Binding of calmodulin to the neuronal cytoskeletal protein kinase type II cooperatively stimulates autophosphorylation.

The kinetics of autophosphorylation of the cytoskeletal form of the neuronal calmodulin-dependent protein kinase type II were studied as a function of calmodulin binding under the same conditions. Whereas calmodulin binding was noncooperative with respect to calmodulin concentration (Hill coefficient = 1), the activation of autophosphorylation and the phosphorylation of exogenous substrates showed marked positive cooperativity (Hill coefficient greater than or equal to 1.6). Reduction of the active calmodulin concentration by the addition of the calmodulin antagonist trifluoperazine confirmed the cooperative nature of enzyme activation, because autophosphorylation was more sensitive to the drug than was binding at high concentrations of calmodulin. At intracellular levels of calmodulin the binding and activation of autophosphorylation were cooperative functions of magnesium and calcium concentration. The calmodulin-dependent cooperative activation seems to be a unique feature of the cytoskeletal, but not the soluble, form of the protein kinase and may result from the supramolecular organization of the cytoskeletal enzyme. These observations suggest that interactions among the subunits of the oligomeric cytoskeletal calmodulin-dependent protein kinase regulate enzyme activation, enhancing the sensitivity of the enzyme to small changes in the intracellular calcium levels that may be particularly relevant to signaling at the synapse.     

Inhibition of calcium/calmodulin kinase II alpha subunit expression results in epileptiform activity in cultured hippocampal neurons

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the alpha subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy.     

Novel and uncommon isoforms of the calcium sensing enzyme calcium/calmodulin dependent protein kinase II in heart tissue

Calcium/calmodulindependent protein kinase type II (CaM kinase II) is an intracellular enzyme discovered several years ago in the brain which is obviously involved in intracellular signal transmission. Meanwhile it was detected in virtually every mammalian tissue. Several isoforms have been found to exist. Most recently the tissue distribution and the molecular structure of these isoforms have suggested that each form entails a particular function. In the present study we describe the identification, cloning, and nucleotide sequencing of two novel CaM kinase II isoforms which we discovered in rat heart. The presence of these additional subtypes makes the heart the organ which possesses the greatest number of different and unusual CaM kinase II isoforms throughout the body except for the brain. The importance of this finding is underscored by the fact that calcium is involved in the regulation of many crucial cardial parameters. The deduced amino acid sequence that we have obtained from the new CaM kinase II isoforms indicates a molecular organization which could make the design of subtype-specific inhibitory drugs for CaM kinase II possible. Such compounds would act similarly to but much more selectively than digitalis glycosides and would be likely to possess less side effects.     

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by calmodulin with two bound calciums

Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, we designed mutant CaMs that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the "closed" Ca2+-free conformation. We have measured their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation. We show that CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. Our results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways.     

Characterization of a calmodulin kinase II inhibitor protein in brain

Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII α and β and potently inhibited kinase activity with an IC₅₀ of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i.e., in the presence of Ca²⁺/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.     

Reduced contractile response to alpha1-adrenergic stimulation in atria from mice with chronic cardiac calmodulin kinase II inhibition

The sustained positive inotropic effect of alpha-adrenoceptor agonists in the heart is associated with a small increase in intracellular Ca(2+) transients together with a larger sensitization of myofilaments to Ca(2+). The multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) could contribute to this effect, either by affecting the Ca(2+) release (ryanodine receptor) or by an uptake mechanism (via phospholamban [PLB] and SR Ca(2+) ATPase). Here we examined the role of CaMKII in the positive inotropic effect of the alpha-adrenoceptor agonist phenylephrine in left atria isolated from a genetic mouse model of cardiac CaMKII inhibition (AC3-I). Compared to atria from wild-type (WT) or AC3-C (scrambled peptide), AC3-I atria showed the following abnormalities. PLB phosphorylation at Thr17, a known CaMKII target, was significantly lower ( approximately 20%). Post-rest (30 s, 1 Hz, 37 degrees C) potentiation of force was absent (AC3-C, 190% of pre-rest amplitude). Basal force was approximately 20% lower at 1.8 mM Ca(2+), but normal at high Ca(2+) concentration (>4.5 mM). The maximal positive inotropic effect of phenylephrine, which was more pronounced at low frequencies in WT and AC3-C atria, lost its frequency dependence (1 Hz to 8 Hz). Thus, the effect of phenylephrine was reduced by approximately 50% at 1 Hz, but was normal at 8 Hz. All three groups showed a negative force-frequency relation, and did not differ in the frequency-dependent acceleration of relaxation. Our data indicate a role of CaMKII in post-rest potentiation and the positive inotropic effect of alpha-adrenergic stimulation at low frequencies.     

T-cell function is partially maintained in the absence of class IA phosphoinositide 3-kinase signaling

The class IA subgroup of phosphoinositide 3-kinase (PI3K) is activated downstream of antigen receptors, costimulatory molecules, and cytokine receptors on lymphocytes. Targeted deletion of individual genes for class IA regulatory subunits severely impairs the development and function of B cells but not T cells. Here we analyze conditional mutant mice in which thymocytes and T cells lack the major class IA regulatory subunits p85alpha, p55alpha, p50alpha, and p85beta. These cells exhibit nearly complete loss of PI3K signaling downstream of the T-cell receptor (TCR) and CD28. Nevertheless, T-cell development is largely unperturbed, and peripheral T cells show only partial impairments in proliferation and cytokine production in vitro. Both genetic and pharmacologic experiments suggest that class IA PI3K signaling plays a limited role in T-cell proliferation driven by TCR/CD28 clustering. In vivo, class IA-deficient T cells provide reduced help to B cells but show normal ability to mediate antiviral immunity. Together these findings provide definitive evidence that class IA PI3K regulatory subunits are essential for a subset of T-cell functions while challenging the notion that this signaling mechanism is a critical mediator of costimulatory signals downstream of CD28.     

Localization of alpha type II calcium calmodulin-dependent protein kinase at glutamatergic but not gamma-aminobutyric acid (GABAergic) synapses in thalamus and cerebral cortex.

The alpha subunit of type II calcium/calmodulin-dependent protein kinase (CAM II kinase-alpha) plays an important role in longterm synaptic plasticity. We applied preembedding immunocytochemistry (for CAM II kinase-alpha) and postembedding immunogold labeling [for glutamate or gamma-aminobutyric acid (GABA)] to explore the subcellular relationships between transmitter-defined axon terminals and the kinase at excitatory and inhibitory synapses in thalamus and cerebral cortex. Many (but not all) axon terminals ending in asymmetric synapses contained presynaptic CAM II kinase-alpha immunoreactivity; GABAergic terminals ending in symmetric synapses did not. Postsynaptically, CAM II kinase-alpha immunoreactivity was associated with postsynaptic densities of many (but not all) glutamatergic axon terminals ending on excitatory neurons. CAM II kinase-alpha immunoreactivity was absent at postsynaptic densities of all GABAergic synapses. The findings show that CAM II kinase-alpha is selectively expressed in subpopulations of excitatory neurons and, to our knowledge, demonstrate for the first time that it is only associated with glutamatergic terminals pre- and postsynaptically. CAM II kinase-alpha is unlikely to play a role in plasticity at GABAergic synapses.     

Norepinephrine-induced inositol 1,4,5-trisphosphate formation in atrial myocytes is regulated by extracellular calcium,protein kinase C,and calmodulin

We investigated whether alteration of extracellular and intracellular Ca2+ concentrations, protein kinase C, and calmodulin modulate norepinephrine (NE)-induced inositol 1,4,5-trisphosphate (IP3) formation in neonatal rat atrial myocytes. NE-induced IP3 production in atrial myocytes was stimulated by elevation of extracellular Ca2+ in a dose-dependent manner. However, TMB-8 (an intracellular calcium antagonist) and A23187 (an intracellular calcium agonist) did not significantly affect NE-induced IP3 production. PMA (a protein kinase C agonist) significantly decreased and staurosporine (a protein kinase C antagonist) significantly stimulated NE-induced IP3 production. W7 (a calmodulin antagonist) significantly increased the NE-induced IP3. In conclusion, elevation of extracellular Ca2+ concentrations affects NE-induced IP3 formation in atrial myocytes. Protein kinase C and calmodulin may control the IP3 response to NE by a negative feedback mechanism.     

Activation of calcium-dependent calmodulin by calcium(II)3(3,5-diisopropylsalicylate)6(H2O)6 decreases thrombin receptor activating peptide-induced P-selectin expression.

We examined the influence of 3,5-diisopropylsalicylic acid (3,5-DIPS) and calcium(II)3 (3,5-diisopropylsalicylate)6 (H2 O)6 [Ca(II)3 (3,5-DIPS)6 ], a new activator of calcium-dependent calmodulin-triggered nitric oxide synthase, on thrombin-induced platelet P-selectin expression. Citrated whole blood samples were incubated with either ethanol vehicle, 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6. These whole blood samples were also co-incubated with thrombin receptor activating peptide (TRAP) or adenosine diphosphate (ADP), to up-regulate P-selectin (CD62P) on platelets. Both TRAP and ADP up-regulated P-selectin on platelets compared with platelets in whole blood samples that were not incubated with either platelet activator. Co-incubation of whole blood samples with TRAP, ADP together with 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6 revealed that Ca(II)3 (3,5-DIPS)6 caused a decrease in platelet P-selectin expression for TRAP, ADP, and no-activator co-incubated samples of whole blood. Incubation of platelets with 3,5-DIPS also caused a decrease in ADP-induced up-regulation of P-selectin but failed to affect TRAP or no-activator-treated platelets. Incubation of whole blood with Ca(II)3 (3,5-DIPS)6 induced some hemolysis. We found that hemolysis increases basal P-selectin expression on platelets. We therefore conclude that Ca(II)3 (3,5-DIPS)6 decreased not only basal, but also hemolysis-induced P-selectin expression on platelets. In contrast, incubation of haemolysed whole blood with SIN-1 (standard nitric oxide-releasing drug) had no effect on P-selectin expression. In summary, Ca(II)3 (3,5-DIPS)6, a new calmodulin-dependent nitric oxide synthase activator, decreases P-selectin expression of human platelets in response to thrombin receptor activation. Improved calcium-dependent calmodulin activators may become useful drugs for the treatment of disorders associated with platelet activation, and P-selectin may decrease expression due to hemolysis.     

Calmodulin-dependent gating of Ca(v)1.2 calcium channels in the absence of Ca(v)beta subunits

It is generally accepted that to generate calcium currents in response to depolarization, Ca(v)1.2 calcium channels require association of the pore-forming alpha(1C) subunit with accessory Ca(v)beta and alpha(2)delta subunits. A single calmodulin (CaM) molecule is tethered to the C-terminal alpha(1C)-LA/IQ region and mediates Ca2+-dependent inactivation of the channel. Ca(v)beta subunits are stably associated with the alpha(1C)-interaction domain site of the cytoplasmic linker between internal repeats I and II and also interact dynamically, in a Ca2+-dependent manner, with the alpha(1C)-IQ region. Here, we describe a surprising discovery that coexpression of exogenous CaM (CaM(ex)) with alpha(1C)/alpha(2)delta in COS1 cells in the absence of Ca(v)beta subunits stimulates the plasma membrane targeting of alpha(1C), facilitates calcium channel gating, and supports Ca2+-dependent inactivation. Neither real-time PCR with primers complementary to monkey Ca(v)beta subunits nor coimmunoprecipitation analysis with exogenous alpha(1C) revealed an induction of endogenous Ca(v)beta subunits that could be linked to the effect of CaM(ex). Coexpression of a calcium-insensitive CaM mutant CaM(1234) also facilitated gating of Ca(v)beta-free Ca(v)1.2 channels but did not support Ca2+-dependent inactivation. Our results show there is a functional matchup between CaM(ex) and Ca(v)beta subunits that, in the absence of Ca(v)beta, renders Ca2+ channel gating facilitated by CaM molecules other than the one tethered to LA/IQ to support Ca2+-dependent inactivation. Thus, coexpression of CaM(ex) creates conditions when the channel gating, voltage- and Ca2+-dependent inactivation, and plasma-membrane targeting occur in the absence of Ca(v)beta. We suggest that CaM(ex) affects specific Ca(v)beta-free conformations of the channel that are not available to endogenous CaM.     

Ca2+/calmodulin-dependent protein kinase II: identification of threonine-286 as the autophosphorylation site in the alpha subunit associated with the generation of Ca2+-independent activity.

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II converts the enzyme to a Ca2+-independent form. The time course for this conversion correlates with the autophosphorylation of a threonine residue located within a thermolytic phosphopeptide common to the alpha and beta/beta' subunits. In the present study, this site was identified in the alpha subunit. After autophosphorylation under conditions that produced near-maximal Ca2+-independent activity, the alpha and beta/beta' subunits were separated by NaDodSO4/PAGE, and the alpha subunit was cleaved with cyanogen bromide. The major phosphopeptide (CB-1), containing phosphothreonine as the only radiolabeled amino acid, was purified by reverse-phase high performance liquid chromatography and subjected to automated gas-phase Edman degradation. The sequence obtained, Xaa-Arg-Gln-Glu-Thr-Val-Asp-Xaa-Leu-Lys-Lys-Phe-Asn-Ala-Arg-Arg-Lys-Leu, represented the NH2-terminal 18 residues (residues 282-299) of a 26-amino acid cyanogen bromide peptide predicted from the deduced primary structure of the alpha subunit and contained a consensus sequence for Ca2+/calmodulin-dependent kinase II phosphorylation that included Thr-286. The sequences obtained for two phosphopeptides derived from secondary chymotryptic digestion of CB-1 confirmed that Thr-286 was the phosphorylated residue.     

The stimulation of MAP kinase by 1,25(OH)(2)-vitamin D(3) in skeletal muscle cells is mediated by protein kinase C and calcium

In previous work we have demonstrated that the steroid hormone 1,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] stimulates in skeletal muscle cells the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2. In the present study we evaluated the involvement of Ca(2+) and protein kinase C (PKC) on 1,25(OH)(2)D(3)-induced activation of MAP kinase. The hormone response was found to depend on PKC stimulation since it was attenuated by the PKC inhibitors calphostin C (100 nM) and bisindolylmaleimide I (30 nM) and PKC downregulation by prolonged treatment with the phorbol ester TPA (1 microM). Removal of external Ca(2+), chelation of intracellular Ca(2+) with BAPTA (5 microM), inhibition of phosphoinositide-phospholipase C (PLC) by neomycin, the calmodulin antagonist fluphenazine (50 microM) and the specific inhibitor of calmodulin kinase II, KN-62 (10 microM), significantly decreased 1,25(OH)(2)D(3)-activation of MAP kinase. In addition, the Ca(2+)-channel blocker verapamil (5 microM) suppressed hormone-induced MAP kinase activity in these cells. Furthermore, the Ca(2+)-mobilizing agent thapsigargin and the Ca(2+)-inophore A23187 paralleled the phosphorylation of MAP kinase observed with 1,25(OH)(2)D(3). Taken together, these results indicate that PKC and Ca(2+) are two upstream activators mediating the effects of 1,25(OH)(2)D(3) on MAP kinase in skeletal muscle cells.     

钙调蛋白激酶Ⅱ抑制剂对心肌肥厚兔室性心律失常的影响

目的观察钙调蛋白激酶Ⅱ抑制剂KN-93对心肌肥厚兔室性心律失常的影响。方法雌性新西兰大白兔随机分为4组：假手术组（Sham组）、心肌肥厚组（LVH组）、心肌肥厚＋KN-93组（KN-93组）、心肌肥厚＋KN-92组（KN-92组）,每组10只。LVH、KN-93及KN-92组通过缩窄腹主动脉制备兔心肌肥厚模型,Sham组仅游离腹主动脉未进行缩窄。8周后制备兔左室楔形心肌块的灌注模型,同步记录心内、外膜动作电位及跨壁心电图,观察低钾（2mmol／L）、低镁（0．25mmol／L）台氏液灌流及慢频率刺激条件下各组早期后除极（EAD）和尖端扭转型室性心动过速（Tap）的发生率,并记录在不同起搏周期下QT间期、动作电位时程（APD）及跨室壁复极离散度（TDa）的变化。结果在低钾、低镁台氏液灌流及2000～4000hi8慢频率刺激下,Sham、LVH、KN-92组（0．5μmol／L）及KN-93组（0．5μmol/L）EAD的发生率分别为0／10、10／10、9／10和5／10,Tdp的发生率分别为0／10、5／10、4／10和1／10;当KN-92组及KN-93组中药物浓度增至1μmol／L时,EAD的发生率分别为9／10和3／10,Tdp的发生率分别为4／10和1／10。而且KN-93组、KN-92组对QT间期、APD及TDR无明显影响（P〉0．05）。结论钙调蛋白激酶Ⅱ特异性抑制剂KN-93能够有效抑制心肌肥厚兔室性心律失常的发生,其主要作用机制是通过减少EAD的发生来实现。     

Platelet glycoprotein IV (CD36) deficiency is associated with the absence (type I) or the presence (type II) of glycoprotein IV on monocytes

Platelet membrane glycoprotein (GP) IV (also called CD36 and GPIIb) deficiency is associated with N(aka)-negative platelets. Using flow- cytometric analysis of cells stained with the monoclonal anti-GPIV antibody OKM5, we have studied the expression of GPIV on the monocytes from 16 healthy Japanese individuals whose platelets were deficient in GPIV. GPIV was absent on the surface of monocytes from 2 platelet GPIV- negative donors (type I), whereas it was present on the monocytes from the remaining 14 platelet GPIV-negative donors (type II). The fluorescent intensity of OKM5-stained type II monocytes was significantly (P < .05) lower than that of normal monocytes derived from platelet GPIV-positive donors, suggesting that the expression of GPIV on the type II monocytes is also abnormally regulated as compared with that on normal monocytes. OKM5 induced an oxidative burst in the type II monocytes as well as in the normal monocytes, but it failed to induce it in the type I monocytes. Because the 2 individuals with the type I deficiency have been healthy and exhibited no immunologic problems, GPIV appears to be not essential for the normal physiologic functions of monocytes. An anti-GPIV antibody was detected in the serum from one of the type I GPIV-deficient women, who had never received any blood transfusions but had given birth to three apparently healthy children. These results suggest that type I GPIV-deficient individuals may be at risk of developing an anti-GPIV isoantibody upon blood transfusion or pregnancy.     

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.