Stimulation of Ca(2+)/calmodulin-dependent protein kinase phosphatase by polycations

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly-L-lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101-109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn(2+). Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.     

Transcriptional regulation of nuclear orphan receptor,NOR-1, by Ca(2+)/calmodulin-dependent protein kinase cascade

It has been proposed that the REV1 protein plays an important role in the induced-mutagenesis pathway. We show that purified REV1 protein inserts dCMP opposite template G, A, T and C, and dGMP and dTMP opposite template G in the presence of magnesium, while in the presence of manganese the specificity for dCMP was found to be relaxed and the REV1 protein acquired the ability to insert dCMP, dGMP, dAMP and dTMP opposite templates G, A, T, and C. Kinetic analysis provided evidence for high affinity for dCTP with template G, suggesting that the REV1 protein is specialized for dCTP and template G.     

Auto-inhibition of Ca(2+)/calmodulin-dependent protein kinase II by its ATP-binding domain

Ca(2+)/calmodulin dependent protein kinase (CaMPK) II is a key enzyme in many physiological processes. The enzyme is inactive unless Ca(2+)/CaM binds to it. In this inactive form CaMPK-II does not bind ATP suggesting that the ATP-binding domain is involved in an intramolecular interaction. We show here that F12, a 12 amino acid long peptide fragment of the ATP-binding domain (CaMPK-II(23-34), GAFSVVRRCVKV) can inhibit the Ca(2+)/CaM-dependent activity (IC(50) of 3 microM) but has no effect on the Ca(2+)/CaM-independent activity of CaMPK-II. Kinetic analysis exhibited mixed inhibition with respect to autocamtide-2 and ATP. The inhibition by F12 showed specificity towards CaMPK-II, but also inhibited CaMPK-I (IC(50) = 12.5 microM), while CaMPK-IV (IC(50) = 85 microM) was inhibited poorly and cAMP-dependent protein kinase (PKA) was not inhibited. Substitution of phenylalanine at position 25 to alanine (A12), had little effect on the inhibition of different Ca(2+)/CaM-dependent protein kinases, suggesting that phenylalanine 25 does not play a crucial role in the interactions involving F12. Thus the molecular interactions involving the ATP-binding domain appears to play a role in the regulation of nonphosphorylated CaMPK-II activity.     

Studies on the phosphorylation of protein kinase B by Ca(2+)/calmodulin-dependent protein kinases

Protein kinase B (PKB) was recently reported to be activated on the phosphorylation of Thr(308) by Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaM-kinase kinase alpha), suggesting that PKB was regulated through not only the phosphoinositide 3-kinase pathway but also the Ca(2+)/calmodulin protein kinase pathway. The activation of PKB by CaM-kinase kinase alpha was as high as 300-fold after incubation for 30 min under the phosphorylation conditions, and still increased thereafter, suggesting that the maximal activation of PKB on phosphorylation of the Thr(308) residue is several hundred fold. On the other hand, the V(max) value of CaM-kinase kinase alpha for the phosphorylation of PKB was more than two orders of magnitude lower than that for CaM-kinase IV, although the K(m) values for PKB and CaM-kinase IV were not significantly different, raising the question of whether or not PKB is a physiological substrate of CaM-kinase kinase alpha. Besides CaM-kinase kinase alpha, CaM-kinase II also remarkably activated PKB. However, the specific activities of CaM-kinase kinase alpha and CaM-kinase II as to the activation of PKB were more than three orders of magnitude lower than that of 3-phosphoinositide-dependent protein kinase 1 (PDK1).     

A possible role for Ca(2+)/calmodulin-dependent protein kinase IV during pancreatic acinar stimulus-secretion coupling

Ca(2+)/calmodulin-dependent protein kinases (CaMKs) are important intracellular mediators in the mediation of stimulus-secretion coupling and excitation-contraction coupling in a wide variety of cell types. We attempted to identify and characterize the functional roles of CaMK in mediating pancreatic enzyme secretion. Immunoprecipitation and immunoblotting studies using a CaMKII or CaMKIV antibody showed that rat pancreatic acini expressed both CaMKII and CaMKIV. Phosphotransferase activities of CaMKs were measured by a radioenzyme assay (REA) using autocamtide II, peptide gamma and myosin P-light chain as substrates. Although CaMKII and CaMKIV use autocamtide II as a substrate, peptide gamma is more efficiently phosphorylated by CaMKIV than by CaMKII. Intact acini were stimulated with cholecystokinin (CCK)-8, carbachol (CCh) and the high-affinity CCK-A receptor agonist, CCK-OPE, and the cell lysates were used for REA. CCK-8, CCh and CCK-OPE caused a concentration-dependent increase in CaMKs activities. When autocamtide II was used, maximal increases were 1.5-1.8-fold over basal (20.2+/-2.0 pmol/min/mg protein), with peaks occurring at 20 min after cell stimulation. In separate studies that used peptide gamma, CCK-8, CCh and CCK-OPE dose-dependently increased CaMKIV activities. Maximal increases were 1.5-2.4-fold over basal (30.7+/-3. 2 pmol/min/mg protein) with peaks occurring at 20 min after cell stimulation. Peak increases after cell stimulation induced by peptide gamma were 1.8-2.8-fold higher than those induced by autocamtide II. CCK-8, CCh and CCK-OPE also significantly increased phosphotransferase activities of myosin light chain kinase (MLCK) substrate (basal: 4.4+/-0.7 pmol/min/mg protein). However, maximal increases induced by MLCK substrate were less than 10% of those occurring in peptide gamma. Characteristics of the phosphotransferase activity were also different between autocamtide II and peptide gamma. When autocamtide II was used, elimination of medium Ca(2+) in either cell lysates or intact cells resulted in a significant decrease in the activity, whereas it had no or little effect when peptide gamma was used. This suggests that Ca(2+) influx from the extracellular space is not fully required for CaMKIV activity and Ca(2+) is not a prerequisite for phosphotransferase activity once CaMKIV is activated by either intracellular Ca(2+) release or intracellular Ca(2+) oscillations. The specific CaMKII inhibitor KN-62 (50 microM) had no effect on the CaMKIV activity and pancreatic enzyme secretion elicited by CCK-8, CCh and CCK-OPE. The specific MLCK inhibitor, ML-9 (10 microM), also did not inhibit CCK-8-stimulated pancreatic amylase secretion. In contrast, wide spectrum CaMK inhibitors, K-252a (1 microM) and KT5926 (3 microM), significantly inhibited CaMKIV activities and enzyme secretion evoked by secretagogues. Thus, CaMKIV appears to be an important intracellular mediator during stimulus-secretion coupling of rat pancreatic acinar cells.     

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.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: II. Mutational analysis

We previously reported that rat brain Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem. Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase alpha by PKA and identified six phosphorylation sites, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA. In the present study, a causal relationship between the phosphorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The following conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr(108) or Ser(458) causes a decrease in the activity; (iii) the inhibitory effect of the phosphorylation of Thr(108) is canceled by the stimulatory effect of the phosphorylation, but that of Ser(458) is not; and (iv) the inhibitory effects of Thr(108) and Ser(458) are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr(108), but not significantly affected by the phosphorylation of Ser(458).     

Mutational analysis of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.     

Expression of Ca(2+)/calmodulin-dependent protein kinase IV (caMKIV) messenger RNA during murine embryogenesis.

Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric, multifunctional serine/threonine protein kinase that is expressed in subanatomic regions of the central and peripheral nervous system, T lymphocytes, and male germ cells. It is frequently localized to the nucleus, where it serves as a mediator of Ca(2+)-dependent gene expression. Although CaMKIV expression in the adult rat central nervous system and thymus has been described, little is known about the embryonic expression of murine CaMKIV. Here we report a thorough embryonic expression study of CaMKIV mRNA from embryonic day 9.5 through postnatal day 1. Expression patterns during embryonic development are significantly different from those of adults, suggesting specific roles for CaMKIV during development. Regions of high CaMKIV mRNA expression include thymic and bone cartilage primordia as well as specific cranial nerve ganglia (trigeminal, vestibulocochlear, and glossopharyngeal), thalamus, and dorsal root ganglia. This pattern of expression is chronologically consistent with periods of extensive cellular differentiation, proliferation, or neuronal survival selection and shows a predilection for neural crest-derived cells. These trends, along with recent studies in the CaMKIV null mouse, suggest that CaMKIV may play an important physiological role in cellular differentiation during embryogenesis.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Dual effect of ATP in the activation mechanism of brain Ca(2+)/calmodulin-dependent protein kinase II by Ca(2+)/calmodulin.

The activation mechanism of Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys(75)-labeled TA-cal [T?r?k, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820] is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin [Drum et al. (2000) J. Biol. Chem. 275, 36334-36340] (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca(2+)/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr(286)-phospho-alphaCaMKII as well as with alphaCaMKII-derived calmodulin-binding peptides in the presence of Ca(2+). These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to alphaCaMKII with estimated interprobe distances of 40 and 22-26 A. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr(286)-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca(2+)/TA-cal with alphaCaMKII is estimated as 2 x 10(7) M(-1) s(-1) and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca(2+)/DA-cal is markedly slower, occurring with a rate constant of 2.5 x 10(6) M(-1) s(-1), suggesting that unproductive complexes may play a role in the activation mechanism.     

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.     

STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase

STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) was synthesized, and its inhibitory properties were investigated both in vitro and in vivo. STO-609 inhibits the activities of recombinant CaM-KK alpha and CaM-KK beta isoforms, with K(i) values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activities. Comparison of the inhibitory potency of the compound against various protein kinases revealed that STO-609 is highly selective for CaM-KK without any significant effect on the downstream CaM kinases (CaM-KI and -IV), and the IC(50) value of the compound against CaM-KII is approximately 10 microg/ml. STO-609 inhibits constitutively active CaM-KK alpha (glutathione S-transferase (GST)-CaM-KK-(84-434)) as well as the wild-type enzyme. Kinetic analysis indicates that the compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 suppresses the Ca(2+)-induced activation of CaM-KIV in a dose-dependent manner. In agreement with this observation, the inhibitor significantly reduces the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 microg/ml (approximately 80% inhibitory rate). Taken together, these results indicate that STO-609 is a selective and cell-permeable inhibitor of CaM-KK and that it may be a useful tool for evaluating the physiological significance of the CaM-KK-mediated pathway in vivo as well as in vitro.     

Ca(2+)/calmodulin-dependent protein kinase IV is expressed in spermatids and targeted to chromatin and the nuclear matrix

Ca(2+)/calmodulin-dependent protein kinase IV and calspermin are two proteins encoded by the Camk4 gene. Both are highly expressed in the testis, where in situ hybridization studies in rat testes have demonstrated that CaMKIV mRNA is localized to pachytene spermatocytes, while calspermin mRNA is restricted to spermatids. We have examined the expression patterns of both CaMKIV and calspermin in mouse testis and unexpectedly find that CaMKIV is expressed in spermatogonia and spermatids but excluded from spermatocytes, while calspermin is found only in spermatids. CaMKIV and calspermin expression in the testis are stage-dependent and appear to be coordinately regulated. In germ cells, we find that CaMKIV is associated with the chromatin. We further demonstrate that a fraction of CaMKIV in spermatids is hyperphosphorylated and specifically localized to the nuclear matrix. These novel findings may implicate CaMKIV in chromatin remodeling during nuclear condensation of spermatids.     

Bistability in the Ca(2+)/calmodulin-dependent protein kinase-phosphatase system

A mathematical model is presented of autophosphorylation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) and its dephosphorylation by a phosphatase. If the total concentration of CaMKII subunits is significantly higher than the phosphatase Michaelis constant, two stable steady states of the CaMKII autophosphorylation can exist in a Ca(2+) concentration range from below the resting value of the intracellular [Ca(2+)] to the threshold concentration for induction of long-term potentiation (LTP). Bistability is a robust phenomenon, it occurs over a wide range of parameters of the model. Ca(2+) transients that switch CaMKII from the low-phosphorylated state to the high-phosphorylated one are in the same range of amplitudes and frequencies as the Ca(2+) transients that induce LTP. These results show that the CaMKII-phosphatase bistability may play an important role in long-term synaptic modifications. They also suggest a plausible explanation for the very high concentrations of CaMKII found in postsynaptic densities of cerebral neurons.     

Ca(2+)/calmodulin-dependent protein kinase II is associated with pelvic pain of neurogenic cystitis

Interstitial cystitis/painful bladder syndrome is a chronic bladder inflammatory disease of unknown etiology that is often regarded as a neurogenic cystitis. Interstitial cystitis is associated with urothelial lesions, voiding dysfunction, and pain in the pelvic/perineal area. In this study, we used a murine neurogenic cystitis model to identify genes participating in the development of pelvic pain. Neurogenic cystitis was induced by the injection of Bartha's strain of pseudorabies virus (PRV) into the abductor caudalis dorsalis (tail base) muscle of female C57BL/6J mice. Mice infected with PRV developed progressive pelvic pain. The sacral spinal cord was harvested on postinfection days (PID) 2 and 4, and gene expression was analyzed by microarrays and confirmed by quantitative RT-PCR. On PID 2, the overall expression profile was similar to that of uninfected sacral spinal cord; by PID 4, there were substantial differences in expression of multiple functional classes of genes, especially inflammation. Analysis of pain-signaling pathways at the dorsal horn suggested that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) contributes to neurogenic cystitis pelvic pain. Consistent with this, CaMKIIδ expression exhibited a mast cell-dependent increase in the sacral spinal cord at the mRNA level, and phospho-CaMKII immunoreactivity in the dorsal horn was increased on postinfection day (PID) 4 during PRV infection. Finally, intrathecal injection of the CaMKII inhibitor KN-93 attenuated the PRV pain response. These data suggest that CaMKII plays a functional role in pelvic pain due to neurogenic cystitis.     

Depolarizing stimuli reduce Ca(2+)/calmodulin-dependent protein kinase II activity in islets of Langerhans

Elevations in intracellular Ca(2+) ([Ca(2+)](i)) initiate insulin secretion from pancreatic beta-cells, but the secretory responses become rapidly desensitised to maintained elevations in [Ca(2+)](i). We have investigated the mechanisms underlying the Ca(2+) desensitization of insulin secretion using electrically permeabilized rat islets of Langerhans. Measurements of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) enzyme activity and immunoreactivity in permeabilized islets demonstrated Ca(2+)-induced reductions in enzyme activity which could not be attributed to reductions in CaMK II immunoreactive protein. Measurements in intact islets demonstrated that the Ca(2+)-induced reduction of CaMK II activity was also operative in intact cells, suggesting that this mechanism may have pathophysiological implications for beta-cell function.     

Dynamics of Ca(2+)/calmodulin-dependent protein kinase II following acute myocardial ischemia-translocation and autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca(2+) concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.     

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.     

Cloning of a Ca(2+)/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides

A Ca(2+)/calmodulin-dependent protein kinase (CaMK) gene was cloned and characterized from Arthrobotrys dactyloides, a nematode-trapping fungus. The resulting 373-amino-acid protein, FCaMK, has significant homology to mammalian CaMKs. FCaMK contains a serine/threonine kinase domain followed by a calmodulin-binding domain. The activation loop in FCaMK (amino acids 184-199) contains a phosphorylation site at threonine-188, which could be the target of a kinase activator. Truncated FCaMK mutants revealed that amino acids 296-324 are essential for calmodulin binding. An oligopeptide designed from residues 297-324 formed a stable peptide-calmodulin complex of 1:1 stoichiometry. Southern blot analysis detected a single copy of the fcamk gene, suggesting that FCaMK plays an important role in Ca(2+)/calmodulin signaling in A. dactyloides.