synaptic proteins may be responsible for producing synapse-specific changes associated with the processing, storage, and retrieval of information in neural networks.     

Binding of protein kinase substrates by fluorescently labeled calmodulin

A synthetic protein kinase substrate, PRO-LEU-SER-ARG-THR-LEU-SER-VAL-SER-SER-NH₂, undergoes calcium-dependent binding by calmodulin. Phosphorylation of the peptide decreases its affinity for calmodulin with the dissociation constant increasing from 2.4 to $\text{ca}$. 7 mM. The results are consistent with the suggestion that calmodulin and the cAMP-dependent protein kinase can act on common recognition sequences.     

Site-specific methionine oxidation in calmodulin affects structural integrity and interaction with Ca2+/calmodulin-dependent protein kinase II

Methionine oxidation in the ubiquitous calcium signaling protein calmodulin (CaM) is known to disrupt downstream signaling and target CaM for proteasomal degradation. The susceptibility of CaM to oxidation in the different conformations that are sampled during calcium signaling is currently not well defined. Using an integrative mass spectrometry (MS) approach, applying both native MS and LC/MS/MS, we unravel molecular details of CaM methionine oxidation in the context of its interaction with the Ca(2+)/CaM-dependent protein kinase II (CaMKII). Sensitivity to methionine oxidation in CaM was found to vary according to the conformational state. Three methionine residues (Met71, 72, 145) show increased reactivity in calcium-saturated CaM (holo-CaM) compared to calcium-free CaM (apo-CaM), which has important consequences for oxidation-targeted proteasomal degradation. In addition, all four methionines in the C-terminal lobe (Met109, 124, 144 and 145) are found to be protected from oxidation in a peptide-based model of the CaMKII-bound conformation (cbp-CaM). We furthermore demonstrate that the oxidation of Met144 and 145 inhibits the interaction of CaM with CaMKII. cbp-CaM, in contrast to apo- and holo-CaM, maintains its ability to bind CaMKII under simulated conditions of oxidative stress and is also protected from oxidation-induced unfolding. Thus, we show that the susceptibility towards oxidation of specific residues in CaM is tightly linked to its signaling state and conformation, which has direct implications for calcium/CaM-CaMKII related signaling.     

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.     

Spectroscopic characterization of the interaction between calmodulin-dependent protein kinase I and calmodulin

Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM-binding region is believed to occur within a 22-residue sequence (amino acids 299-320). This sequence was chemically synthesized and utilized for CaM interaction studies. Gel band shift assays and densitometry experiments with intact CaM kinase I and the CaM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that conformational changes in CaM on binding CaM kinase I or CaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of CaM, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of Met mutants of CaM showed that the Trp-containing N-terminal end of CaMKIp was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicate that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase.     

Calmodulin-dependent multiprotein kinase and protein kinase C phosphorylate the same site on HMG-CoA reductase as the AMP-activated protein kinase

Calmodulin-dependent multiprotein kinase and protein kinase C phosphorylate and inactivate both intact, microsomal HMG-CoA reductase, and the purified 53 kDa catalytic fragment. Isolation of the single phosphopeptide produced by combined cleavage with cyanogen bromide and Lys-C proteinase reveals that this is due to phosphorylation of a single serine residue near the C-terminus, corresponding to serine-872 in the human enzyme. This is identical with the single serine phosphorylated by the AMP-activated protein kinase. The nature of the protein kinase responsible for phosphorylation of this site in vivo is discussed.     

Phosphorylation of microtubule-associated protein STOP by calmodulin kinase II

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.     

Interactions of calmodulin with death-associated protein kinase peptides: experimental and modeling studies

We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM-DAPK2 S308D mutant peptide is also reported. The structures of CaM-DAPK peptide complexes present a mode of CaM-kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the largest contribution coming from the Trp305. About two-thirds of the buried surface area corresponds to nonpolar residues, showing that hydrophobic interactions play an important role in these CaM-peptide complexes. The simulation results agree with the experimental data in predicting a small effect of the S308D mutation on CaM interactions with DAPK2, suggesting that this mutation is not a good model for the S308 phosphorylation.     

Protein-peptide interaction studies demonstrate the versatility of calmodulin target protein binding

Calmodulin (CaM) is a prototypical Ca2+-sensor protein that can control many important biological functions by binding to hundreds of target proteins. To gain insight into the versatility of CaM-target recognition, we have analyzed the complex structures for many types of CaM-binding peptides and some target proteins. In particular, some recently reported novel complex structures reveal that the versatile target binding of CaM is accommodated by its flexible domain arrangement and the malleability of its interfaces.     

Immunohistochemical detection of calmodulin and calmodulin-dependent protein kinase II in the mouse testis

We reported previously that in mouse testis calmodulin-dependent protein phosphatase (calcineurin) is localised in the nuclei of round and elongating spermatids (Cell Tissue Res. 1995; 281: 273-81). In this study, we studied the immunohistochemical localisation of calcium/calmodulin-dependent protein kinase (CaM kinase II) using antibodies against CaM kinase IIgamma from chicken gizzard and specific antibodies raised against the amino acid sequence Ileu480-Ala493 of this enzyme, and compared it with the distribution of calmodulin. Indirect immunofluorescence was most concentrated in early spermatocytes and localised in the outermost layer of seminiferous tubules where the calmodulin level was relatively low. Measurements of immuno-gold particle densities on electron micrographs revealed that CaM kinase II is transiently increased in the nucleus of zygotene spermatocytes. These observations suggest the involvement of CaM kinase II in the meiotic chromosomal pairing process. An extremely high concentration of calmodulin in spermatogenic cells undergoing meiosis may not be directly related to activation of calmodulin-dependent kinases and phosphatases.