Evidence that the major postsynaptic density protein is a component of a Ca2+/calmodulin-dependent protein kinase.

Polypeptides of Mr 50,000 and 60,000 in isolated synaptic junctions have been compared to polypeptides of corresponding molecular weight in Ca2+/calmodulin-dependent protein kinase II. The polypeptides of corresponding molecular weight from the two preparations were shown by several criteria to be indistinguishable. These criteria included 125I-labeled tryptic/chymotryptic peptide patterns, 32P-labeled proteolytic peptide maps, and crossreactivity on immunoblots using polyclonal and monoclonal antibodies. Furthermore, studies examining the phosphorylation of substrate proteins, by the endogenous synaptic junction kinase and by Ca2+/calmodulin-dependent protein kinase II, indicated that the two enzymes have similar substrate specificities. Since the Mr 50,000 polypeptide present in synaptic junctions is known to be the major postsynaptic density protein, the present results indicate that the major postsynaptic density protein is a component of Ca2+/calmodulin-dependent protein kinase II.     

Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II.

Ca2+/calmodulin-dependent protein kinase II contains two subunits, alpha (Mr 50,000) and beta (Mr 60,000/58,000), both of which undergo Ca2+/calmodulin-dependent autophosphorylation. In the present study, we have studied the mechanism of this autophosphorylation reaction and its effect on the activity of the enzyme. Both subunits are autophosphorylated through an intramolecular mechanism. Using synapsin I as substrate, Ca2+/calmodulin-dependent protein kinase II, in its unphosphorylated form, was totally dependent on Ca2+ and calmodulin for its activity. Preincubation of the enzyme with Ca2+, calmodulin, and ATP, under conditions where autophosphorylation of both subunits occurred, converted the enzyme to one that was only partially dependent on Ca2+ and calmodulin for its activity. No change in the total activity, measured in the presence of Ca2+ and calmodulin, was observed. The nonhydrolyzable ATP analog adenosine 5'-[beta, gamma-imido] triphosphate did not substitute for ATP in the preincubation. Moreover, dephosphorylation of autophosphorylated Ca2+/calmodulin-dependent protein kinase II with protein phosphatase 2A resulted in an enzyme that was again totally dependent on Ca2+ and calmodulin for its activity. We propose that autophosphorylation and dephosphorylation reversibly regulate the Ca2+ and calmodulin requirement of Ca2+/calmodulin-dependent protein kinase II.     

Ca2+/calmodulin-dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor

The cardiac ryanodine receptor (RyR2)/calcium release channel on the sarcoplasmic reticulum is required for muscle excitation-contraction coupling. Using site-directed mutagenesis, we identified the specific Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation site on recombinant RyR2, distinct from the site for protein kinase A (PKA) that mediates the "fight-or-flight" stress response. CaMKII phosphorylation increased RyR2 Ca2+ sensitivity and open probability. CaMKII was activated at increased heart rates, which may contribute to enhanced Ca2+-induced Ca2+ release. Moreover, rate-dependent CaMKII phosphorylation of RyR2 was defective in heart failure. CaMKII-mediated phosphorylation of RyR2 may contribute to the enhanced contractility observed at higher heart rates. The full text of this article is available online at http://circres.ahajournals.org.     

Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates Ca2+/calmodulin-independent activity.

We have examined the role of Thr-286 autophosphorylation in the autoregulation of Ca2+/calmodulin-dependent protein kinase II. Using site-directed mutagenesis, we have substituted alanine or serine for Thr-286, or isoleucine for Arg-283, in the 50-kDa subunit of the kinase and expressed each protein in bacteria. Activation and autophosphorylation of all four enzymes were stringently dependent on Ca2+/calmodulin, indicating that neither Arg-283 nor Thr-286 is an absolute requirement for the pseudosubstrate inhibition of the enzyme. Autophosphorylation of the Ile-283 or Ala-286 enzyme generated little, if any, Ca2+/calmodulin-independent kinase activity, unlike the parent (Thr-286) or Ser-286 enzyme. The enzymes expressed in bacteria are predominantly monomeric, indicating that the generation of Ca2+/calmodulin-independent activity does not require the cooperative interactions of subunits normally present in the brain holoenzyme.     

Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence.

Ca2+/calmodulin-dependent protein kinase II contains two types of subunit, alpha (Mr 50,000) and beta (Mr 60,000/58,000), both of which undergo Ca2+/calmodulin-dependent autophosphorylation. Autophosphorylation is known to convert the enzyme to a Ca2+/calmodulin-independent form. In the present study, we have characterized the autophosphorylation sites on rat forebrain Ca2+/calmodulin-dependent protein kinase II that are most likely to be responsible for the generation of Ca2+/calmodulin-independence. Under conditions (0 degree C, low concentrations of ATP) sufficient to generate close to maximal Ca2+/calmodulin-independence, only a few of the phosphorylatable sites on the enzyme became phosphorylated. These autophosphorylation sites were examined by phospho amino acid analysis, two-dimensional thermolytic phosphopeptide mapping, and high-performance liquid chromatography. The time course of phosphorylation of threonine in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Moreover, the time course of phosphorylation of one set of peptides, referred to as peptide 1/1', present in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Threonine was the only amino acid phosphorylated in peptide 1/1'. An additional peptide, referred to as peptide 2, was phosphorylated in the beta subunit. The time course of phosphorylation of peptide 2, which also contained only phosphothreonine, did not parallel the time course of the generation of Ca2+/calmodulin-independence. It is likely that the phosphorylation of a threonine residue on peptide 1/1' is responsible for the generation of Ca2+/calmodulin-independence of Ca2+/calmodulin-dependent protein kinase II.     

Dynamic properties of the Ca2+/calmodulin-dependent protein kinase in Drosophila: identification of a synapsin I-like protein.

Visual adaptation with blue light induces a change in a special light/dark choice behavior in Drosophila. On the molecular level adaptation induces long-term modulation of the in vitro autophosphorylation capacity of a Ca2+/calmodulin-dependent protein kinase. Here I describe a Drosophila phosphoprotein that is a substrate of this protein kinase. The molecular mass and phosphopeptide composition of this protein are similar to those of rat synapsin I. Furthermore, the Drosophila protein shows immunological cross-reactivity with monoclonal antibodies against rat synapsin I. I conclude that this 86-kDa protein in Drosophila is homologous to the vertebrate synapsin I.     

Ca/calmodulin-dependent protein kinase II contributes to intracellular pH recovery from acidosis via Na/H exchanger activation

The Na⁺/H⁺ exchanger (NHE-1) plays a key role in pH_i recovery from acidosis and is regulated by pH_i and the ERK1/2-dependent phosphorylation pathway. Since acidosis increases the activity of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in cardiac muscle, we examined whether CaMKII activates the exchanger by using pharmacological tools and highly specific genetic approaches. Adult rat cardiomyocytes, loaded with the pH_i indicator SNARF-1/AM were subjected to different protocols of intracellular acidosis. The rate of pH_i recovery from the acid load (dpH_i/dt)—an index of NHE-1 activity in HEPES buffer or in NaHCO₃ buffer in the presence of inhibition of anion transporters—was significantly decreased by the CaMKII inhibitors KN-93 or AIP. pH_i recovery from acidosis was faster in CaMKII-overexpressing myocytes than in overexpressing β-galactosidase myocytes (dpH_i/dt: 0.195 ± 0.04 vs. 0.045 ± 0.010 min^− 1, respectively, n = 8) and slower in myocytes from transgenic mice with chronic cardiac CaMKII inhibition (AC3-I) than in controls (AC3-C). Inhibition of CaMKII and/or ERK1/2 indicated that stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 cascade. In vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C-terminal domain of NHE-1 indicate that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites for the kinase (Ser648, Ser703, and Ser796). These results provide new mechanistic insights and unequivocally demonstrate a role of the already multifunctional CaMKII on the regulation of the NHE-1 activity. They also prove clinically important in multiple disorders which, like ischemia/reperfusion injury or hypertrophy, are associated with increased NHE-1 and CaMKII.     

Acquisition and loss of a neuronal Ca2+/calmodulin-dependent protein kinase during neuronal differentiation.

Calcium ions play a critical role in neural development. Insights into the ontogeny of Ca(2+)-signaling pathways were gained by investigating the developmental expression of granule cell-enriched Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) in the cerebellum and hippocampus of the rat. Neurons of these brain regions displayed characteristic schedules by which they acquired and lost CaM kinase-Gr during differentiation. In the cerebellum, granule cells did not begin to express CaM kinase-Gr until after birth when they migrated into the granule cell layer, and this expression persisted in the adult. Purkinje cells expressed CaM kinase-Gr prenatally and lost this expression by postnatal day 14. In contrast, the granule and pyramidal cells of the hippocampus expressed the enzyme prenatally and in the adult. Moreover, CaM kinase-Gr was localized to the processes and nuclei of developing neurons. This subcellular localization together with the scheduled expression of CaM kinase-Gr can serve to regulate a developing neuron's sensitivity to Ca2+ at different subcellular levels.     

Activity of cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human hearts.

OBJECTIVES: A hallmark of human heart failure is prolonged myocardial relaxation. Although the intrinsic mechanism of phospholamban coupling to the Ca(2+)-ATPase is unaltered in normal and failed human hearts, it remains possible that regulation of phospholamban phosphorylation by cAMP-dependent mechanisms or other second messenger pathways could be perturbed, which may account partially for the observed dysfunctions of the sarcoplasmic reticulum (SR) associated with this disease. METHODS: cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase) were characterized initially by DEAE-Sepharose chromatography in hearts from patients with end-stage dilated cardiomyopathy. We measured the activity of PKA and CaM kinase in left ventricular tissue of failing (idiopathic dilated cardiomyopathy; ischemic heart disease) and nonfailing human hearts. RESULTS: Basal PKA activity was not changed between failing and nonfailing hearts. One major peak of CaM kinase activity was detected by DEAE-Sepharose chromatography. CaM kinase activity was increased almost 3-fold in idiopathic dilated cardiomyopathy. In addition, hemodynamical data (left ventricular ejection fraction, cardiac index) from patients suffering from IDC positively correlate with CaM kinase activity. CONCLUSIONS: Increased CaM kinase activity in hearts from patients with dilated cardiomyopathy could play a role in the abnormal Ca2+ handling of the SR and heart muscle cell.     

Role of Ca2+/calmodulin-dependent protein kinase (CaMK) in excitation-contraction coupling in the heart

Calcium (Ca(2+)) is the central second messenger in the translation of electrical signals into mechanical activity of the heart. This highly coordinated process, termed excitation-contraction coupling or ECC, is based on Ca(2+)-induced Ca(2+) release from the sarcoplasmic reticulum (SR). In recent years it has become increasingly clear that several Ca(2+)-dependent proteins contribute to the fine tuning of ECC. One of these is the Ca(2+)/calmodulin-dependent protein kinase (CaMK) of which CaMKII is the predominant cardiac isoform. During ECC CaMKII phosphorylates several Ca(2+) handling proteins with multiple functional consequences. CaMKII may also be co-localized to distinct target proteins. CaMKII expression as well as activity are reported to be increased in heart failure and CaMKII overexpression can exert distinct and novel effects on ECC in the heart and in isolated myocytes of animals. In the present review we summarize important aspects of the role of CaMKII in ECC with an emphasis on recent novel findings.     

Muscle-specific activation of Ca2+/calmodulin-dependent protein kinase IV increases whole-body insulin action in mice

Aims/hypothesis

Aerobic exercise increases muscle glucose and improves insulin action through numerous pathways, including activation of Ca²⁺/calmodulin-dependent protein kinases (CAMKs) and peroxisome proliferator γ coactivator 1α (PGC-1α). While overexpression of PGC-1α increases muscle mitochondrial content and oxidative type I fibres, it does not improve insulin action. Activation of CAMK4 also increases the content of type I muscle fibres, PGC-1α level and mitochondrial content. However, it remains unknown whether CAMK4 activation improves insulin action on glucose metabolism in vivo.

Methods

The effects of CAMK4 activation on skeletal muscle insulin action were quantified using transgenic mice with a truncated and constitutively active form of CAMK4 (CAMK4^●) in skeletal muscle. Tissue-specific insulin sensitivity was assessed in vivo using a hyperinsulinaemic–euglycaemic clamp and isotopic measurements of glucose metabolism.

Results

The rate of insulin-stimulated whole-body glucose uptake was increased by ～25% in CAMK4^● mice. This was largely attributed to an increase of ～60% in insulin-stimulated glucose uptake in the quadriceps, the largest hindlimb muscle. These changes were associated with improvements in insulin signalling, as reflected by increased phosphorylation of Akt and its substrates and an increase in the level of GLUT4 protein. In addition, there were extramuscular effects: CAMK4^● mice had improved hepatic and adipose insulin action. These pleiotropic effects were associated with increased levels of PGC-1α-related myokines in CAMK4^● skeletal muscle.

Conclusions/interpretation

Activation of CAMK4 enhances mitochondrial biogenesis in skeletal muscle while also coordinating improvements in whole-body insulin-mediated glucose.     

Identification of a Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in non-N-methyl-D-aspartate glutamate receptors.

Glutamate receptor ion channels are colocalized in postsynaptic densities with Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), which can phosphorylate and strongly enhance non-N-methyl-D-aspartate (NMDA) glutamate receptor current. In this study, CaM-kinase II enhanced kainate currents of expressed glutamate receptor 6 in 293 cells and of wild-type glutamate receptor 1, but not the Ser-627 to Ala mutant, in Xenopus oocytes. A synthetic peptide corresponding to residues 620-638 in GluR1 was phosphorylated in vitro by CaM-kinase II but not by cAMP-dependent protein kinase or protein kinase C. The 32P-labeled peptide map of this synthetic peptide appears to be the same as the two-dimensional peptide map of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptors phosphorylated in cultured hippocampal neurons by CaM-kinase II described elsewhere. This CaM-kinase II regulatory phosphorylation site is conserved in all AMPA/kainate-type glutamate receptors, and its phosphorylation may be important in enhancing postsynaptic responsiveness as occurs during synaptic plasticity.     

Possible role for calmodulin and the Ca2+/calmodulin-dependent protein kinase II in postsynaptic neurotransmission.

The theory presented here is based on results from in vitro experiments and deals with three proteins in the postsynaptic density/membrane-namely, calmodulin, the Ca2+/calmodulin-dependent protein kinase, and the voltage-dependent Ca2+ channel. It is visualized that, in vivo in the polarized state of the membrane, calmodulin is bound to the kinase; upon depolarization of the membrane and the intrusion of Ca2+, Ca2(+)-bound calmodulin activates the autophosphorylation of the kinase. Calmodulin is visualized as having less affinity for the phosphorylated form of the kinase and is translocated to the voltage-dependent Ca2+ channel. There, with its bound Ca2+, it acts as a Ca2+ sensor, to close off the Ca2+ channel of the depolarized membrane. At the same time, it is thought that the configuration of the kinase is altered by its phosphorylated states; by interacting with Na+ and K+ channels, it alters the electrical properties of the membrane to regain the polarized state. Calmodulin is moved to the unphosphorylated kinase to complete the cycle, allowing the voltage-dependent Ca2+ channel to be receptive to Ca2+ flux upon the next cycle of depolarization. Thus, the theory tries to explain (i) why calmodulin and the kinase reside at the postsynaptic density/membrane site, and (ii) what function autophosphorylation of the kinase may play.