Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The paired helical filaments (PHFs) found in Alzheimer's disease (AD) brains are composed primarily of the microtubule-associated protein tau. PHF-tau is in a hyperphosphorylated state and is unable to promote microtubule assembly. We investigated whether the inhibition of tau binding to microtubules is increased when tau is phosphorylated by different kinases in combination with GSK-3. We found that when tau was first phosphorylated by A-kinase, C-kinase, cdk5, or CaM kinase II and then by GSK-3, its binding to microtubules was inhibited by 45, 61, 78, and 79%, respectively. Further, the kinase combinations cdk5/GSK-3 and CaM kinase II/GSK-3 rapidly phosphorylated the sites Thr 231 and Ser 235. When these sites were individually replaced by Ala and the phosphorylation experiments repeated, tau binding to microtubules was inhibited by 54 and 71%, respectively. By comparison, when Ser 262 was replaced by Ala, tau binding to microtubules was inhibited by only 8% after phosphorylation by CaM kinase II. From these observations we estimate that the phosphorylation of Thr 231, Ser 235, and Ser 262 contributes approximately 26, approximately 9, and approximately 33%, respectively, of the overall inhibition of tau binding to microtubules. Together, our results indicate that the binding of tau to microtubules is controlled by the phosphorylation of several sites, among which are Thr 231, Ser 235, and Ser 262.
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PMID:Phosphorylation of tau at both Thr 231 and Ser 262 is required for maximal inhibition of its binding to microtubules. 973 71

Phosphorylation of neuronal nitric-oxide synthase (nNOS) by Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) including CaM kinase Ialpha (CaM-K Ialpha), CaM kinase IIalpha (CaM-K IIalpha), and CaM kinase IV (CaM-K IV), was studied. It was found that purified recombinant nNOS was phosphorylated by CaM-K Ialpha, CaM-K IIalpha, and CaM-K IV at Ser847 in vitro. Replacement of Ser847 with Ala (S847A) prevented phosphorylation by CaM kinases. Phosphorylated recombinant wild-type nNOS at Ser847 (approximately 0.5 mol of phosphate incorporation into nNOS) exhibited a 30% decrease of Vmax with little change of both the Km for L-arginine and Kact for CaM relative to unphosphorylated enzyme. The activity of mutant S847D was decreased to a level 50-60% as much as the wild-type enzyme. The decreased NOS enzyme activity of phosphorylated nNOS at Ser847 and mutant S847D was partially due to suppression of CaM binding, but not to impairment of dimer formation which is thought to be essential for enzyme activation. Inactive nNOS lacking CaM-binding ability was generated by mutation of Lys732-Lys-Leu to Asp732-Asp-Glu (Watanabe, Y., Hu, Y., and Hidaka, H. (1997) FEBS Lett. 403, 75-78). It was phosphorylated by CaM kinases, as was the wild-type enzyme, indicating that CaM-nNOS binding was not required for the phosphorylation reaction. We developed antibody NP847, which specifically recognize nNOS in its phosphorylated state at Ser847. Using the antibody NP847, we obtained evidence that nNOS is phosphorylated at Ser847 in rat brain. Thus, our results suggest that CaM kinase-induced phosphorylation of nNOS at Ser847 alters the activity control of this enzyme.
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PMID:Regulation of neuronal nitric-oxide synthase by calmodulin kinases. 1040 Jun 90

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.
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PMID:Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins. 1056 54

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.
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PMID:Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. 1061 55

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.
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PMID:Inhibition of neuronal nitric-oxide synthase by calcium/ calmodulin-dependent protein kinase IIalpha through Ser847 phosphorylation in NG108-15 neuronal cells. 1087 31

Stimulation of RBL-2H3 m1 mast cells through the IgE receptor with antigen, or through a G protein-coupled receptor with carbachol, leads to the rapid appearance of phosphothreonine in nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this results from phosphorylation of Thr-1940 by calcium/calmodulin-dependent protein kinase II (CaM kinase II), activated by increased intracellular calcium. The phosphorylation site in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA distal to the coiled-coil region, and identified as Thr-1940 by site-directed mutagenesis. A fusion protein containing the NMHC-IIA carboxyl terminus was phosphorylated by CaM kinase II in vitro, while mutation of Thr-1940 to Ala eliminated phosphorylation. In contrast to rodents, in humans Thr-1940 is replaced by Ala, and human NMHC-IIA fusion protein was not phosphorylated by CaM kinase II unless Ala-1940 was mutated to Thr. Similarly, co-transfected Ala --> Thr-1940 human NMHC-IIA was phosphorylated by activated CaM kinase II in HeLa cells, while wild type was not. In RBL-2H3 m1 cells, inhibition of CaM kinase II decreased Thr-1940 phosphorylation, and inhibited release of the secretory granule marker hexosaminidase in response to carbachol but not to antigen. These data indicate a role for CaM kinase stimulation and resultant threonine phosphorylation of NMHC-IIA in RBL-2H3 m1 cell activation.
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PMID:Calcium-dependent threonine phosphorylation of nonmuscle myosin in stimulated RBL-2H3 mast cells. 1094 86

We investigated the involvement of Ca(2+)-independent activity of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) in stimulation of neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the alpha isoform of CaM kinase II (Nb2a/alpha cells) were stimulated by plating, they changed shape from round to flattened, and began to form neurites within 15 min. Numbers of cells bearing neurites increased from 15 min to about 2 h. Neurite length increased markedly from 30 min to 2 h after stimulation. Ca(2+)-independent activity of CaM kinase II increased immediately after stimulation, peaked at about 30 min, and then gradually decreased. Autophosphorylation of Thr-286 followed the same time course as the increase in Ca(2+)-independent activity. The autophosphorylation and appearance of Ca(2+)-independent activity preceded the formation of neurites. The effect of mutation of the autophosphorylation site in the kinase whose Thr-286 was replaced with Ala (alphaT286A kinase) or Asp (alphaT286D kinase) was examined. alphaT286A kinase was not converted to a Ca(2+)-independent form, and alphaT286D kinase had Ca(2+)-independent activity significantly as an autophosphorylated kinase. Cells expressing alphaT286A kinase did not form neurites, and were indistinguishable from control Nb2a cells. Cells expressing alphaT286D kinase had much longer neurites than Nb2a/alpha cells expressing the wild type kinase, although the initiation of neurite outgrowth was very late. These results indicated that Ca(2+)-independent activity of the kinase autophosphorylated at Thr-286 involves for neurite outgrowth.
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PMID:Ca(2+)-independent activity of Ca(2+)/calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in neuroblastoma cells. 1103 55

Previous studies have demonstrated that vestibular compensation, the process of behavioural recovery which occurs following unilateral deafferentation of the vestibular labyrinth (UVD), is correlated with changes in in vitro phosphorylation of various protein substrates in the brainstem vestibular nucleus complex (VNC). The aim of the present study was to investigate the possible causal relationship between protein kinase activity and the induction of the vestibular compensation process, by delivering inhibitors of protein kinase C (PKC) or Ca(2+)/calmodulin-dependent kinase II (CaMKII) into the ipsilateral VNC at the time of the UVD and determining their effects on three static symptoms of UVD, spontaneous nystagmus (SN), yaw head tilt (YHT) and roll head tilt (RHT) in guinea pigs. Infusion of the PKC inhibitor, 3-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrr ole-2,5-dione, HCl (bisindolylmaleimide I, HCl/GF 109203X, HCl) ('Bis I'), at a concentration of 5 or 50 microM, significantly increased SN frequency at the earliest time points (6 and 8 h post-UVD) compared to vehicle controls and the less selective analogue, 2,3-bis(1H-indol-3-yl)-N-methylmaleimide (bisindolylmaleimide V) ('Bis V'). However, the compensation of YHT and RHT was unaffected by the PKC inhibitor. By contrast, the cell-permeable CaMKII inhibitor, myristoylated autocamtide-2 related inhibitory peptide (N-Myr-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Ala-Val-Asp-Ala-Leu-OH) ('myr-AIP') or the cell-impermeable analogue, autocamtide-2 related inhibitory peptide (N-Lys-Lys-Ala-Leu-Arg-Arg-Cln-Glu-Ala-Val-Asp-Ala-Leu-OH) ('AIP'), failed to alter the compensation of SN, YHT or RHT at any dose compared to vehicle controls. These results implicate PKC-, but not CaMKII-, signal transduction pathways in the initiation of SN compensation in guinea pig.
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PMID:The effects of protein kinase C and calmodulin kinase II inhibitors on vestibular compensation in the guinea pig. 1105 83

Previous studies utilizing inhibitors of the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) to address the role of this enzyme in insulin secretion have produced contradictory results. In the current study, these inconsistencies have been addressed by evaluating the effect of various CaM kinase II inhibitors to decrease Ca(2+)-induced insulin secretion from permeabilized beta-cells. KN-93 (2-[N-(2-hydroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlo rocinnamyl)-N-methylbenzylamine) markedly inhibited both CaM kinase II activation and insulin secretion in parallel in alpha-toxin-permeabilized beta-cells. These effects were specific since they were not mimicked by the inactive analog, KN-92 (2-[N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methy lbenzylamine). In contrast, KN-62 (1-[N, O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine) , while reported to be similar to KN-93 with respect to mechanism of action, did not inhibit Ca(2+)-induced activation of CaM kinase II or insulin secretion in these cell preparations. All three agents suppressed Ca(2+) influx in intact beta-cells induced by depolarization in the presence of elevated extracellular potassium although to different extents. The synthetic peptide inhibitors of CaM kinase II, [Ala(286)]CaMK 281-302 and AIP (autocamtide-2-related inhibitory peptide), strongly inhibited Ca(2+)-induced insulin secretion from electropermeabilized islets, an effect that also correlated with an equivalent inhibition of CaM kinase II activation. This re-evaluation (i) explains a lack of effect of KN-62 on insulin secretion from permeabilized cells based on its inability to inhibit CaM kinase II activation in these preparations; (ii) has revealed that CaM inhibitors, either chemical or peptide in nature, that are capable of preventing enzyme activation uniformly suppress Ca(2+)-sensitive insulin secretion; and (iii) cautions the use of KN-62/93/92 as selective inhibitors of CaM kinase II in intact cell studies. These observations reinforce the suggestion that CaM kinase II plays an important role in insulin exocytosis in the beta-cell.
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PMID:Dependence of insulin secretion from permeabilized pancreatic beta-cells on the activation of Ca(2+)/calmodulin-dependent protein kinase II. A re-evaluation of inhibitor studies. 1107 48

Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) plays important roles in the control of nerve functions in response to intracellular Ca(2+) (for reviews [Annu. Rev. Physiol. 57 (1995) 417-445; Trends Neurosci. 17 (1994) 406-412]). Brief Ca(2+) signals activate CaM kinase II, and stimulate an autophosphorylation of Thr-286 which allows the kinase to maintain its activated state even after the Ca(2+) concentration has returned to basal levels [J. Biol. Chem. 264 (1989) 16759-16763; Neuron 3 (1989) 59-70; J. Biochem. 109 (1991) 137-143]. Autophosphorylation of CaM kinase II occurs in situ, but it occurs relatively quickly, within just a few minutes [Endocrinology 134 (1994) 2245-2250; J. Biol. Chem. 268 (1993) 7863-7867; J. Biol. Chem. 265 (1990) 18055-18058]. In the present study, we investigated the involvement of the autophosphorylated/Ca(2+)-independent form of CaM kinase II in neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the alpha isoform of CaM kinase II (Nb2a/alpha cells) were stimulated by plating, they formed neurites. The autophosphorylation of Thr-286 and appearance of Ca(2+)-independent activity preceded the neurite formation. The effect of mutating of the kinase autophosphorylation site replacing Thr-286 with Ala (alpha T286A kinase) or Asp (alpha T286D kinase) was examined. alpha T286A kinase was not converted to a Ca(2+)-independent form, and alpha T286D kinase had Ca(2+)-independent activity significantly as an autophosphorylated kinase. Cells expressing alpha T286D kinase had much longer neurites than Nb2a/alpha cells, whereas cells with alpha T286A kinase did not form neurites. These results indicated that the Ca(2+)-independent form of CaM kinase II autophosphorylated at Thr-286 is involved in neurite outgrowth.
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PMID:Investigation of the Ca(2+)-independent form of Ca(2+)/calmodulin-dependent protein kinase II in neurite outgrowth. 1173 91


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