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.     

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。     

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.     

Multifunctional Ca2+/calmodulin-dependent protein kinase

Multifunctional Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) is a prominent mediator of neurotransmitters which elevate Ca²⁺. It coordinates cellular responses to external stimuli by phosphorylating proteins involved in neurotransmitter synthesis, neurotransmitter release, carbohydrate metabolism, ion flux and neuronal plasticity. Structure/function studies of CaM kinase have provided insights into how it decodes Ca²⁺ signals. The kinase is kept relatively inactive in its basal state by the presence of an autoinhibitory domain. Binding of Ca²⁺/calmodulin eliminates this inhibitory constraint and allows the kinase to phosphorylate its substrates, as well as itself. This autophosphorylation significantly slows dissociation of calmodulin, thereby trapping calmodulin even when Ca²⁺ levels are subthreshold. The kinase may respond particularly wel to multiple Ca²⁺ spikes since trapping may enable a spike frequency-dependent recruitment of calmodulin with each successive Ca²⁺ spike leading to increased activation of the kinase. Once calmodulin dissociates, CaM kinase remains partially active until it is dephosphorylated, providing for an additional period in which its response to brief Ca²⁺ transients is potentiated.Special issue dedicated to Dr. Paul Greengard.     

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.     

Regulation of Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II

The 63-kDa subunit, but not the 60-kDa subunit, of brain calmodulin-dependent cyclic nucleotide phosphodiesterase was phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II. When calmodulin was bound to the phosphodiesterase, 1.33 +/- 0.20 mol of phosphate was incorporated per mol of the 63-kDa subunit within 5 min with no significant effect on enzyme activity. Phosphorylation in the presence of low concentrations of calmodulin resulted in a phosphorylation stoichiometry of 2.11 +/- 0.21 and increased about 6-fold the concentration of calmodulin necessary for half-maximal activation of the phosphodiesterase. Peptide mapping analyses of complete tryptic digests of the 63-kDa subunit revealed two major (P1, P4) and two minor (P2, P3) 32P-peptides. Calmodulin-binding to the phosphodiesterase almost completely inhibited phosphorylation of P1 and P2 with reduced phosphorylation rates of P3 and P4, suggesting the affinity change of the enzyme for calmodulin may be caused by phosphorylation of P1 and/or P2. When Ca2+/calmodulin-dependent protein kinase II was added without prior autophosphorylation, there was no phosphorylation of the 63-kDa phosphodiesterase subunit or of the kinase itself in the presence of a low concentration of calmodulin, and with excess calmodulin the phosphodiesterase subunit was phosphorylated only at P3 and P4. Thus the 63-kDa subunit of phosphodiesterase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II and blocked by Ca2+/calmodulin binding to the subunit.     

Regulation of Ca2+/calmodulin-dependent protein kinase II by brain gangliosides

Purified rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 microM. Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 microM) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis. CaMK 281-309 strongly inhibited kinase activity (IC50 = 0.2 microM). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

Regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase

Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of alphaCaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca(2+)/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile(441) is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu(440), exhibited enhanced Ca(2+)/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile(441) remained Ca(2+)/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile(441) for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp(444)) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.     

Tyrosine kinase activity of a Ca2+/calmodulin-dependent protein kinase II catalytic fragment

A 30-kDa fragment of Ca²⁺/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX₄₀-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca²⁺/calmodulin-dependent protein kinase I (CX₄₀) were fused to the N-terminal end of 30K-CaMKII. Although CX₄₀-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX₄₀ region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.     

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.     

Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation

A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.     

A novel glutamate-mediated inhibitory mechanism linked with Ca2+/calmodulin-dependent protein kinase II in identified Euhadra neurons

The underlying mechanism(s) of the glutamate (Glu)-induced membrane hyperpolarizing response in identified Euhadra neurons was investigated using the voltage-clamp technique, pressure injection method, and pharmacologic agents. Under voltage-clamp conditions, bath-applied Glu elicits a slow outward potassium current (Glu current) accompanied by an increase in membrane conductance whose amplitude is dose dependent. Of the agonists tested, the Glu current was mimicked only by quisqualate (QA); its potency was approximately 10 times greater than that of Glu. Typical antagonists for the ionotropic type of Glu receptors and G protein inhibitors do not block this current. The Glu current is markedly enhanced by a specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII), KN-62 (1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) in a dose-dependent manner, while intracellularly injected CaM-KII suppresses the current. The potent protein kinase A inhibitors, H-8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) and H-89 (N-[2-(p - bromocinnamylamino)ethyl] - 5 - isoquinolinesulfonamide) or the specific protein kinase C inhibitors staurosporine and K-252b had no effect on the Glu current. These results suggest the presence of a novel subtype of Glu receptor in Euhadra neurons, which may be coupled to the activation of potassium channels normally suppressed by CaM-KII. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 139–149, 1997.     

Regulation of Kv4.3 currents by Ca2+/calmodulin-dependent protein kinase II

The voltage-dependent K+ channel 4.3 (Kv4.3) is one of the major molecular correlates encoding a class of rapidly inactivating K+ currents, including the transient outward current in the heart (Ito) and A currents (IA) in neuronal and smooth muscle preparations. Recent studies have shown that Ito in human atrial myocytes and IA in murine colonic myocytes are modulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII); however, the molecular target of CaMKII in these studies has not been elucidated. We performed experiments to investigate whether CaMKII could regulate Kv4.3 currents directly. Inclusion of the autothiophosphorylated form of CaMKII in the patch pipette (10 nM) prolonged Kv4.3 currents such that the time required to reach 50% inactivation from peak more than doubled, with positive shifts in voltage dependence of both activation and inactivation. In contrast, the rate of recovery from inactivation was accelerated under these conditions. CaMKII-inhibitory peptide or KN-93 produced effects opposite to that above; thus the rate of inactivation was increased, and recovery from inactivation decreased. A number of mutagenesis experiments were conducted on the three candidate CaMKII consensus sequence sites on the channel. Mutations at S550A, located at the COOH-terminal region of the channel, resulted in currents that inactivated more rapidly but recovered from inactivation at a slower rate than that of wild-type controls. In addition, these currents were unaffected by dialysis with either autothiophosphorylated CaMKII or the specific inhibitory peptide of CaMKII, suggesting that CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of Kv4.3 currents by a direct effect at S550A, located at the COOH-terminal region of the channel.     

Pressure overload selectively up-regulates Ca2+/calmodulin-dependent protein kinase II in vivo

Signals transduced by the multifunctional calcium/calmodulin-dependent protein kinases (CaMKs), have been suggested to regulate the development of hypertrophy. We address the role of the three multifunctional CaMKs, CaMK I, II, and IV, in this process using transverse aortic constriction (TAC) to induce cardiac hypertrophy in mice. We find a 33% increase in total CaMK activity 7 d after TAC. However, there are no changes in the levels of CaMKI, which is expressed in the ventricles, or CaMKIV, which is not detectable in the ventricles. Moreover, mice null for the CaMKIV gene develop ventricular hypertrophy and induce the expression of selected hypertrophy marker mRNAs, indicating that CaMKIV is not required at any time during the development of hypertrophy. On the other hand, TAC does increase both mRNA and protein levels of specific isoforms of CaMKII derived from both gamma and delta genes. Included among these isoforms are those that localize to both cytoplasm and nucleus. Collectively, the increased levels of CaMKII isoforms result in a constitutive increase in the Ca(2+)/calmodulin-independent activity of CaMKII in the ventricles. We conclude that CaMKII is the multifunctional CaMK most likely to mediate Ca(2+)- dependent protein phosphorylation events in response to TAC-induced cardiac hypertrophy.     

Functional analysis of Ca2+/calmodulin-dependent protein kinase II expressed in bacteria

The cDNA encoding the 50-kDa subunit of Ca2+/calmodulin (CaM)-dependent protein kinase II from adult rat brain was cloned into the bacterial expression vector pK223-2 and produced in bacteria. Extensive modification of the cDNA was required to express detectable levels of enzyme. The activity of the bacterially expressed kinase was stringently dependent on Ca2+/CaM but did not exhibit cooperative activation kinetics characteristic of the forebrain enzyme and required 10-fold greater amounts of CaM for half-maximal activation. The bacterially expressed enzyme displayed an apparent Km for a synthetic peptide substrate similar to that of the forebrain enzyme (12 and 10 microM, respectively). Limited proteolysis maps of autophosphorylated peptides, and Western blot analysis demonstrated that the bacterially expressed enzyme was structurally and immunologically indistinguishable from the 50-kDa subunit of the rat forebrain holoenzyme. The bacterially expressed enzyme became Ca2+/CaM-independent after Ca2+/CaM-dependent autophosphorylation in a fashion identical to the forebrain enzyme.