Chromosomal localization of the human gene for brain Ca2+/calmodulin-dependent protein kinase type IV

Cloned cDNAs have been identified as corresponding to a new brain Ca2+/calmodulin-dependent protein kinase. On the basis of structural and immunological features, we refer to this new kinase as CaM Kinase IV. Two cDNA clones were used to identify CaM Kinase IV: The downstream clone, lambda ICM-1, contains the sequence encoding the calmodulin-binding site and the second clone, lambda ICM-2, encodes a partial amino acid sequence similar to the catalytic domain of several known protein kinases. Within the calmodulin-binding site a stretch of 8 amino acids (and 9 of 10) is identical to the corresponding site in the subunits of CaM Kinase II. Southern blot analysis shows the CaM Kinase IV gene is single copy in the mouse and human genomes. Synteny analysis of Southern blot data of DNA from hamster--human hybrid cells shows that the gene is present in human chromosome 5. Hybridization of cDNA probes to metaphase spreads of human chromosomes indicates that the gene is most likely located within the region of bands q21 to q23 of chromosome 5.     

Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV

Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly-L-Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase alpha, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 microM CaM was about 10 microg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly-L-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly-L-Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr44. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.     

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.     

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.     

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.     

Regulation of microtubule dynamics by Ca2+/calmodulin-dependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18.

Oncoprotein 18 (Op18; also termed p19, 19K, p18, prosolin, and stathmin) is a regulator of microtubule (MT) dynamics and is phosphorylated by multiple kinase systems on four Ser residues. In addition to cell cycle-regulated phosphorylation, external signals induce phosphorylation of Op18 on Ser-25 by the mitogen-activated protein kinase and on Ser-16 by the Ca2+/calmodulin-dependent kinase IV/Gr (CaMK IV/Gr). Here we show that induced expression of a constitutively active mutant of CaMK IV/Gr results in phosphorylation of Op18 on Ser-16. In parallel, we also observed partial degradation of Op18 and a rapid increase of total cellular MTs. These results suggest a link between CaMK IV/Gr, Op18, and MT dynamics. To explore such a putative link, we optimized a genetic system that allowed conditional coexpression of a series of CaMK IV/Gr and Op18 derivatives. The result shows that CaMK IV/Gr can suppress the MT-regulating activity of Op18 by phosphorylation on Ser-16. In line with these results, by employing a chemical cross-linking protocol, it was shown that phosphorylation of Ser-16 is involved in weakening of the interactions between Op18 and tubulin. Taken together, these data suggest that the mechanism of CaMK IV/Gr-mediated suppression of Op18 activity involves both partial degradation of Op18 and direct modulation of the MT-destabilizing activity of this protein. These results show that Op18 phosphorylation by CaMK IV/Gr may couple alterations of MT dynamics in response to external signals that involve Ca2+.     

Mechanism of the generation of autonomous activity of Ca2+/calmodulin-dependent protein kinase IV

Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is phosphorylated at Thr196 by Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), resulting in induction of both autonomous activity and a high level of Ca2+/CaM-dependent activity. We have shown that the kinetics of Thr196 phosphorylation of CaM-KIV by CaM-KK is well correlated with the generation of its autonomous activity, although Thr177 phosphorylation of CaM-KI does not induce its autonomous activity. The activities of CaM-KI chimera mutants fused with C-terminal regions (residues 296-469 and 296-350) of CaM-KIV are completely dependent on Ca2+/CaM, which is also the case for CaM-KI. Unlike wild-type CaM-KI, however, phosphorylation of Thr177 in the chimera mutants by CaM-KK resulted in generation of significant autonomous activities, indicating that the phosphorylation of Thr in the activation loop is sufficient to partially release the autoinhibitory region of CaM-KIV from the catalytic core. Indeed, the CaM-KIV peptide (residues 304-325) containing minimum autoinhibitory sequences (residues 314-321) suppressed the activity of non-phosphorylated CaM-KIV with an IC50 of approximately 50 microm, and this suppression was competitive with respect to the peptide substrate; however, the CaM-KIV peptide was not capable of inhibiting Thr196-phosphorylated CaM-KIV. Taken together, these results indicated that the Thr196 phosphorylation of CaM-KIV by CaM-KK reduced the interaction of the catalytic core with the autoinhibitory region, resulting in generation of the autonomous activity.     

Molecular cloning of Ca2+/calmodulin-dependent protein kinase phosphatase.

Calmodulin-dependent protein kinase (CaM-kinase) phosphatase dephosphorylates and concomitantly deactivates CaM-kinase II activated upon autophosphorylation, and CaM-kinases IV and I activated upon phosphorylation by CaM-kinase kinase [Ishida, I., Okuno, S., Kitani, T., Kameshita, I., and Fujisawa, H. (1998) Biochem. Biophys. Res. Commun. 253, 159-163], suggesting that CaM-kinase phosphatase plays important roles in the function of Ca2+ in the cell, because the three multifunctional CaM-kinases (CaM-kinases I, II, and IV) are thought to be the key enzymes in the Ca2+-signaling system. In the present study, cDNA for CaM-kinase phosphatase was cloned from a rat brain cDNA library. The coded protein consisted of 450 amino acids with a molecular weight of 49, 165. Western blot analysis showed the ubiquitous tissue distribution of CaM-kinase phosphatase. Immunocytochemical analysis revealed that CaM-kinase phosphatase is evenly distributed outside the nucleus in a cell.     

Nuclear calpain regulates Ca2+-dependent signaling via proteolysis of nuclear Ca2+/calmodulin-dependent protein kinase type IV in cultured neurons

Accumulating evidence indicates that calpains can reside in or translocate to the cell nucleus, but their functions in this compartment remain poorly understood. Dissociated cultures of cerebellar granule cells (GCs) demonstrate improved long-term survival when their growth medium is supplemented with depolarizing agents that stimulate Ca(2+) influx and activate calmodulin-dependent signaling cascades, notably 20 mm KCl. We previously observed Ca(2+)-dependent down-regulation of Ca(2+)/calmodulin-dependent protein kinase (CaMK) type IV, which was attenuated by calpain inhibitors, in GCs supplemented with 20 mm KCl (Tremper-Wells, B., Mathur, A., Beaman-Hall, C. M., and Vallano, M. L. (2002) J. Neurochem. 81, 314-324). CaMKIV is highly enriched in the nucleus and thought to be critical for improved survival. Here, we demonstrate by immunolocalization/confocal microscopy and subcellular fractionation that the regulatory and catalytic subunits of m-calpain are enriched in GC nuclei, including GCs grown in medium containing 5 mm KCl. Calpain-mediated proteolysis of CaMKIV is selective, as several other nuclear and non-nuclear calpain substrates were not degraded under chronic depolarizing culture conditions. Depolarization and Ca(2+)-dependent down-regulation of CaMKIV were associated with significant alterations in other components of the Ca(2+)-CaMKIV signaling cascade: the ratio of phosphorylated to total cAMP response element-binding protein (a downstream CaMKIV substrate) was reduced by approximately 10-fold, and the amount of CaMK kinase (an upstream activator of CaMKIV) protein and mRNA was significantly reduced. We hypothesize that calpain-mediated CaMKIV proteolysis is an autoregulatory feedback response to sustained activation of a Ca(2+)-CaMKIV signaling pathway, resulting from growth of cultures in medium containing 25 mm KCl. This study establishes nuclear m-calpain as a regulator of CaMKIV and associated signaling molecules under conditions of sustained Ca(2+) influx.     

Differential regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase isoforms.

We have previously demonstrated that the alpha isoform of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KKalpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca(2+)/CaM [Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R. (2000) J. Biol. Chem. 275, 20090-20095]. In this study, we find that rat brain extract contains Ca(2+)/CaM-independent CaM-KK activity. This result is consistent with an enhanced Ca(2+)/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KKbeta isoform. By using various truncation mutants of CaM-KKbeta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity. A CaM-KKbeta deletion mutant of this domain shows a significant increase of Ca(2+)/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity. The activities of CaM-KKalpha and CaM-KKbeta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca(2+)/CaM, suggesting that the autoinhibitory regions of CaM-KKalpha and CaM-KKbeta are functional. These results establish for the first time that residues 129-151 of CaM-KKbeta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity.     

Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase

To search for the downstream target protein kinases of Ca (2+)/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKalpha catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5'-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr (189) in the activation loop. Phosphorylation of Thr (189) by CaMKKalpha induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr (189) phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKalpha (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKalpha is capable of activating SAD-B through phosphorylation of Thr (189) both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.     

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.     

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。     

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase

Brain type II Ca²⁺/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca²⁺/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca ²⁺/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg²⁺-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca²⁺/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC₂₀ Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC₁₇ Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid     

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.     

Multifunctional Ca2+/calmodulin-dependent protein kinase made Ca2+ independent for functional studies

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) that is transiently expressed in COS-7 cells is essentially inactive when assayed without Ca2+. Physiological activation of the kinase occurs by binding of Ca2+/calmodulin near a putative autoinhibitory subdomain that contains the sequence His282-Arg-Gln-Glu-Thr286. We have markedly increased the Ca2(+)-independent activity of CaM kinase by altering the charge of this sequence by site-directed mutagenesis. The mutant containing Asp282-Gly-Glu-Glu-Thr286 is 67% Ca2+ independent. We also mimicked the effect of autophosphorylation at Thr286 by the mutant containing His282-Arg-Gln-Glu-Asp286, which is 36% Ca2+ independent. In addition to delineating the autoinhibitory domain by use of mutations that disable it, these constructs are of immediate practical value for simulating CaM kinase action in vivo without elevating Ca2+. To this end, we show that nuclear microinjection of cDNA of a constitutive mutant, but not of the wild-type kinase, initiates maturation of Xenopus oocytes.     

Activation of multifunctional Ca2+/calmodulin-dependent kinase in intact hippocampal slices.

In vitro phosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) converts it to a form that is independent of Ca2+. We demonstrate that significant Ca(2+)-independent CaM kinase activity is present in untreated hippocampal slices. Two manipulations that produce a long-lasting enhancement of neuronal activity in hippocampal slices, elevated extracellular Ca2+ or depolarization with high K+, generate additional Ca(2+)-independent activity. This increase is dependent on extracellular Ca2+ and is correlated with an increased phosphorylation of CaM kinase. In contrast, CaM kinase in posterior pituitary, a brain structure that is not thought to be involved in memory-related processes, is not modulated by depolarization. These results suggest that the Ca(2+)-independent form of CaM kinase may modulate neuronal activity in the hippocampus.