Zinc inhibits calcineurin activity in vitro by competing with nickel

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.     

Structure-activity relationship of ghrelin: pharmacological study of ghrelin peptides

Ni(2+), a toxic and carcinogenic pollutant and one of the leading causes of contact dermatitis, is shown to inhibit neuronal nitric oxide synthase (nNOS) in a competitive, reversible manner with respect to the substrate l-arginine (K(i) = 30 +/- 4 microM). The IC(50) values were dependent on calmodulin (CaM) concentration, but proved independent of Ca(2+), tetrahydrobiopterin (BH(4)) and other essential cofactors. Ni(2+) also inhibited CaM-dependent cytochrome c reduction, NADPH oxidation, and H(2)O(2) production by nNOS. Overall, the action profile of Ni(2+) was suggestive of an unusual, double-acting inhibitor of nNOS affecting l-arginine-binding and Ca(2+)/CaM-dependent enzyme activation.     

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

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

Non-catalytic domains of subunit A negatively regulate the activity of calcineurin

Calcineurin is composed of a catalytic subunit A (CNA) and a regulatory subunit B (CNB). In addition to the catalytic core, CNA further contains three non-catalytic domains--CNB binding domain (BBH), calmodulin binding domain (CBD), and autoinhibitory domain (AI). To investigate the effect of these three domains on the activity of CNA, we have constructed domain deletion mutants CNAa (catalytic domain only), CNAac (CNAa and CBD), and CNAaci (CNAa, CBD and AI). By using p-nitrophenylphosphate and (32)P-labeled R(II) peptide as substrates, we have systematically examined the phosphatase activities, kinetics, and regulatory effects of Mn(2+)/Ni(2+) and Mg(2+). The results show that the catalytic core has the highest activity and the order of activity of the remaining constructs is CNAac>CNAaci>CNA. Sequential removal of the non-catalytic domains corresponds to concurrent increases of the phosphatase activity assayed under several conditions. This observation clearly demonstrates that non-catalytic domains negatively regulate the enzyme activity and act as intra-molecular inhibitors, possibly through restraining the conformation elasticity of the catalytic core required for optimal catalysis or interfering with substrate access. The sequential domain deletion favors activation of the enzyme by Mn(2+)/Ni(2+) but not by Mg(2+) (except for CNAa), suggesting that enzyme activation by Mn(2+)/Ni(2+) is mainly mediated via the catalytic domain, whereas activation by Mg(2+) is via both the catalytic core and non-catalytic domains.     

Insights into the interaction of human liver arginase with tightly and weakly bound manganese ions by chemical modification and site-directed mutagenesis studies

Diethyl pyrocarbonate (DEPC) caused a loss in the ability of inactive subunits of wild-type and H141F mutant human liver arginase (EC 3.5.3.1) to be reactivated by Mn(2+). The effect was reversed by hydroxylamine and involved a residue with a pK(a) of 6.5+/-0.1. Half activation with Mn(2+) was sufficient for total resistance of H141F and full activation was not impeded by a previous incubation of the half-active species with DEPC. The H101N and H126N mutants expressed 60 and 82% of the wild-type activity, respectively, without changes in K(m) for arginine or K(i) for lysine inhibition. After dialysis against EDTA, H126N was inactive in the absence of added Mn(2+) and contained <0.1 Mn(2+)/subunit, whereas H101N was half active and contained 1.2+/-0.1 Mn(2+)/subunit. Results support the concept that a weakly bound metal ion is needed only for conversion of active species to a more active active state.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca(2+)/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca(2+)/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca(2+) chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca(2+)- and/or Ca(2+)/CaM-dependent EGFR regulators, pointing to a direct effect of Ca(2+)/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca(2+)/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.     

Kaempferol: a new immunosuppressant of calcineurin

Calcineurin (CN), the Ca(2+)/calmodulin (CaM)-dependant protein phosphatase, is the target for immunosuppressive drugs cyclosporine A (CsA) and FK506. These immunosuppressants can inhibit CN activity after binding with respective immunophilins. Based on the model of screening by using p-nitrophenyl phosphate as a substrate for preliminary screening and (32)P-labeled 19-residue phosphopeptide as a specific substrate for final determination, we found Kaempferol, a natural flavonol, could inhibit CN activity in purified enzyme and Jurkat T-cells. Unlike CsA and FK506, CN inhibition by kaempferol is independent of matchmaker protein and the inhibitory manner is noncompetitive. Through investigation of inhibitions for CNA and a series of its truncated mutants, we suggested that Kaempferol could directly act on the catalytic domain. Data also indicated that the CN inhibition by kaempferol could be enhanced when the enzyme was activated in the presence of CaM and CNB. CNB is necessary for mediating inhibition of enzyme by kaempferol. The result of RT-PCR also indicated that kaempferol had an inhibitory activity against IL-2 gene expression in activated Jurkat cells. All data suggested that kaempferol could be a new immunosuppressant of CN.     

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation

Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10(-6) M Ca(2+). Motility features changed when Ca(2+) concentrations were increased from 10(-6) to 10(-5) M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca(2+). Thus, it is possible that Ca(2+) binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca(2+)-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by (45)Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca(2+)-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca(2+)/CaM-dependent manner for regulating the dynein activity. A (32)P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca(2+)/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10(-5) M Ca(2+). It is possible that an increase in Ca(2+) induces Ca(2+)/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme.     

Calmodulin kinase II chimeras used to investigate the structural requirements for smooth muscle myosin light chain kinase autoinhibition and calmodulin-dependent activation

Segments of the autoregulatory domain of MK, a catalytically active fragment of the monomeric smooth muscle myosin light chain kinase (smMLCK) (residues 472-972), were replaced with their counterparts from a homologous but multimeric enzyme, calmodulin-dependent protein kinase II (CaM KII). Chimeric proteins in which both the autoregulatory and oligomerization domains of CaM KII (residues 281-478) were substituted for residues 781-972 of smMLCK, MK(CK281-478), or only the autoregulatory domain of CaM KII (residues 281-315) was exchanged for residues 781-813 of smMLCK, MK(CK281-315), exhibited significant enzymatic activity in the absence of Ca(2+)/CaM. In contrast, both MK and a chimeric protein in which the C-terminal half of the autoregulatory domain of smMLCK was replaced with CaM KII residues 301-315, MK(CK301-315), were inactive in the absence of Ca(2+)/CaM. These results indicate that the sequence of the N-terminal half of the autoregulatory domain of smMLCK is important for complete autoinhibition of its enzymatic activity. All proteins bound to Ca(2+)/CaM, and the chimeric proteins MK(CK281-478) and MK(CK281-315) were activated by Ca(2+)/CaM with activation constants (K(CaM)) and maximal enzymatic activities comparable to those of the wild-type MK enzyme. This demonstrates that the entire autoregulatory domain of CaM KII can replace that of smMLCK in its ability to promote efficient CaM-dependent activation of the smMLCK enzyme. However, the inability of the chimeric protein MK(CK301-315) to be activated by Ca(2+)/CaM suggests that replacement of only the C-terminal half of the autoregulatory domain of smMLCK, while still retaining the ability to bind Ca(2+)/CaM, also substitutes residues that prevent activation of the enzyme by Ca(2+)/CaM.     

Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.     

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.     

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

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

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.     

Nitric oxide activation of soluble guanylyl cyclase reveals high and low affinity sites that mediate allosteric inhibition by calcium

Cyclic GMP (cGMP) and Ca(2+) regulate opposing mechanisms in (patho)physiological processes reflected in the reciprocal regulation of their intracellular concentrations. Although mechanisms by which cGMP regulates [Ca(2+)](i) have been described, those by which Ca(2+) regulates [cGMP](i) are less well understood. In the present study, Ca(2+) inhibited purified sGC activated by sodium nitroprusside (SNP), a precursor of nitric oxide (NO), employing Mg-GTP as substrate in a concentration-dependent fashion, but was without effect on basal enzyme activity. Ca(2+) inhibited sGC stimulated by protoporphyrin IX or YC-1 suggesting that inhibition was not NO-dependent. In contrast, Ca(2+) was without effect on sGC activated by SNP employing Mn-GTP as substrate, demonstrating that inhibition did not reflect displacement of heme from sGC. Ligand activation of sGC unmasked negative allosteric sites of high (K(i) similar 10(-7) M) and low (K(i) approximately 10(-5) M) affinity for Ca(2+) that mediated noncompetitive and uncompetitive inhibition, respectively. Free Mg(2+) in excess of substrate did not alter the concentration-response relationship of Ca(2+) inhibition at high affinity sites, but produced a rightward shift in that relationship at low affinity sites. Similarly, Ca(2+) inhibition at high affinity sites was noncompetitive, whereas inhibition at low affinity sites was competitive, with respect to free Mg(2+). Purified sGC specifically bound (45)Ca(2+) in the presence of a 1000-fold excess of Mg(2+) and in the absence of activating ligands. These data suggest that sGC is a constitutive Ca(2+) binding protein whose allosteric function is conditionally dependent upon ligand activation.     

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.