Activation Of CaM Kinase Research Articles

Modulation of skeletal and cardiac voltage‐gated sodium channels by calmodulin

Calmodulin (CaM) has been shown to modulate different ion channels, including voltage-gated sodium channels (NaChs). Using the yeast two-hybrid assay, we found an interaction between CaM and the C-terminal domains of adult skeletal (NaV1.4) and cardiac (NaV1.5) muscle NaChs. Effects of CaM were studied using sodium channels transiently expressed in CHO cells. Wild type CaM (CaM(WT)) caused a hyperpolarizing shift in the voltage dependence of activation and inactivation for NaV1.4 and activation for NaV1.5. Intracellular application of CaM caused hyperpolarizing shifts equivalent to those seen with CaM(WT) coexpression with NaV1.4. Elevated Ca2+ and CaM-binding peptides caused depolarizing shifts in the inactivation curves seen with CaM(WT) coexpression with NaV1.4. KN93, a CaM-kinase II inhibitor, had no effect on NaV1.4, suggesting that CaM acts directly on NaV1.4 and not through activation of CaM-kinase II. Coexpression of hemi-mutant CaMs showed that an intact N-terminal lobe of CaM is required for effects of CaM upon NaV1.4. Mutations in the sodium channel IQ domain disrupted the effects of CaM on NaV1.4: the I1727E mutation completely blocked all calmodulin effects, while the L1736R mutation disrupted the effects of Ca2+-calmodulin on inactivation. Chimeric channels of NaV1.4 and NaV1.5 also indicated that the C-terminal domain is largely responsible for CaM effects on inactivation. CaM had little effect on NaV1.4 expressed in HEK cells, possibly due to large differences in the endogenous expression of beta-subunits between CHO and HEK cells. These results in heterologous cells suggest that Ca2+ released during muscle contraction rapidly modulates NaCh availability via CaM.

Nuclear Ca2+ and CaM kinase IV specify hormonal‐ and Notch‐responsiveness

Many neuronal processes require gene activation by synaptically evoked Ca(2+) transients. Ca(2+)-dependent signal pathways activate some transcription factors outright, but here we report that such signals also potentiate the activation of nuclear receptors by their cognate hormone, and of CBF1 by Notch, transcription factors hitherto not thought to be Ca(2+)-responsive. This potentiation is occluded by histone deacetylase inhibition, indicating a mechanism involving inactivation of co-repressors associated with these transcription factors. Synaptic activity, acting via the nuclear Ca(2+)-dependent activation of CaM kinase IV, triggers the disruption of subnuclear domains containing class II histone deacetylases (HDACs) and silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), a broad-specificity co-repressor which represses nuclear hormone receptors and CBF1. The sequential loss of class II HDACs and SMRT from the subnuclear domains, followed by nuclear export, is associated with disruption of SMRT interaction with its target transcription factors and sensitization of these factors to their activating signal. Counterbalancing these changes, protein phosphatase 1 promotes nuclear localization of SMRT and inactivation of nuclear receptors and CBF1. Thus, the synaptically controlled kinase-phosphatase balance of the neuron determines the efficacy of SMRT-mediated repression and the signal-responsiveness of a variety of transcription factors.

Activity-Dependent Long-Term Potentiation of Intrinsic Excitability in Hippocampal CA1 Pyramidal Neurons

The efficiency of neural circuits is enhanced not only by increasing synaptic strength but also by increasing intrinsic excitability. In contrast to the detailed analysis of long-term potentiation (LTP), less attention has been given to activity-dependent changes in the intrinsic neuronal excitability. By stimulating hippocampal CA1 pyramidal neurons with synaptic inputs correlating with postsynaptic neuronal spikes, we elicited an LTP of intrinsic excitability (LTP-IE) concurring with synaptic LTP. LTP-IE was manifested as a decrease in the action potential threshold that was attributable to a hyperpolarized shift in the activation curve of voltage-gated sodium channels (VGSCs) rather than activity-dependent changes in synaptic inputs or A-type K+ channels. Cell-attached patch recording of VGSC activities indicated such an activity-dependent change in VGSCs. Induction of LTP-IE was blocked by the NMDA receptor antagonist APV, intracellular BAPTA, the CaM kinase inhibitors KN-62 and autocamtide-2-related inhibitory peptide, and the protein synthesis inhibitors emetine and anisomycin. The results suggest that induction of LTP-IE shares a similar signaling pathway with the late phase of synaptic LTP and requires activation of the NMDA glutamate receptor subtype, Ca2+ influx, activity of CaM kinase II, and function of the protein synthesis. This new form of hippocampal neuronal plasticity could be a cellular correlate of learning and memory besides synaptic LTP.

Redox Regulation of the Calcium/Calmodulin-dependent Protein Kinases

Reactive oxygen intermediates (ROI) have been viewed traditionally as damaging to the cell. However, a predominance of evidence has shown that ROI can also function as important activators of key cellular processes, and ROI have been shown to play a vital role in cell signaling networks. The calcium/calmodulin-dependent protein kinases (CaM kinases) are a family of related kinases that are activated in response to increased intracellular calcium concentrations. In this report we demonstrate that hydrogen peroxide treatment results in the activation of both CaM kinase II and IV in Jurkat T lymphocytes. Surprisingly, this activation occurs in the absence of any detectable calcium flux, suggesting a novel means for the activation of these kinases. Treatment of Jurkat cells with phorbol 12-myristate 13-acetate (PMA), which does not cause a calcium flux, also activated the CaM kinases. The addition of catalase to the cultures inhibited PMA-induced activation of the CaM kinases, suggesting that similar to hydrogen peroxide, PMA also activates the CaM kinases via the production of ROI. One mechanism by which this likely occurs is through oxidation and consequential inactivation of cellular phosphatases. In support of this concept, okadaic acid and microcystin-LR, which are inhibitors of protein phosphatase 2A (PP2A), induced CaM kinase II and IV activity in these cells. Overall, these results demonstrate a novel mechanism by which ROI can induce CaM kinase activation in T lymphocytes.

Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulin-dependent protein kinase II.

Phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) regulates protein synthesis in hippocampal dendrites. CPEB binds the 3' untranslated region (UTR) of cytoplasmic mRNAs and, when phosphorylated, initiates mRNA polyadenylation and translation. We report that, of the protein kinases activated in the hippocampus during synaptic plasticity, calcium/calmodulin-dependent protein kinase II (CaMKII) robustly phosphorylated the regulatory site (threonine 171) in CPEB in vitro. In postsynaptic density fractions or hippocampal neurons, CPEB phosphorylation increased when CaMKII was activated. These increases in CPEB phosphorylation were attenuated by a specific peptide inhibitor of CaMKII and by the general CaM-kinase inhibitor KN-93. Inhibitors of protein phosphatase 1 increased basal CPEB phosphorylation in neurons; this was also attenuated by a CaM-kinase inhibitor. To determine whether CaM-kinase activity regulates CPEB-dependent mRNA translation, hippocampal neurons were transfected with luciferase fused to a 3' UTR containing CPE-binding elements. Depolarization of neurons stimulated synthesis of luciferase; this was abrogated by inhibitors of protein synthesis, mRNA polyadenylation, and CaMKII. These results demonstrate that CPEB phosphorylation and translation are regulated by CaMKII activity and provide a possible mechanism for how dendritic protein synthesis in the hippocampus may be stimulated during synaptic plasticity.

Calcium activation of ERK mediated by calmodulin kinase I.

Elevated intracellular Ca(2+) triggers numerous signaling pathways including protein kinases such as the calmodulin-dependent kinases (CaMKs) and the extracellular signal-regulated kinases (ERKs). In the present study we examined Ca(2+)-dependent "cross-talk" between these two protein kinase families. Using a combination of pharmacological inhibitors and dominant-negative kinases (dnKinase), we identified a requirement for CaMKK acting through CaMKI in the stimulation of ERKs upon depolarization of the neuroblastoma cell line, NG108. Depolarization stimulated prolonged ERK and JNK activation that was blocked by the CaMKK inhibitor, STO-609; this inhibition of ERK activation by STO-609 was rescued by expression of a STO-609-insensitive mutant of CaMKK. However, activation of ERK by epidermal growth factor or carbachol were not suppressed by inhibition of CaMKK, indicating specificity for this "cross-talk." To identify the downstream target of CaMKK that mediated ERK activation upon depolarization, dnKinases were expressed. The dnCaMKI completely suppressed ERK2 activation whereas dnAKT/PKB or nuclear-targeted dnCaMKIV, other substrates for CaMKK, were not inhibitory. ERK activation upon depolarization or transfection with constitutively active (ca) CaMKI was blocked by dnRas. Additionally, depolarization of NG108 cells promoted neurite outgrowth, and this effect was blocked by inhibition of either CaMKK (STO-609) or ERK (UO126). Co-transfection with caCaMKK plus caCaMKI also stimulated neurite outgrowth that was blocked by inhibition of ERK (UO126). These data are the first to suggest that ERK activation and neurite outgrowth in response to depolarization are mediated by CaMKK activation of CaMKI.

Novel mechanisms for regulating the TGF‐β‐/Smad‐signalling pathway

Introduction Aberrant transforming growth factor‐β (TGF‐β) signalling is responsible for a number of developmental disorders, human cancers and fibrotic disease. Signalling is accomplished via cell‐surface serine/threonine kinase receptors and intracellular Smad transcription factors. A key regulatory step in the pathway involves phosphorylation of Smads by Ca2+‐dependent kinases such as Cam kinase II (Wicks et al. 2000; Abdel‐Wahab et al. 2001). In addition, specific ubiquitination by Smurfs and other ubiquitin ligases mediates the proteosomal degradation of Smads. The aims of this study were to functionally analyse the regulation of Smads by phosphorylation in response to decorin‐mediated Ca2+ mobilization and to use modified yeast‐2 hybrid systems for the identification of novel Smad regulatory proteins.Methods Phosphorylation sites in Smad‐2 were mapped in vitro using Smad‐2–GST fusion proteins incubated with purified kinases in the presence of 32P‐γ‐ATP. Sites were confirmed by site‐directed mutagenesis, and phosphopeptide‐specific antisera were used to study in vivo phosphorylation in decorin‐treated human mesangial cells. Yeast‐2 hybrid screening was performed using Smad31−240 as bait and a mouse brain cDNA library. Interactions were then verified by co‐expression and co‐immunoprecipitation of epitope‐tagged proteins in HEK‐293 cells.Results In this study, we provide evidence that decorin, a naturally occurring inhibitor of the TGF‐β‐dependent fibrotic response, can disrupt glucose‐ and TGF‐β/Smad‐dependent transcriptional events in human mesangial cells through a mechanism that involves an increase in Ca2+ signalling, the activation of Cam kinase II and ensuing phosphorylation of Smad‐2 at serine‐240. We show that decorin also induces serine‐240 phospho‐Smad hetero‐oligomerization with Smad‐4 and the nuclear localization of this complex, independently of TGF‐β receptor activation. Thus, in human mesangial cells, the mechanism of decorin‐mediated inhibition of TGF‐β signalling may involve activation of Ca2+ signalling, the subsequent phosphorylation of Smad‐2 at a key regulatory site and the sequestration of Smad‐4 in the nucleus. In other studies, we have used two‐hybrid screening approaches to identify Smad‐specific regulatory proteins. Data will be presented on a novel interaction between Smad‐3 and a ubiquitin c‐termninal hydrolase that is likely to compete with Smurf‐mediated ubiquitination and subsequent Smad turnover.Conclusions In summary, we conclude that an important mechanism by which decorin can play a functional role in regulating TGF‐β signalling is through decorin‐induced Ca2+ signalling and subsequent inhibition of the Smad pathway. In addition, we have identified a novel interaction that occurs between the Smad‐3 transcription factor and a ubiquitin C‐terminal hydrolase that could lead to stabilization of the Smad‐3 protein and potentiation of TGF‐β signalling by reversal of ubiquitin‐mediated proteosomal degradation. Overall, our present findings may lead to the development of new approaches in the treatment of fibrotic diseases.

Ser16-, but not Thr17-phosphorylation of phospholamban influences frequency-dependent force generation in human myocardium.

Beta-adrenoceptor/cAMP-dependent Ser16-phosphorylation as well as Ca(2+)-dependent Thr17-phosphorylation of phospholamban (PLB) influences SERCA 2a activity and thus myocardial contractility. To determine the cross-signaling between Ca2+ and cAMP pathways, the phosphorylation of Ser16-PLB and Thr17-PLB was studied at increasing stimulation frequencies as well as in the presence of beta-adrenergic stimulation in isolated ventricular trabeculae from failing (dilative cardiomyopathy, DCM, heart transplants, n=9) and non-failing human myocardium (donor hearts, NF, n=9). In addition, we measured the intracellular Ca(2+)-transient (fura-2) at increasing stimulation frequencies (0.5-3.0 Hz). Protein expression of SERCA 2a and phospholamban was similar in DCM and NF. In DCM, diastolic [Ca2+]i was increased and systolic [Ca2+]i as well as Ser16 PLB-phosphorylation were decreased as compared to NF at 0.5 Hz. The positive force-frequency relationship in human non-failing myocardium was accompanied by a frequency-dependent increase in Ser16-PLB, but not Thr17-PLB phosphorylation. In DCM, Ser16-PLB as well as Thr17-PLB phosphorylation were not altered at higher stimulation frequencies. After application of isoprenaline (1 microM), a profound increase in Ser16-PLB phosphorylation was accompanied by a small increase in Thr17-PLB phosphorylation, only in NF. The frequency-dependent phosphorylation of Ser16-PLB may favor an increase in Ca2+ transient and force generation in humans. Cross talk signaling of Ser16/Thr17-PLB phosphorylation after beta-adrenergic stimulation exists in non-failing, but not in failing human myocardium. The Ca(2+)-dependent CaM-kinase activity may be altered in human heart failure.

Inhibition of Ca2+/calmodulin-dependent protein kinase or epidermal growth factor receptor tyrosine kinase abolishes lysophosphatidic acid-mediated DNA-synthesis in human myometrial smooth muscle cells.

Human myometrial smooth muscle cells (SMCs) were used to evaluate the proliferative activity of lysophosphatidic acid (LPA). This study specifically focuses on the role of Ca(2+)/calmodulin-dependent protein (CaM) kinase and epidermal growth factor (EGF) receptor tyrosine kinase. Myometrial SMCs were cultured from biopsies taken at Cesarean sections. The expression of LPA receptors was determined by reverse transcriptase polymerase chain reaction (RT-PCR), and DNA-synthesis was measured by [3H]thymidine incorporation. LPA(1), LPA(2), and LPA(3)receptor subtypes were detected in the SMCs using RT-PCR. KN-62, an inhibitor of CaM kinase, and Tyrphostin AG 1478, an inhibitor of EGF receptor tyrosine kinase, dose-dependently decreased LPA-stimulated [3H]thymidine incorporation. Furthermore, BB-3103, an inhibitor of matrix metalloproteinases (MMPs), also reduced DNA-synthesis induced by LPA in these cells. The results show, for the first time, that human myometrial SMCs express all three known LPA receptor subtypes. Growth stimulatory effects of LPA on myometrial SMCs seems to be mediated by several pathways, where transactivation of EGF receptors through MMPs appears to be of importance. Furthermore, CaM kinase activity may be critical for LPA signaling since inhibition of CaM kinase totally abolish the proliferative effect of LPA.

The involvement of Ca(2+) gradients, Ca(2+) fluxes, and CaM kinase II in polarization and germination of Silvetia compressa zygotes.

Previous work has shown that distinct Ca(2+) gradients precede and predict the loci of germination of the zygotes of the brown alga, Silvetia compressa (J. Agardh) E. Serrão, T.O. Cho, S.M. Boo et Brawley, that are polarized by unilateral blue light. We show here that dark-grown S. compressa zygotes also form cytosolic Ca(2+) gradients prior to germination and then germinate from the site of elevated Ca(2+). In no case did germination occur without a prior formation of a Ca(2+) gradient. Using the self-referencing Ca(2+)-selective probe, we measured highly localized influx of Ca(2+) during photopolarization, indicating that extracellular stores supply at least some of the Ca(2+) needed to construct a gradient. Finally, we find that germination was inhibited by a bath-applied inhibitor of calcium/calmodulin-dependent kinase II (CaM kinase II), KN-93 (but not by its inactive analog, KN-92), and by an injected inhibitory peptide for the kinase. KN-93 did not interfere with the photopolarization of the zygotes, consistent with the view that calmodulin is not involved in the initial response to light. The KN-93 results indicate that the requirement for active CaM kinase II for germination ends about 2 h before overt germination. We conclude that Ca(2+) gradients, generated in part by localized calcium entry from the seawater, are an essential part of the process of polarity development and expression in these cells, regardless of the nature of the external cue that directs the orientation of the axis. Calmodulin and CaM kinase II are involved in interpreting (but not in establishing) the calcium gradient, allowing germination to occur at the site of elevated calcium, but CaM kinase II appears not to be involved in the initiation of germination.

Synaptic Mechanism for Pain Sensitization

Injured spinal nerves, caused by trauma or inflammation, can cause chronic pain that is not blunted by conventional analgesics. Two groups investigated mechanisms underlying this hyperalgesic response. Garry et al. examined the molecular mechanisms that account for sensitization of behavioral reflexes in response to painful stimuli in mice with a chronic constriction injury to the common sciatic nerve. The adaptor protein PSD-95 was localized in spinal cord neurons likely to influence pain sensitivity. In transgenic mice expressing a truncated form of PSD-95, the enhanced sensitivity to pain normally caused by the constriction injury was reduced or absent, whereas a control response to pain caused by inflammation was not altered. One role of PSD-95 is to localize Ca 2+ -calmodulin-dependent protein kinase II (CaM kinase II) to complexes with N -methyl-D-aspartate (NMDA) receptors. In wild-type mice, pharmacological inhibitors of CaM kinase II also reduced sensitization to pain after injury, and the amount of phosphorylated active CaM kinase II associated with NMDA receptors in spinal neurons was increased after nerve injury. Although the results do not rule out roles for other targets of PSD-95, organization of CaM kinase II in complexes with NMDA receptors appears to be a key aspect of increased sensitivity after neuronal damage. In fact, high concentrations of NMDA still activated CaM kinase II in PSD-95-mutant animals, even though injury-induced activation of the kinase was blocked. Thus, the response to neuronal injury may critically depend on a particular subcellular population of the enzyme, likely that tethered to NMDA receptors through PSD-95. Ikeda et al. analyzed the induction of long-term potentiation (LTP) in neurons of lamina 1 of the spinal cord evoked by pain-transmitting C fibers. Only those neurons that expressed neurokinin 1 receptors, which is activated by substance P, exhibited LTP. Additionally, LTP was blocked if the NMDA receptors were inhibited or if increases in the concentration of intracellular Ca 2+ were blocked by inhibition of release from intracellular stores or inhibition of T-type Ca 2+ channels. Thus, these two reports address mechanisms underlying the hyperalgesic response to nociceptive stimuli. E. M. Garry, A. Moss, A. Delaney, F. O'Neill, J. Blakemore, J. Bowen, H. Husi, R. Mitchell, S. G. N. Grant, S. M. Fleetwood-Walker, Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice. Curr. Biol. 13 , 321-328 (2003). [Online Journal] H. Ikeda, B. Heinke, R. Ruscheweyh, J. Sandkühler, Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia. Science 299 , 1237-1240 (2003). [Abstract] [Full Text]

Insulin-Stimulated Cyclic Guanosine Monophosphate Inhibits Vascular Smooth Muscle Cell Migration by Inhibiting Ca/Calmodulin-Dependent Protein Kinase II

Insulin resistance is associated with vascular disease. Physiological concentrations of insulin inhibit cultured vascular smooth muscle cell (VSMC) migration in the presence of nitric oxide, and the failure to do so in insulin-resistant states may aggravate vascular disease. We sought to determine the molecular mechanisms by which insulin inhibits VSMC migration. Insulin at 1 nmol/L stimulated cGMP production in cultured rat VSMCs that were induced to express inducible nitric oxide synthase (iNOS). VSMC migration was measured in a wound-closure assay, and the platelet-derived growth factor-AB (PDGF-AB)-stimulated component of VSMC migration after wounding was inhibited by insulin, 8-Br-cGMP, and 1-[N-0-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a selective inhibitor of calcium/calmodulin-dependent protein kinase II (CaM kinase II). Wounding alone or incubating cells with only PDGF-AB stimulated CaM kinase II activity in an insulin- and 8-Br-cGMP-inhibitable manner. Transfecting VSMCs with a constitutively active CaM kinase II mutant blocked the inhibition by insulin of both wound-induced and wound plus PDGF-AB-induced VSMC migration. High intracellular Ca2+ ([Ca]i)-stimulated CaM kinase II activity was inhibited by 8-Br-cGMP by an okadaic acid-sensitive mechanism. We conclude that in cultured rat VSMCs expressing iNOS, insulin, via stimulation of cGMP production, inhibits both wound alone-induced and the PDGF-AB-stimulated component of VSMC migration by inhibiting CaM kinase II activity. cGMP inhibits CaM kinase II at a post-[Ca]i step by a protein phosphatase-dependent mechanism.

Neuropathic Sensitization of Behavioral Reflexes and Spinal NMDA Receptor/CaM Kinase II Interactions Are Disrupted in PSD-95 Mutant Mice

Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depend on the NMDA receptor [1, 2]. We reveal complexes of NMDA receptors with the multivalent adaptor protein PSD-95 [3, 4] in the dorsal horn of spinal cord and show that PSD-95 plays a key role in neuropathic reflex sensitization. Using mutant mice expressing a truncated form of the PSD-95 molecule [5], we show their failure to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain [6], but normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes, elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr286 CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice.

CAMP-dependent Protein Kinase and Ca2+Influx through L-type Voltage-gated Calcium Channels Mediate Raf-independent Activation of Extracellular Regulated Kinase in Response to Glucagon-like Peptide-1 in Pancreatic β-Cells

Glucagon like peptide-1 (GLP1) is a G(s)-coupled receptor agonist that exerts multiple effects on pancreatic beta-cells, including the stimulation of insulin gene expression and secretion. In this report, we show that treatment of the mouse pancreatic beta-cell line MIN6 with GLP1 leads to the glucose-dependent activation of Erk. These effects are mimicked by forskolin, a direct activator of adenylate cyclase, and blocked by H89, an inhibitor of cAMP-dependent protein kinase. Additionally, we provide evidence that GLP1-stimulated activation of Erk requires an influx of calcium through L-type voltage-gated calcium channels and the activation of calcium/calmodulin-dependent protein kinase II. GLP1-stimulated activation of Erk is blocked by inhibitors of MEK, but GLP1 does not induce the activation of A-Raf, B-Raf, C-Raf, or Ras. Additionally, dominant negative forms of Ras(N17) and Rap1(N17) fail to block GLP1-stimulated activation of Erk. In conclusion, our results indicate that, in the presence of stimulatory concentrations of glucose, GLP1 stimulates the activation of Erk through a mechanism dependent on MEK but independent of both Raf and Ras. This requires 1) the activation of cAMP-dependent protein kinase, 2) an influx of extracellular Ca(2+) through L-type voltage-gated calcium channels, and 3) the activation of CaM kinase II.

Phosphorylation of calmodulin by Ca 2+/calmodulin-dependent protein kinase IV

Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly- l-Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase α, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 μM CaM was about 10 μg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly- l-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly- l-Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr 44. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca 2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.

Ca2+/calmodulin-dependent protein kinase II-dependent long-term potentiation in the rat suprachiasmatic nucleus and its inhibition by melatonin.

We recently reported that Ca(2+)/calmodulin-dependent protein (CaM) kinase II is involved in light-induced phase delays and Per gene induction in the suprachiasmatic nucleus (SCN). To clarify the activation mechanisms of CaM kinase II by glutamate receptor stimulation in the SCN, we documented CaM kinase II activation following induction of long-term potentiation (LTP) in the rat SCN. High-frequency stimulation (100 Hz, 1 sec) applied to the optic nerve resulted in LTP of a postsynaptic field potential in the rat SCN. Unlike LTP in the hippocampal CA1 region, LTP onset in the SCN was slow and partly dependent on N-methyl-D-aspartate receptor activation. LTP induction in the SCN was completely inhibited by treatment with a nitric oxide synthase inhibitor or with a specific CaM kinase II inhibitor. Immunoblotting analysis using phosphospecific antibodies against autophosphorylated CaM kinase II revealed that LTP induction was accompanied by an increase in autophosphorylation. After high-frequency stimulation, we could visualize activation of CaM kinase II in vasoactive intestinal polypeptide-positive neurons in the SCN by immunohistochemistry. Treatment with cyclosporin A, a calcineurin inhibitor, potentiated LTP induction in the rat SCN. Interestingly, treatment with melatonin totally prevented LTP induction, without changes in basal synaptic transmission. Analyses of phosphorylation of CaM kinase II, mitogen-activated protein kinase, and cAMP-responsive element binding protein revealed that stimulatory and inhibitory effects on CaM kinase II autophosphorylation underlie the effects of cyclosporin A and melatonin, respectively. These results suggest that CaM kinase II plays critical roles in LTP induction in the SCN and that melatonin has inhibitory effects on synaptic plasticity through CaM kinase II.

Possible role for Ca2+ calmodulin-dependent protein kinase II as an effector of the fertilization Ca2+ signal in mouse oocyte activation

The present study shows that Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) is physiologically activated in fertilized mouse oocytes and is involved in the Ca(2+) response pathways that link the fertilization Ca(2+) signal to meiosis resumption and cortical granule (CG) exocytosis. After 10 min of insemination, CaM kinase II activity increased transiently, then peaked at 1 h and remained elevated 30 min later when most of the oocytes had completed the emission of the second polar body. In contrast, in ethanol-activated oocytes the early transient activation of CaM kinase II in response to a monotonic Ca(2+) rise was not followed by any subsequent increase. Inhibition of CaM kinase II by 20 micromol/l myristoylated-AIP (autocamtide-2-related inhibitory peptide) negatively affected MPF (maturing promoting factor) inactivation, cell cycle resumption and CG exocytosis in both fertilized and ethanol-activated oocytes. These results indicate that the activation of CaM kinase II in mouse oocytes is differently modulated by a monotonic or repetitive Ca(2+) rise and that it is essential for triggering regular oocyte activation.

Activation of Ca2+/calmodulin-dependent protein kinase I in cultured rat hippocampal neurons.

We have focused on activation mechanisms of calcium/calmodulin-dependent protein kinase (CaM) kinase I in the hippocampal neurons and compared them with that of CaM kinase IV. Increased activation of CaM kinase I occurred by stimulation with glutamate and depolarization in cultured rat hippocampal neurons. Similar to CaM kinases II and IV, CaM kinase I was essentially activated by stimulation with the NMDA receptor. Although both CaM kinases I and IV seem to be activated by CaM kinase kinase, the activation of CaM kinase I was persistent during stimulation with glutamate in contrast to a transient activation of CaM kinase IV. In addition, CaM kinase I was activated in a lower concentration of glutamate than that of CaM kinase IV. Depolarization-induced activation of CaM kinase I was also evident in the cultured neurons and was largely blocked by nifedipine. In the experiment with 32P-labeled cells, phosphorylation of CaM kinase I was stimulated by glutamate treatment and depolarization. The glutamate- and depolarization-induced phosphorylation was inhibited by the NMDA receptor antagonist and nifedipine, respectively. These results suggest that, although CaM kinases I and IV are activated by the NMDA receptor and depolarization stimulation, these kinase activities are differently regulated in the hippocampal neurons.

Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium.

Compromised SERCA 2a activity is a key malfunction leading to the Ca(2+) cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) but alterations of the CaM-kinase pathway regarding SERCA 2a in heart failure are unresolved. Therefore we investigated the CaM-kinase and phosphatase calcineurin mediated regulation of SERCA 2a in failing and non-failing human myocardium. We studied human myocardial preparations from explanted hearts from non-failing organ donors (NF, n=8) and from patients with terminal heart failure undergoing cardiac transplantation (dilated cardiomyopathy, DCM, n=8). SERCA 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by(45)Ca(2+) uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/microg protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma -(32)P] (expressed as pmol(32)P/mg). Maximal(45)Ca(2+) uptake (in pmol/mg/min) (NF: 3402+/-174; DCM: 2488+/-189) and maximal SERCA 2a activity were reduced in DCM compared to NF (V(max): NF: 125+/-9; DCM: 98+/-5). The V(max) reduction could be mimicked by calcineurin in vitro in NF (NF(control): 72.1+/-3.7; NF(+calcineurin): 49.8+/-2.9) and restored in DCM by CaM-kinase in vitro (DCM(control): 98+/-5; DCM(+CaM-kinase): 120+/-6). Protein expression of SERCA 2a, phospholamban and calsequestrin remained similar, but calcineurin expression was significantly increased in failing human hearts (NF: 11.6+/-1.5 v DCM: 17.1+/-1.6). Although the capacity of endogenous CaM-kinase to phosphorylate PT17 was significantly higher in DCM (DCM(control): 128+/-36; DCM(+endogenous CaM-kinase): 205+/-20) compared to NF myocardium (NF(control): 273+/-37; NF(+endogenous CaM-kinase): 254+/-31), net phosphorylation at threonine-17 phospholamban was significantly lower in DCM (DCM 130+/-11 v NF 170+/-11). A calcineurin-dependent dephosphorylation of phospholamban could be mimicked in vitro by incubation of NF preparations with calcineurin (NF(control) 80.7+/-4.4 v NF(+calcineurin) 30.7+/-4.1, P<0.05). In human myocardium, the V(max) of SERCA 2a and the phosphorylation of phospholamban is modulated by CaM-kinase and calcineurin, at least in vitro. In failing human myocardium, despite increased CaM-kinase activity, calcineurin dephosphorylation leads to decreased net phosphorylation of threonine-17 phospholamban in vivo. Increased calcineurin activity contributes to the impaired V(max) of SERCA 2a in failing human myocardium and the disorder in Ca(2+)-handling in heart failure.

Baclofen is neuroprotective and prevents loss of calcium/calmodulin-dependent protein kinase II immunoreactivity in the ischemic gerbil hippocampus.

Excessive release of glutamate during transient cerebral ischemia initiates a cascade of events that leads to the delayed and selective death of neurons located in the hippocampus. Activity of calcium calmodulin kinase II (CaM kinase), a protein kinase critical to neuronal functioning, disappears following ischemia. The in vivo link between glutamate excitoxicity and alterations in CaM kinase activity has not been extensively studied. Baclofen, a selective gamma-aminobutyric acid (GABA)(B) receptor agonist, has been shown to inhibit glutamate release. The present study evaluated the neuroprotective efficacy of this compound and assessed early changes in hippocampal-dependent behaviors and CaM kinase immunoreactivity following transient cerebral ischemia. Baclofen (50 mg/kg) prevented both the loss of hippocampal CA1 pyramidal cells and the reduction in hippocampal CaM kinase immunoreactivity observed in control animals following ischemic insult. Cerebral ischemia produced a significant increase in working memory errors; however, baclofen failed to attenuate this memory deficit. Results confirm that baclofen is neuroprotective and support a link between glutamate excitotoxicity and reductions in CaM kinase immunoreactivity.