Calvasculin, an encoded protein from mRNA termed pEL-98, 18A2, 42A, or p9Ka, is secreted by smooth muscle cells in culture and exhibits Ca(2+)-dependent binding to 36-kDa microfibril-associated glycoprotein.

Calvasculin, an EF-hand protein with a molecular mass of 11 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is present abundantly in bovine aorta (Watanabe, Y., Kobayashi, R., Ishikawa, T., and Hidaka, H. (1992) Arch. Biochem. Biophys. 292, 563-569). This protein is synthesized constitutively by bovine aortic smooth muscle (BASM) cells and rat embryo fibroblast 3Y1 cells in culture. We discovered that calvasculin was secreted by BASM cells and 3Y1 cells. Immunofluorescence staining of BASM cells showed a granular distribution for calvasculin that was typical of a secreted protein. This protein bound with an extracellular matrix protein, 36-kDa microfibril-associated glycoprotein (36-kDa MAP), in a Ca(2+)-dependent manner in vitro. A stoichiometry analysis showed that the 36-kDa MAP bound 2.2 calvasculin eq/mol of protein. Solid-phase binding assays indicated a preferential affinity of native calvasculin for 36-kDa MAP among the extracellular matrices in a Ca(2+)-dependent manner. These results suggest that calvasculin, intracellular Ca(2+)-binding protein, is released to the extracellular space and binds with 36-kDa MAP.     

Calcium ion regulates the release of lipase of Fusarium oxysporum.

The lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl- L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4 had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 microM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes.     

Phosphorylation of Purified Rat Striatal Tyrosine Hydroxylase by Ca2+/Calmodulin-Dependent Protein Kinase II: Effect of an Activator Protein

The phosphorylation of tyrosine hydroxylase, purified from rat striatum, was investigated using purified Ca2+/calmodulin (CaM)-dependent protein kinase II. This kinase catalyzed the Ca2+-dependent incorporation of up to 0.8 mol 32PO4/mol tyrosine hydroxylase subunit (62 kilodaltons). Reverse-phase high-performance liquid chromatography mapping of tryptic 32P-peptides established that the Ca2+/CaM-dependent protein kinase II phosphorylated a different serine residue than was phosphorylated by the cyclic AMP-dependent protein kinase. Limited proteolysis sequentially reduced the subunit Mr from 62 to 59 kilodaltons and finally to 57 kilodaltons, resulting in loss of the site phosphorylated by the Ca2+/CaM-dependent protein kinase II, but not the site phosphorylated by the cyclic AMP-dependent protein kinase. Phosphorylation by the Ca2+/CaM-dependent protein kinase II had little direct effect on the kinetic properties of tyrosine hydroxylase, but did convert it to a form that could be activated twofold by addition of an activator protein. This heat-labile activator protein increased the Vmax without affecting the Km for the pterin cofactor. This effect was specific in that the activator protein was without effect on nonphosphorylated tyrosine hydroxylase or on tyrosine hydroxylase phosphorylated by the cyclic AMP-dependent protein kinase. These results are consistent with the hypothesis that the "Vmax-type" activation of tyrosine hydroxylase observed upon depolarization of neural and adrenal tissues may be mediated by the Ca2+/CaM-dependent protein kinase II.     

Calmodulin-Dependent Protein Phosphorylation in Synaptic Junctions

Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.     

牛脑Ca~(2+)/CaM依赖性蛋白激酶Ⅱ的纯化和鉴定

通过一系列层析法,首次从牛脑纯化得到胶凝电泳匀一的Ca~(2+)/CaM PKⅡ。凝胶过滤法测定全酶分子量为550kD,SDS-PAGE法测定亚基分子量为55kD,推测牛脑Ca~(2+)/CaM PK Ⅱ由十个相同的亚基组成。该酶活性绝对依赖于Ca~(2+)和CaM,以63kD PDE同工酶为底物,其AC_(50)分别为0.85μmol/L和0.18μmol/L;以酪蛋白为底物,其AC_(50)分别为0.22μmol/L和0.06μmol/L。牛脑Ca~(2+)/CaM PK Ⅱ旣能催化63kD PDE同工酶等多种蛋白或酶磷酸化,又能进行自身磷酸化。该酶催化63kD PDE同工酶最大磷酸参入量为1mol/mol亚基。磷酸化型63kD PDE同工酶的Ca~(2+)的AC_(50)高于非磷酸化型。     

Calcium/Calmodulin-Dependent Protein Kinase II in Squid Synaptosomes

The Ca2+/calmodulin (CaM)-dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM-dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high-speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM-dependent protein kinase II from its calcium-dependent form to a calcium-independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5'-O-(3-thiotriphosphate) for ATP. When [gamma-32P]ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins greater than 100 kDa were rapidly 32P-labeled in a calcium-dependent manner. Major 125I-CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM-dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54- and 58-60-kDa subunits. The purified kinase, like Ca2+/CaM-dependent protein kinase II from rat brain, catalyzed autophosphorylation associated with formation of the calcium-independent form. These studies, characterizing the Ca2+/CaM-dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium-dependent synaptic functions.     

Nerve growth factor treatment or cAMP elevation reduces Ca2+/calmodulin-dependent protein kinase III activity in PC12 cells

Ca2+/calmodulin-dependent protein kinase III (Ca2+/CaM kinase III) phosphorylates a protein of Mr = 100,000 (the 100-kDa protein), a major substrate for Ca2+/CaM-dependent protein phosphorylation found in many mammalian tissues and cell lines (Nairn, A.C., Baghat, B., and Palfrey, H.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7939-7943). Treatment of PC12 cells with nerve growth factor (NGF) or forskolin resulted in a decrease in the depolarization-dependent phosphorylation of the 100-kDa protein in intact cells and in a decrease in the Ca2+/CaM-dependent phosphorylation of the 100-kDa protein in cytosolic extracts. In experiments using cytosolic extracts, the initial effect of NGF on the phosphorylation of the 100-kDa protein was observed in less than 1 h, was maximal (70% decrease) after 12 h, and began to recover after 24 h. The effect of forskolin was more rapid and the maximal effect was greater (90-95% decrease). Decreased Ca2+/CaM kinase III activity was also found in PC12 cells treated with epidermal growth factor, 2-chloroadenosine plus isobutylmethylxanthine, or dibutyryl cAMP. The effect of forskolin did not reverse unless it was removed. Cycloheximide blocked the recovery of Ca2+/CaM kinase III activity observed following the removal of forskolin but did not affect the ability of forskolin to reduce kinase activity. Short-term treatment with phorbol ester had little effect on Ca2+/CaM kinase III activity; long-term treatment with phorbol ester, which results in the disappearance of enzymatically detectable protein kinase C, had no effect on the ability of NGF or 2-chloroadenosine to reduce Ca2+/CaM kinase III activity. The level of the 100-kDa protein as determined by immunological techniques was not changed by any treatment. These results suggested that the effect of treatment of PC12 cells with NGF or forskolin was to reduce the level of Ca2+/CaM kinase III per se.     

Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme.

The mechanism for the generation of the Ca2+/calmodulin (CaM)-independent activity of calmodulin-dependent protein kinase II (CaM-kinase II) by autophosphorylation was studied by characterizing the autothiophosphorylated enzyme, which is resistant to hydrolysis. When CaM-kinase II was incubated with adenosine 5'-O-(thiotriphosphate) at 5 degrees C, the incorporation of thiophosphate into the enzyme occurred rapidly, reaching a maximum level within a few minutes, in parallel with increase in Ca2+/CaM-independent activity. The maximum level was 1 mol of thiophosphate per mol of subunit of the enzyme, and the thiophosphorylation occurred exclusively at Thr286 in the alpha subunit and Thr287 in the other subunits of the enzyme. These results, taken together, indicate that the autothiophosphorylation of Thr286/Thr287 of each subunit is involved in the generation of the Ca2+/CaM-independent activity. The activity of the autothiophosphorylated enzyme, when assayed in the presence of Ca2+/CaM, showed the same kinetic properties as did the Ca2+/CaM-dependent activity of the original non-phosphorylated enzyme, but when assayed in the absence of Ca2+/CaM, it showed the same Vmax as the Ca2+/CaM-dependent activity but higher Km values for protein substrates. Thus, the phosphorylation of Thr286/Thr287 of the subunit of the enzyme by autophosphorylation appears to not only enhance the affinity of its substrate-binding site for the protein substrate, although it is lower than that of the enzyme activated by the binding of CaM, but also convert the active site to the fully active state.     

Ca(2+)/calmodulin-mediated regulation of the desensitizing process in G(q) protein-coupled histamine H(1) receptor-mediated Ca(2+) responses in human U373 MG astrocytoma cells

We investigated Ca(2+)/calmodulin (CaM)-mediated regulation of the desensitizing process of the histamine H(1) receptor-mediated increase in intracellular Ca(2+) concentration in human U373 MG astrocytoma cells. The desensitizing process was evaluated by measuring the histamine-induced Ca(2+) responses in cells pretreated with histamine for 15 s-30 min under various conditions. Under normal physiological conditions, desensitization developed with three successive phases : a fast desensitization within 15 s, a transient resensitization at 45 s, and a prompt and sustained redesensitization from 1 to 30 min. Similar processes of desensitization/resensitization occurred even under hypertonic conditions, where histamine-mediated internalization of the histamine H(1) receptor is inhibited. The transient resensitization phase was selectively prevented by deprivation of extracellular Ca(2+) and, even more strikingly, by the presence of W-7 (a CaM antagonist). FK506 and cyclosporin A, Ca(2+)/CaM-dependent protein phosphatase (PP2B) inhibitors, mimicked such effects. In the presence of KN-62, a Ca(2+)/CaM-dependent protein kinase II (CaM kinase II) inhibitor, the early development of desensitization disappeared, allowing a slow and simple development of desensitization. The early processes of desensitization and resensitization were unaffected by W-5, okadaic acid, and KN-04 (less potent inhibitors against CaM, PP2B, and CaM kinase II, respectively) or by GF109203X and chelerythrine (protein kinase C inhibitors). The high-affinity site for histamine was converted to a lower-affinity site by histamine treatment, which also showed a transient restoration phase at 45 s in a manner sensitive to KN-62 and FK506. These results provide the first evidence that Ca(2+)/CaM plays a crucial role in determining the early phase of the desensitizing process via activation of CaM kinase II and PP2B, by regulating agonist affinity for histamine H(1) receptors.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

Na+-H+ antiport during fertilization of the sea urchin egg is blocked by W-7 but is insensitive to K252a and H-7

Fertilization of the sea urchin egg initiates or accelerates a number of metabolic activities, which have been causally linked to a rise in cytoplasmic pH due to increased Na+-H+ antiport. Two possible regulatory pathways linking sperm-egg fusion to the activity of the antiporter are activation of protein kinase C (PKC) and Ca2+, calmodulin (CaM)-dependent kinase. This report presents the effects of protein kinase inhibitors on antiporter activation during fertilization and treatment with PKC agonists, dioctanoylglycerol or phorbol diester. Protein kinase inhibitors, K252a and H-7 blocked the action of PKC agonists, without inhibiting cytoplasmic alkalinization during fertilization. In contrast, W-7 blocked fertilization-induced rise in cytoplasmic pH, without altering the actions of PKC agonists. These results suggest that the Na+-H+ antiporter may be regulated by PKC or Ca2+, CaM-dependent kinase activities, but activation of the antiporter during fertilization is Ca2+, CaM-dependent, despite production of diacylglycerols by hydrolysis of phosphatidylinositols.     

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.     

Calcium-dependent interactions of an ionophore A23187 with calmodulin

We found that ionophore A23187 interacted reversibly with calmodulin (CaM), in a calcium-dependent fashion. It was found that A23187 interacts selectively with CaM, among calcium binding proteins (such as troponin C and S-100 protein) and other proteins. However, apparently differing from W-7, A23187 did not suppress CaM-dependent enzyme activity such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase. Our observations suggest that there are novel calcium-dependent regions of CaM which can be monitored using ionophore A23187 and may not be related to enzyme activation.     

Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.     

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.     

Phosphorylation of calcineurin: effect on calmodulin binding

The effect of phosphorylation of calcineurin on calmodulin (CaM) binding was examined using a synthetic peptide which contains the CaM-binding domain and the serine phosphorylation site. The peptide, corresponding to residues 391-414 of brain calcineurin A subunit, was rapidly phosphorylated by protein kinase C and Ca2+/CaM-dependent protein kinase II but not by cAMP-dependent protein kinase. Phosphorylation of peptide 391-414 did not significantly alter the binding of CaM when compared to the non-phosphorylated peptide.     

P2X1-mediated ERK2 activation amplifies the collagen-induced platelet secretion by enhancing myosin light chain kinase activation

The ATP-gated P2X1 ion channel is the only P2X subtype expressed in human platelets. Via transmission electron microscopy, we found that P2X1 mediates fast, reversible platelet shape change, secretory granule centralization, and pseudopodia formation. In washed human platelets, the stable P2X1 agonist alpha,beta-methylene ATP (alpha,beta-meATP) causes rapid, transient (2-5 s), and dose-dependent myosin light chain (MLC) phosphorylation, requiring extracellular Ca2+. Phosphorylation was inhibited by the calmodulin (CaM) inhibitor W-7, but not by the Rho kinase inhibitor HA-1077, i.e. it is exclusively regulated by Ca2+/CaM-dependent MLC kinase. Correspondingly, the P2X1-induced platelet shape change was inhibited by W-7 and by the MLC kinase inhibitor ML-7 but not by HA-1077. W-7, ML-7, the protein kinase C inhibitor GF109203-X, and the Src family kinase inhibitor PP1 inhibited the collagen and convulxin-induced early platelet degranulation, shape change, and subsequent aggregation, indicating a role for Ca2+/CaM and MLC kinase in these glycoprotein VI-related platelet responses. The secreted ATP-mediated P2X1-dependent ERK2 activation induced by low collagen concentrations contributes to MLC kinase activation since P2X1 desensitization or blockade of ERK2 phosphorylation by U0126 strongly attenuated MLC phosphorylation, degranulation, and aggregation. We therefore conclude that at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent degranulation. Rapidly released ATP triggers P2X1 -mediated Ca2+ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation.     

A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin-dependent kinase kinase.

The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+/CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy. In this complex, the CaMKK peptide forms a fold comprising an alpha-helix and a hairpin-like loop whose C-terminus folds back on itself. The binding orientation of this CaMKK peptide by the two CaM domains is opposite to that observed in all other CaM-target complexes determined so far. The N- and C-terminal hydrophobic pockets of Ca2+/CaM anchor Trp 444 and Phe 459 of the CaMKK peptide, respectively. This 14-residue separation between two key hydrophobic groups is also unique among previously determined CaM complexes. The present structure represents a new and distinct class of Ca2+/CaM target recognition that may be shared by other Ca2+/CaM-stimulated proteins.     

Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate

A cDNA clone for the alpha subunit of mouse brain Ca2+/CaM-dependent protein kinase II (CaM-kinase II) was transcribed in vitro and translated in a rabbit reticulocyte lysate system. Inclusion of [35S]methionine in the translation system yielded a single 35S-polypeptide of about 50 kDa. When the translation system was assayed for CaM-kinase II activity, there was a 5-10-fold enrichment of kinase activity which was totally dependent on Ca2+/calmodulin (CaM). Both the 50-kDa 35S-polypeptide and the Ca2+/CaM-dependent protein kinase activity were quantitatively immunoprecipitated by rat brain CaM-kinase II antibody. When the translated wild-type kinase was subjected to autophosphorylation conditions in the presence of Ca2+, CaM, Mg2+, and ATP, the Ca2+-independent activity (assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) increased from 5.8 +/- 0.7 to 26.5 +/- 2.1% of total activity (assayed in the presence of Ca2+/CaM). These properties confirm the identity of the kinase translated in vitro as CaM-kinase II. The role of Thr-286 autophosphorylation in formation of the Ca2+-independent activity was investigated by site-directed mutation of Thr-286 to Ala (Ala-286 kinase) and to Asp (Asp-286 kinase). The Ala-286 kinase was completely dependent on Ca2+/CaM for activity prior and subsequent to autophosphorylation. The Asp-286 kinase exhibited 21.9 +/- 0.8% Ca2+-independent activity, and this was not increased by autophosphorylation. These results establish that introduction of negative charge(s) at residue 286, either by autophosphorylation of Thr or by mutation to Asp, is sufficient and necessary to generate the partially Ca2+-independent form of CaM-kinase II.     

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.