Rebekah S. Vest

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca(2+) signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca(2+)-stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T305. However, T286 autophosphorylation (the autophosphorylation generating autonomous activity) was only mildly affected. Two mechanisms can explain this unusual differential inhibitor effect. First, CNs inhibited activity by interacting with the CaMKII T-site (and thereby also interfered with NMDA-type glutamate receptor binding to the T-site). Because of this, the CaMKII region surrounding T286 competed with CNs for T-site interaction, whereas other substrates did not. Second, the intersubunit T286 autophosphorylation requires CaM binding both to the "kinase" and the "substrate" subunit. CNs dramatically decreased CaM dissociation, thus facilitating the ability of CaM to make T286 accessible for phosphorylation. Tat-fusion made CN21 cell penetrating, as demonstrated by a strong inhibition of filopodia motility in neurons and insulin secrection from isolated Langerhans' islets. These results reveal the inhibitory mechanism of CaM-KIIN and establish a powerful new tool for dissecting CaMKII function.

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling involved in higher brain functions. Here, we show CaMKII involvement in pathological glutamate signaling relevant in stroke. The novel inhibitor tatCN21 was neuroprotective even when added hours after glutamate insults. By contrast, the "traditional" inhibitor KN93 attenuated excitotoxicity only when present during the insult. Both inhibitors efficiently blocked Ca(2+)/CaM-stimulated CaMKII activity, CaMKII interaction with NR2B and aggregation of CaMKII holoenzymes. However, only tatCN21 but not KN93 blocked the Ca(2+)-independent "autonomous" activity generated by Thr-286 autophosphorylation, the hallmark feature of CaMKII regulation. Mutational analysis further validated autonomous CaMKII activity as the drug target crucial for post-insult neuroprotection. Overexpression of CaMKII wild type but not the autonomy-deficient T286A mutant significantly increased glutamate-induced neuronal death. Maybe most importantly, tatCN21 also significantly reduced infarct size in a mouse stroke model (middle cerebral arterial occlusion) when injected (1 mg/kg intravenously) 1 h after onset of arterial occlusion. Together, these data demonstrate that inhibition of autonomous CaMKII activity provides a promising therapeutic avenue for post-insult neuro-protection after stroke.

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A2 (sPLA2). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA2. Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A2 was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA2is a consequence of increased order of membrane lipids. Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A2 (sPLA2). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA2. Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A2 was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA2is a consequence of increased order of membrane lipids. secretory phospholipase A2 balanced salt solution monomeric aspartate 49 phospholipase A2 from the venom of A. piscivorus piscivorus acrylodan-labeled fatty acid-binding protein 6-dodecanoyl-2-dimethylaminonaphthalene (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide generalized polarization methyl arachidonyl fluorophosphonate E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one Under normal conditions, healthy cell membranes resist catalysis by secretory phospholipase A2(sPLA2)1 (1Fourcade O. Simon M.-F. Viode C. Rugani N. Leballe F. Ragab A. Fournie B. Sarda L. Chap H. Cell. 1995; 80: 919-927Abstract Full Text PDF PubMed Scopus (492) Google Scholar, 2Kudo I. Murakami M. Hara S. Inoue K. Biochim. 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Full Text PDF PubMed Scopus Google Scholar). were with the in for hydrolysis was to the the of the in the were with for an and a was to of in a microscopy were an to and were In some were to the of the with laurdan for and laurdan was by were in of and to the microscopy the to were was to the and were was and the of changes in laurdan fluorescence was by of of the In that from the as the in the from a of hydrolysis among some of the and were by of by for the of was was not possible to in the of increased the of that by of by the were with the with ionomycin using for that were by the of with of the of and in was in the were significant the was to In the of was and the were as significant to of on the of ionomycin to induce of were in the of the the in of from that to ionomycin phenylhydrazine to that that from ionomycin by of by the of both under and were from of for the of the and incubation of merocyanine on susceptibility to sPLA2. of erythrocyte phospholipids by was in the of incubation to as in and of for of hydrolysis with merocyanine was from the by of were by for and they were in were to the of an by for the were for using with the of as the to were with the in these cases, a was to the as to the the of the for the of of intracellular phospholipase A2 does not the of ionomycin to induce susceptibility. were for in the of under the for the thin-layer chromatography was and the incubation for of was to a cell and susceptibility was using as under was by the for to that with ionomycin and by as under in the of fatty acid release from erythrocyte membranes in the of was enhanced by a incubation of the with ionomycin. among in in was by in the revealed that and the for ionomycin were to the cell of the Experiments in the of incubation with ionomycin was revealed that the effect a of and a In to with H.A. Waldrip J.B. Nielson K.H. Judd A.M. Han S.K. Cho W. Sims P.J. Bell J.D. J. Biol. Chem. 1999; 274: 11494-11504Abstract Full Text Full Text PDF PubMed Scopus (38) Google the hydrolysis was not to the and no was the of the In order to the of hydrolysis of the were cell in the of Under such conditions, the of of the effect of ionomycin was such that the for hydrolysis was release of in the of ionomycin was chromatography hydrolysis of erythrocytes in the of ionomycin revealed no significant hydrolysis of phosphatidylcholine phosphatidylethanolamine a In both were erythrocytes were with ionomycin were with ionomycin in the of J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google R.W. J. Biol. Chem. 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