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.     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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).     

Identification of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II in the postsynaptic density by protein sequencing and mass spectrometry.

Previously we detected more than 28 PSD proteins to be phosphorylated by CaM kinase II, and identified 14 protein substrates (Yoshimura, Y., Aoi, T., Yamauchi, T., Mol. Brain Res. 81, 118-128, 2000). In the present study, the remaining substrates were analyzed by protein sequencing and mass spectrometry. We found 6 proteins not previously known to be substrates of CaM kinase II, namely PSD95-associated protein, SAP97, TOAD-64, TNF receptor-associated protein, insulin-receptor tyrosine kinase 58/53 kDa substrate, and homer 1b.     

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.     

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.     

Investigation of neuronal cell type-specific gene expression of Ca2+/calmodulin-dependent protein kinase II

The promoter activity of the rat Ca²⁺/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5′-flanking region of α and β isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5′-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types. Published: April 12, 2002