Phosphorylation and activation of Ca2+/calmodulin-dependent protein kinase phosphatase by Ca2+/calmodulin-dependent protein kinase II.

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKPase) is a protein phosphatase which dephosphorylates autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) and deactivates the enzyme (Ishida, A., Kameshita, I. and Fujisawa, H. (1998) J. Biol. Chem. 273, 1904-1910). In this study, a phosphorylation-dephosphorylation relationship between CaMKII and CaMKPase was examined. CaMKPase was not significantly phosphorylated by CaMKII under the standard phosphorylation conditions but was phosphorylated in the presence of poly-L-lysine, which is a potent activator of CaMKPase. The maximal extent of the phosphorylation was about 1 mol of phosphate per mol of the enzyme and the phosphorylation resulted in an about 2-fold increase in the enzyme activity. Thus, the activity of CaMKPase appears to be regulated through phosphorylation by its target enzyme, CaMKII.     

Phospholipase C, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditis elegans

Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.     

Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by alpha-lipoic acid in C2C12 myotubes

alpha-Lipoic acid (ALA) widely exists in foods and is an antidiabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by the activation of AMP-activated protein kinase (AMPK) in skeletal muscle, but the underlying mechanism for AMPK activation is unknown. Here, we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. Incubation of C2C12 myotubes with 200 and 500 microM ALA increased the activity and phosphorylation of the AMPK alpha-subunit at Thr(172). Phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC), at Ser(79) was also increased. No difference in ATP, AMP, and the calculated AMP-to-ATP ratio was observed among the different treatment groups. Since the upstream AMPK kinase, LKB1, requires an alteration of the AMP-to-ATP ratio to activate AMPK, this data showed that LKB1 might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased the intracellular Ca(2+) concentration measured by fura-2 fluorescent microscopy (P < 0.05), showing that ALA may activate AMPK through enhancing Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) signaling. Indeed, chelation of intracellular free Ca(2+) by loading cells with 25 microM BAPTA-AM for 30 min abolished the ALA-induced activation of AMPK and, in turn, phosphorylation of ACC at Ser(79). Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA-stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser(79). In addition, ALA treatment increased the association of AMPK with CaMKK. To further show the role of CaMKK in AMPK activation, short interfering RNA was used to silence CaMKK, which abolished the ALA-induced AMPK activation. These data show that CaMKK is the kinase responsible for ALA-induced AMPK activation in C2C12 myotubes.     

Ca2+/calmodulin-dependent protein kinase kinase is not involved in hypothalamic AMP-activated protein kinase activation by neuroglucopenia

Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK) activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca(2+)/Calmodulin-dependent protein kinase kinase (CaMKK), which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO), a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG)-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV) in rats 30 min before 2DG ICV injection (40 μmol) to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol) did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK.     

Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase.

Protein kinase C (PKC) exhibits both negative and positive cross-talk with multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in PC12 cells. PKC effects negative cross-talk by inhibiting the mobilization of intracellular Ca2+ stores and by inhibiting Ca2+ influx through voltage-sensitive Ca2+ channels. In the absence of cross-talk, Ca2+ influx induced by depolarization with 56 mM K+ stimulates CaM kinase and its autophosphorylation and converts up to 50% of the enzyme to a Ca(2+)-independent or autonomous species. Acute treatment with phorbol myristate acetate (PMA) elicits a parallel reduction in depolarization-induced Ca2+ influx and in generation of autonomous CaM kinase. Negative cross-talk also occurs during stimulation of the phosphatidylinositol signaling system with bradykinin, which activates both PKC and CaM kinase. The extent of CaM kinase activation is attenuated by the simultaneous activation of PKC; it is enhanced by prior down-regulation of PKC. PKC also exhibits positive cross-talk with CaM kinase. Submaximal activation of CaM kinase by ionomycin is potentiated by concurrent activation of PKC with PMA. Such PMA treatment is found to increase the level of cytosolic calmodulin. Enhanced activation of CaM kinase by PKC may result from PKC-mediated phosphorylation of calmodulin-binding proteins, such as neuromodulin and MARCKS, and the subsequent increase in the availability of previously bound calmodulin for activation of CaM kinase.     

钙离子／钙调素依赖性蛋白激酶Ⅱ及其功能

所有引起细胞内钙离子浓度升高的激素或神经递质都可通过不同的钙离子／钙调素依赖性蛋白激酶达到调节细胞生理功能的作用。在神经元活动、细胞分泌、平滑肌缩等 细胞活动中起重要作用。     

Phospholipase D isozymes mediate epigallocatechin gallate-induced cyclooxygenase-2 expression in astrocyte cells

Little is known about the effect of epigallocatechin-3 gallate (EGCG), a major constituent of green tea, on the expression of cyclooxygenase (COX)-2. Here, we studied the role of phospholipase D (PLD) isozymes in EGCG-induced COX-2 expression. Stimulation of human astrocytoma cells (U87) with EGCG induced formation of phosphatidylbutanol, a specific product of PLD activity, and synthesis of COX-2 protein and its product, prostaglandin E(2) (PGE(2)). Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed EGCG-induced COX-2 expression and PGE synthesis. Furthermore, evidence that PLD was involved in EGCG-induced COX-2 expression was provided by the observations that COX-2 expression was stimulated by overexpression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid (PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2 expression induced by EGCG. EGCG induced activation of p38 mitogen-activated protein kinase (p38 MAPK), and specific inhibition of p38 MAPK dramatically abolished EGCG-induced PLD activation, COX-2 expression, and PGE(2) formation. Moreover, protein kinase C (PKC) inhibition suppressed EGCG-induced p38 MAPK activation, COX-2 expression, and PGE(2) accumulation. The same pathways as those obtained (2)in the astrocytoma cells were active in primary rat astrocytes, suggesting the relevance of the findings. Collectively, our results demonstrate for the first time that PLD isozymes mediate EGCG-induced COX-2 expression through PKC and p38 in immortalized astroglial line and normal astrocyte cells.     

The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases

The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKalphaThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB(-/-) mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), treatment activates AMPK by alphaThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, are largely inhibited by the Ca(2+)/ calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKalpha and CaMKKbeta. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, alphaThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKalpha and CaMKKbeta. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1(-/-) murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo.     

Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals

The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.     

Regulation of Ca2+/calmodulin-dependent protein kinase II by Ca2+/calmodulin-independent autophosphorylation

The autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) results in the generation of kinase activity that is largely Ca2+/CaM-independent. We report that continued Ca2+/CaM-independent autophosphorylation of CaM-KII results in the generation of distinct phosphopeptides as identified by high performance liquid chromatography and enzymatic properties that are different than those observed for Ca2+/CaM-dependent autophosphorylation. These Ca2+/CaM-independent properties include (a) increased catalytic activity, (b) higher substrate affinity for the phosphorylation of synapsin I, and (c) decreased CaM-binding to both CaM-KII subunits as analyzed by gel overlays. Our results indicate that the autophosphorylation of only one subunit per holoenzyme is required to generate the Ca2+/CaM-independent CaM-KII. We suggest a two-step process by which autophosphorylation regulates CaM-KII. Step I requires Ca2+/CaM and underlies initial kinase activation. Step II involves continued autophosphorylation of the Ca2+/CaM-independent kinase and results in increased affinity for its substrate synapsin I and decreased affinity for calmodulin. These results indicate a complex mechanism through which autophosphorylation of CaM-KII may regulate its activity in response to transient fluctuations in intracellular calcium.     

Eph and NMDA receptors control Ca2+/calmodulin-dependent protein kinase II activation during C. elegans oocyte meiotic maturation

Fertilization in the female reproductive tract depends on intercellular signaling mechanisms that coordinate sperm presence with oocyte meiotic progression. To achieve this coordination in Caenorhabditis elegans, sperm release an extracellular signal, the major sperm protein (MSP), to induce oocyte meiotic maturation and ovulation. MSP binds to multiple receptors, including the VAB-1 Eph receptor protein-tyrosine kinase on oocyte and ovarian sheath cell surfaces. Canonical VAB-1 ligands called ephrins negatively regulate oocyte maturation and MPK-1 mitogen-activated protein kinase (MAPK) activation. Here, we show that MSP and VAB-1 regulate the signaling properties of two Ca2+ channels that are encoded by the NMR-1 N-methyl D-aspartate type glutamate receptor subunit and ITR-1 inositol 1,4,5-triphosphate receptor. Ephrin/VAB-1 signaling acts upstream of ITR-1 to inhibit meiotic resumption, while NMR-1 prevents signaling by the UNC-43 Ca2+/calmodulin-dependent protein kinase II (CaMKII). MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation, and UNC-43 acts redundantly in oocytes to promote oocyte maturation and MAPK activation. Our results support a model in which VAB-1 switches from a negative regulator into a redundant positive regulator of oocyte maturation upon binding to MSP. NMR-1 mediates this switch by controlling UNC-43 CaMKII activation at the oocyte cortex.     

Ischemia-elicited oxidative modulation of Ca2+/calmodulin-dependent protein kinase II

Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) plays a critical role in neuronal signal transduction and synaptic plasticity. Here, we showed that this kinase was very susceptible to oxidative modulation. Treatment of mouse brain synaptosomes with H2O2, diamide, and sodium nitroprusside caused aggregation of CaMKII through formation of disulfide and non-disulfide linkages, and partial inhibition of the kinase activity. These CaMKII aggregates were found to associate with the post synaptic density. However, treatment of purified CaMKII with these oxidants did not replicate those effects observed in the synaptosomes. Using two previously identified potential mediators of oxidants in the brain, glutathione disulfide S-monoxide (GS-DSMO) and glutathione disulfide S-dioxide (GS-DSDO), we showed that they oxidized and inhibited CaMKII in a manner partly related to those of the oxidant-treated synaptosomes as well as the ischemia-elicited oxidative stress in the acutely prepared hippocampal slices. Interestingly, the autophosphorylated and activated CaMKII was relatively refractory to GS-DSMO- and GS-DSDO-mediated aggregation. Short term ischemia (10 min) caused a depression of basal synaptic response of the hippocampal slices, and re-oxygenation (after 10 min) reversed the depression. However, oxidation of CaMKII remained at above the pre-ischemic level throughout the treatment. Oxidation of CaMKII also prevented full recovery of CaMKII autophosphorylation after re-oxygenation. Subsequently, the high frequency stimulation-mediated synaptic potentiation in the hippocampal CA1 region was significantly reduced compared with the control without ischemia. Thus, ischemia-evoked oxidation of CaMKII, probably via the action of glutathione disulfide S-oxides or their analogues, may be involved in the suppression of synaptic plasticity.     

Ca2+/calmodulin-dependent protein kinase II is required for microcystin-induced apoptosis.

The potent natural toxins microcystin, nodularin, and okadaic acid act rapidly to induce apoptotic cell death. Here we show that the apoptosis correlates with protein phosphorylation events and can be blocked by protein kinase inhibitors directed against the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The inhibitors used comprised a battery of cell-permeable protein kinase antagonists and CaMKII-directed peptide inhibitors introduced by microinjection or enforced expression. Furthermore, apoptosis could be induced by enforced expression of active forms of CaMKII but not with inactive CaMKII. It is concluded that the apoptogenic toxins, presumably through their known ability to inhibit serine/threonine protein phosphatases, can cause CaMKII-dependent phosphorylation events leading to cell death.     

Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons.

In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA receptor antagonists but was partially blocked by DL-2-amino-3-phosphonopropionate. Quisqualate or trans-1-amoinocyclopentane-trans1,3-dicarboxylate produced a similar effect on the translocation of PKC. In the experiments with 32P-labeled cells, the phosphorylation of microtuble-associated protein 2 and synapsin I, as well as autophosphorylation of CaM kinase II, were found to be stimulated by exposure to glutamate. These results suggest that glutamate can activate CaM kinase II through the ionotropic NMDA receptor, which in turn increases the phosphorylation of microtuble-associated protein 2 and synapsin I. PKC was activated through the metabotropic glutamate receptor in the hippocampal neurons.     

Cloning, expression and chromosomal localization of human Ca2+/calmodulin-dependent protein kinase kinase

A human cDNA clone encoding the calcium/calmodulin-dependent protein kinase kinase (CaMKK) was isolated by RT-PCR amplification of the fragment corresponding to the conserved kinase catalytic domain followed by rapid amplification of cDNA ends and cDNA library screening. Compilation of nucleotide sequencing data yielded a consensus cDNA sequence of 1.9 kb with an open reading frame of 1,251 nucleotides in length which translates to a polypeptide of 417 amino acids (47 kd). It showed significant homology to the rat brain CaMKK isozymes. The human CaMKK, which was expressed as a Flag-tagged protein in human non-small cell lung cancer H-1299 cells followed by immunoprecipitation with anti-Flag antibody, was shown to phosphorylate recombinant human CaMK I in a calcium/CaM-dependent fashion. Northern blot analysis revealed that human CaMKK is ubiquitously expressed, with brain showing the highest level of expression. The CaMKK gene is localized to human chromosome 12. The presence of cDNA clones with divergent 3' terminal sequences suggests a family of CaMKK variants which may arise from alternative splicing.     

Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells. Identification of a novel neuronal calmodulin-dependent protein kinase

Ca2+-sensitive protein kinases are thought to play a pivotal role in Ca2+-mediated neuronal communication. We describe here the cloning, purification, and characterization of a major Ca2+/calmodulin-dependent, brain-specific protein kinase which is particularly enriched in cerebellar granule cells. The enzyme is comprised of Mr 65,000 and 67,000 polypeptides which copurify to homogeneity and phosphorylate synapsin I. The protein kinase is coded for by two poly(A+) RNAs of 2.0 and 3.5 kilobases which probably derive from a single gene. Two cDNA inserts, one of 198 base pairs and one of 1225 base pairs, contain a total of 677 base pairs of the protein coding sequence which includes sequences homologous to other calmodulin-dependent protein kinases including part of the calmodulin-binding domain. The surprising presence of extended sequences which are enriched in glutamate residues may influence the subcellular distribution of this kinase. Immunohistochemical localization with an affinity-purified antibody reveals that whereas the enzyme is expressed in several neuronal subpopulations, it is exceptionally enriched in the granule cells of the cerebellum. The relevance of the biochemical, molecular, and histologic properties of this enzyme is discussed in the context of neuronal Ca2+ signaling.     

Ca2+/calmodulin-dependent protein kinase in Saccharomyces cerevisiae

Ca²⁺-dependent chromatography of soluble cytosolic proteins on calmodulin-Sepharose gave a fraction that exhibited Ca²⁺- and calmodulin-dependent phosphorylation of several polypeptides, including 60, 56 and 45 kDa species. At 0.2 μM beef calmodulin the phosphorylation was optimal at 3 μM free Ca²⁺, and at 80 μM free Ca²⁺ it was half-maximal at about 0.1 μM beef calmodulin. It is concluded that the fraction contains calmodulin-dependent protein kinase(s) which is (are) autophosphorylated or associated with substrates.