Possible role for Ca(2+) calmodulin-dependent protein kinase II as an effector of the fertilization Ca(2+) 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.     

CaMKII in the cardiovascular system: sensing redox states

The multifunctional Ca(2+)- and calmodulin-dependent protein kinase II (CaMKII) is now recognized to play a central role in pathological events in the cardiovascular system. CaMKII has diverse downstream targets that promote vascular disease, heart failure, and arrhythmias, so improved understanding of CaMKII signaling has the potential to lead to new therapies for cardiovascular disease. CaMKII is a multimeric serine-threonine kinase that is initially activated by binding calcified calmodulin (Ca(2+)/CaM). Under conditions of sustained exposure to elevated Ca(2+)/CaM, CaMKII transitions into a Ca(2+)/CaM-autonomous enzyme by two distinct but parallel processes. Autophosphorylation of threonine-287 in the CaMKII regulatory domain "traps" CaMKII into an open configuration even after Ca(2+)/CaM unbinding. More recently, our group identified a pair of methionines (281/282) in the CaMKII regulatory domain that undergo a partially reversible oxidation which, like autophosphorylation, prevents CaMKII from inactivating after Ca(2+)/CaM unbinding. Here we review roles of CaMKII in cardiovascular disease with an eye to understanding how CaMKII may act as a transduction signal to connect pro-oxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of cardiovascular disease.     

Heparin inhibits phosphorylation and autonomous activity of Ca(2+)/calmodulin-dependent protein kinase II in vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) hyperproliferation is a characteristic feature of both atherosclerosis and restenosis seen after vascular surgery. A number of studies have shown that heparin inhibits VSMC proliferation in vivo and in culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering Ca(2+) regulated pathways involved in mitogenic signaling in VSMC, we analyzed the effect of heparin on multifunctional Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II) which is abundantly expressed in VSMC. Using activity assays, radioactive labeling, and immunoprecipitation it was found that heparin inhibits the overall phosphorylation of the delta-subunit of CaM kinase II which is consistent with inhibition of autophosphorylation-dependent, Ca(2+)/calmodulin-independent CaM kinase II activity. This effect was less evident in heparin-resistant cells, consistent with a role for CaM kinase II in mediating the antiproliferative effect of heparin. Finally, the effects of pharmacological inhibitors of phosphatases like okadaic acid, calyculin, and tautomycin suggest that heparin inhibits CaM kinase II phosphorylation by activating protein phosphatases 1 and 2A. These findings support the hypothesis that alterations in calcium-mediated mitogenic signaling pathways may be involved in the antiproliferative mechanism of action of heparin.     

Changes in Ca(2+)/calmodulin-dependent protein kinase II activity and its relation to performance in passive avoidance response and long-term potentiation formation in mice prenatally exposed to diethylstilbestrol

We investigated the effects of prenatal exposure to diethylstilbestrol (DES), an endocrine disrupter on learning behavior and synaptic functions. Specifically, we determined the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and related kinases that play an essential role in long-term potentiation (LTP) in the hippocampus in mice that were prenatally exposed to DES. Treatment with DES resulted in increased CaMKII autophosphorylation and Ca(2+)-independent activity in the hippocampus and cortex of male mice. Impaired passive avoidance correlated with this increased CaMKII autophosphorylation, as did the enhanced early phase of LTP (E-LTP) in hippocampus. These data suggest that prenatal exposure to DES induces deficits in passive avoidance responses as a result of increased CaMKII activity and hippocampal LTP.     

Calcium/calmodulin-dependent kinase IV in immune and inflammatory responses: novel routes for an ancient traveller

Ca(2+) is a pivotal second messenger controlling the activation of lymphocytes. Crucial events in the social life of immunocytes are regulated by the calcium/calmodulin complex (Ca(2+)/CaM), which controls the activation status of many enzymes, including the Ca(2+)/CaM-dependent Ser-Thr kinases (CaMK) I, II and IV. Although CaMKI and CaMKII are expressed ubiquitously, CaMKIV is found predominately in cells of the nervous and immune systems. To be active, CaMKIV requires binding of Ca(2+)/CaM and phosphorylation by CaMKKalpha or beta. The requirement of two CaM kinases in the same signalling pathway led to the concept of a CaM kinase cascade. In this review, we focus on the roles of CaMKK and CaMKIV cascades in immune and inflammatory responses.     

CaMKII,an emerging molecular driver for calcium homeostasis,arrhythmias, and cardiac dysfunction

Maintenance of cytoplasmic calcium homeostasis is critical for all cells. An exciting field has emerged in elucidating the multiple roles that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays in regulating Ca(2+) cycling in normal cardiac myocytes and in pathophysiological states. Moreover, CaMKII was recently identified as a potential drug target in cardiac disease. This work has given us a closer view of the complexity and therapeutic possibilities of CaMKII regulation of Ca(2+) signaling in cardiac myocytes.     

Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.     

Cellular roles of neuronal calcium sensor-1 and calcium/calmodulin-dependent kinases in fungi

The neuronal calcium sensor-1 (NCS-1) possesses a consensus signal for N-terminal myristoylation and four EF-hand Ca(2+)-binding sites, and mediates the effects of cytosolic Ca(2+). Minute changes in free intracellular Ca(2+) are quickly transformed into changes in the activity of several kinases including calcium/calmodulin-dependent protein kinases (Ca(2+)/CaMKs) that are involved in regulating many eukaryotic cell functions. However, our current knowledge of NCS-1 and Ca(2+)/CaMKs comes mostly from studies of the mammalian enzymes. Thus far very few fungal homologues of NCS-1 and Ca(2+)/CaMKs have been characterized and little is known about their cellular roles. In this minireview, we describe the known sequences, interactions with target proteins and cellular roles of NCS-1 and Ca(2+)/CaMKs in fungi.     

Ca2+-dependent inward current induced by nicotinic receptor activation depends on Ca2+/calmodulin-CaMKII pathway in dopamine neurons

It is well known that midbrain dopamine (DA) neurons receive massive projection from cholinergic neurons in the brainstem. In our preceding report, we showed that Ca(2+)-influx through nicotinic acetylcholine (ACh) receptors in the DA neurons subsequently activated an inward current that was sensitive to fulfenamic acid (FFA) and phenytoin, presumably a Ca(2+)-activated non-selective cation current. The FFA-sensitive current exhibited a negative slope conductance and predominantly enhanced the depolarizing responses of DA neurons. In this study, we showed that the inward FFA-sensitive current was eliminated by antagonists of Ca(2+)/calmodulin (Ca(2+)/CaM), N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7; 1 microM), trifluoperazine (TFP; 1.5 microM) and calmidazolium (100 nM). Application of W-7 and TFP reduced the ACh-induced inward current and the current component suppressed by these drugs exhibited negative slope conductance, as well as the FFA-sensitive current. Further, intracellular application of KN-93, an antagonist of Ca(2+)/CaM-dependent protein kinase II (CaMKII), but not KN-92 eliminated the FFA-sensitive current. All these results suggest that Ca(2+)/CaM-CaMKII pathway is involved in an activation of the FFA-sensitive current.     

Distinct roles of CaM and Ca(2+)/CaM -dependent protein kinase II in Ca(2+) -dependent facilitation and inactivation of cardiac L-type Ca(2+) channels

L-type Ca(2+) channels have two opposing forms of autoregulatory feedback, Ca(2+) -dependent facilitation (CDF) and Ca(2+) -dependent inactivation (CDI), in response to increases in intracellular Ca(2+) concentration. Calmodulin (CaM) has been reported to mediate the two feedbacks. Although both the direct binding of CaM and the phosphorylation mediated by Ca(2+)/CaM -dependent protein kinase II (CaMKII) have been suggested as underlying mechanisms, the detailed features remain to be clarified. In this study, we investigated the effects of CaM and CaMKII inhibitors on CDF and CDI with patch clamp cell-attached recordings in guinea-pig ventricular myocytes. We confirmed that a high-K(+) and high-Ca(2)(+) could induce an increase of the intracellular Ca(2+) concentration and subsequent CDF and CDI. We then found that CDF and CDI were both depressed and were finally abolished by treatment with a CaM inhibitor chlorpromazine (1-100 microM) in a concentration-dependent manner. Another CaM antagonist calmidazolium (1 microM) showed a similar effect. In contrast, CaMKII inhibitors, KN-62 (0.1-3 microM) and autocamtide 2 -related inhibitory peptide (1 microM), delayed the development of CDF and CDI significantly, but they did not depress either CDF or CDI. These results imply that CaM is necessary and possibly sufficient for the two mechanisms. We propose a hypothesis that CaM is a key molecule to bifurcate the Ca(2+) signal to CDF and CDI and that CaMKII plays a modulatory role in them both.     

Comparison of Ca2+ and CaMKII responses in IVF and ICSI in the mouse

Novel methods of egg activation in human assisted reproductive technologies and animal somatic cell nuclear transfer are likely to alter the signalling process that occurs during normal fertilization. Intracytoplasmic sperm injection (ICSI) bypasses the normal processes of the acrosome reaction, sperm-egg fusion, and processing of the sperm plasma membrane, as well as alters some parameters of intracellular calcium ([Ca(2+)](i)) dynamics (reported previously by Kurokawa and Fissore (2003)). Herein, we extend these studies to determine if ICSI alters the activity of the Ca(2+)-dependent protein, Ca(2+)/calmodulin-dependent kinase II (CaMKII), which is responsible for the completion of meiosis in vertebrate eggs. After ICSI or in vitro fertilization (IVF), individual mouse eggs were monitored for their relative changes in both [Ca(2+)](i) and CaMKII activity during the first [Ca(2+)](i) rise and a subsequent rise associated with second polar body extrusion. The duration of the first [Ca(2+)](i) rise was greater in ICSI than in IVF, but the amplitude of the rise was transiently higher for IVF than ICSI. However, a similar mean CaMKII activity was observed in both procedures. During polar body extrusion, the amplitude and duration of the Ca(2+) rises were increased by a small amount in ICSI compared with IVF, whereas the CaMKII activities were similar. Thus, compared with IVF, ICSI is not associated with decreased or delayed CaMKII activity in response to these Ca(2+) signals in the mouse.     

Mechanisms of mycobacterial persistence in tuberculosis

Tuberculosis is one of the world's most devastating diseases, with more than two million deaths and eight million new cases occurring annually. Mycobacterium tuberculosis evades the innate antimicrobial defenses of macrophages by inhibiting the maturation of its phagosome to a bactericidal phagolysosome. Phagosome maturation is dependent on macrophage Ca(2+) signaling, which results in the recruitment of cytosolic calmodulin (CaM) to the phagosome membrane and subsequent focal activation of CaM kinase II (CaMKII). M. tuberculosis blocks this process via inhibition of a macrophage enzyme, sphingosine kinase, which is a proximal generator of Ca(2+) signaling during phagocytosis. This results in a failure of assembly of the Ca(2+)/CaM/CaMKII signaling complex on the membrane of the mycobacterial phagosome and the bacilli's persistence and replication in a protective intracellular niche. Pharmacologic or physiologic reversal of this inhibition of macrophage Ca(2+) signaling restores the normal sequence of phagosome maturation, resulting in decreased intracellular viability of M. tuberculosis.     

Characterization of Plasmodium falciparum protein kinase 2

A sustained elevation of free Ca(2+) is observed on the rupture and release of merozoites of Plasmodium falciparum from the erythrocytes. The immunoelectron micrographs demonstrate that calmodulin is localized in merozoites. To elucidate the Ca(2+) signal of P. falciparum invasion, we attempted to characterize P. falciparum protein kinase 2 (PfPK2), which is homologous to human calcium calmodulin-dependent protein kinase (CaMK). PfPK2 was purified as a fusion protein that was labeled with [gamma-(32)P]ATP; this labeling was then eliminated by phosphatase. This phosphorylation was eliminated when the putative catalytic lysine residue of PfPK2 was replaced with alanine. PfPK2 phosphorylated histone II(AS) as a representative substrate in a Ca(2+)- and calmodulin-dependent manner. Calmodulin antagonists inhibited the phosphorylation of PfPK2 in vitro and markedly decreased the parasitemia of ring forms in an invasion assay, whereas CaMKII-specific inhibitors had no effect. PfPK2 was localized in the merozoites in the culture of P. falciparum. Thus, purified PfPK2 possesses protein kinase activity in a Ca(2+)- and calmodulin-dependent manner and the catalytic lysine of this protein was determined. These data suggest that PfPK2 is the Plasmodium protein kinase expressed in the merozoites during the invasion stage.     

钙调蛋白与肥厚心肌的心律失常

钙调蛋白(calmodulin,CaM)是广泛存在于真核生物体内的一种多功能蛋白质,是Ca2+在体内的一个重要受体。CaM通过Ca2+/CaM依赖的钙调神经磷酸酶及CaMKⅡ,调节心肌细胞肥大相关基因的表达;通过心肌细胞膜上钙通道的磷酸化,造成钙超载,进而诱导心肌肥厚及肥厚心肌心律失常的发生。钙调蛋白在肥厚心肌心律失常机制的阐述,对指导临床治疗和开发新的、更有效的药物具有重要的意义。     

Serine/threonine protein kinases synergistically regulate phospholipase D1 and 2 and secretion in RBL-2H3 mast cells

The role of phospholipase (PL) D in secretion was examined in RBL-2H3 mast cells which contain both PLD1 and 2. The effects of pharmacologic stimulants and inhibitors of Ca(2+)/calmodulin-dependent kinase II, protein kinase C, and protein kinase A suggested that all three kinases synergistically stimulate PLD and, when associated with a calcium signal, secretion as well to indicate a possible linkage between these two events. Overexpression of either PLD1 or 2 markedly enhanced the activation of PLD by pharmacologic stimulants as well as antigen and both isoforms thus appear co-ordinately regulated. As the expressed PLD1 was associated with secretory granules and PLD2 with the plasma membrane, the two isoforms may serve distinct but complementary functions in secretion.     

Intracellular injection of synapsin I induces neurotransmitter release in C1 neurons of Helix pomatia contacting a wrong target

The contact with the postsynaptic target induces structural and functional modifications in the serotonergic cell C1 of Helix pomatia. In previous studies we have found that the presence of a non-physiological target down-regulates the number of presynaptic varicosities formed by cultured C1 neurons and has a strong inhibitory effect on the action potential-evoked Ca(2+) influx and neurotransmitter release at C1 terminals. Since a large body of experimental evidence implicates the synapsins in the development and functional maturation of synaptic connections, we have investigated whether the injection of exogenous synapsin I into the presynaptic neuron C1 could affect the inhibitory effect of the wrong target on neurotransmitter release. C1 neurons were cultured with the wrong target neuron C3 for three to five days and then injected with either dephosphorylated or Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated Cy3-labeled synapsin I. The subcellular distribution of exogenous synapsin I, followed by fluorescence videomicroscopy, revealed that only synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II diffused in the cytoplasm and reached the terminal arborizations of the axon, while the dephosphorylated form did not diffuse beyond the cell body. Evoked neurotransmitter release was measured during C1 stimulation using a freshly dissociated neuron B2 (sniffer) micromanipulated in close contact with the terminals of C1. A three-fold increase in the amplitude of the sniffer depolarization with respect to the pre-injection amplitude (190+/-29% increase, n=10, P<0.006) was found 5 min after injection of Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated synapsin I that lasted for about 30 min. No significant change was observed after injection of buffer or dephosphorylated synapsin I.These data indicate that the presence of synapsin I induces a fast increase in neurotransmitter release that overcomes the inhibitory effect of the non-physiological target and suggest that the expression of synapsins may play a role in the modulation of synaptic strength and neural connectivity.     

Constitutive activity of inwardly rectifying K+ channel at physiological [Ca]i is mediated by Ca2+/CaMK II pathway in opossum kidney proximal tubule cells

Using patch-clamp technique, we studied the role of the Ca(2+)/calmodulin kinase II (CaMK II)-mediated phosphorylation process on the K(+) channel with an inward conductance of 90 pS in opossum kidney proximal tubule cells (OKPCs). The intracellular Ca(2+) concentration ([Ca](i)) was measured by use of the fluorescent dye fura 2. The following results were obtained: (i) In cell-attached patches, the channel activity was inhibited by a decrease in [Ca](i) induced by perfusion with low Ca(2+) (10(-8) M), La(3+) (100 microM), or EGTA/AM (100 microM) contained in the bath solution. The application of KN-62 (10 microM) or KN-93 (5 microM), inhibitors of CaMK II, also inhibited the channel activity. (ii) The membrane potential measured with nystatin-perforated patches was significantly decreased by the fall in [Ca](i) induced by the perfusion with EGTA- or La(3+)-containing solution. Also, the application of KN-62 (10 microM) or KN-93 (5 microM) to the bath significantly decreased the membrane potential. (iii) In inside-out patches, the channel activity was significantly stimulated by the application of CaMK II (300 pM) at 10(-7) M Ca(2+) in the bath. Furthermore, the application of KN-62 (10 microM) to the bath significantly decreased the channel activity. Our findings show that the constitutive activity of inwardly rectifying K(+) channel at physiological [Ca](i) is mediated by the Ca(2+)/CaMK II pathway in OKPCs.     

CaM kinase II-alpha activity,levels and Ca/calmodulin dependent phosphorylation of substrate proteins in mice brain during fatal murine cerebral malaria

The activity and levels of CaM kinase II-alpha was investigated in the cytosolic and membrane fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). In parallel, Ca(2+)/Calmodulin dependent phosphorylation of target substrate proteins was studied using syntide-2 as substrate. Pathology of FMCM resulted in decreased CaM kinase-II activity in both cortex and cerebellum though western analysis revealed no appreciable changes in the levels of CaM kinase-II alpha in cytosol and membrane fractions from control and cerebral malaria infected brain. Given the abundant expression of Cam kinase-II in neuronal tissue, its significance in neurotransmitter release and synthesis and signal transduction during apoptosis, decreased levels of enzyme activity and altered phosphorylation of substrate proteins by CaM kinase II may serve as important cues in understanding the CaM kinase signal transduction events central to neurological disorders during FMCM.