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Characterization of Ca2+/calmodulin-dependent protein kinase II from smooth muscle.

ABSTRACT: We have characterized chicken gizzard smooth muscle Ca2+/calmodulin-dependent protein kinase II (CaM-PKII) with particular focus on its autophosphorylation. The autophosphorylation of smooth muscle CaMPKII produced a partially constitutively active enzyme, as occurs with the alpha- and beta-isoforms of this enzyme, but the autophosphorylation kinetics were significantly slower. Phosphorylation during the initial rapid phase coincided with the production of constitutively active enzyme. The phosphorylation was on both serine and threonine residues, which is distinct from the brain enzyme where threonine phosphorylation is much faster and more prevalent than serine phosphorylation. The major autophosphorylation sites identified were different from the known autophosphorylation sites of the alpha- and beta-isoforms. During the initial autophosphorylation phase Ser-319, Ser-352 and a Thr residue within residues 345-368 were found to be phosphorylated. During the subsequent gradual phase two serine residues in the variable region and Ser-280 were phosphorylated, but Thr-286 and Thr-305, which are the known major autophosphorylation sites for the alpha- and beta-isoforms, were not detected as the major autophosphorylation sites of smooth muscle CaMPKII. By comparing the phosphopeptide sequence with the known sequences of various isoforms, we concluded that isoform gamma-b, which contains a unique insertion and two deletions at the C-terminal side of the calmodulin binding domain, is the dominant CaMPKII isoform in smooth muscle. The molecular mass of smooth muscle CaMPKII was estimated to be 240 kDa which would comprise four subunits, fewer than in the alpha- and beta-isoforms. The results show that smooth muscle CaMPKII is functionally distinct from the alpha- and beta-isoforms of this enzyme, which might be crucial for its physiological relevance.

SUBMITTER: Zhou ZH

PROVIDER: S-EPMC1135925 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Project description:OBJECTIVE:The main objective of this study is to define the mechanisms by which mitochondria control vascular smooth muscle cell (VSMC) migration and impact neointimal hyperplasia. APPROACH AND RESULTS:The multifunctional CaMKII (Ca2+/calmodulin-dependent kinase II) in the mitochondrial matrix of VSMC drove a feed-forward circuit with the mitochondrial Ca2+ uniporter (MCU) to promote matrix Ca2+ influx. MCU was necessary for the activation of mitochondrial CaMKII (mtCaMKII), whereas mtCaMKII phosphorylated MCU at the regulatory site S92 that promotes Ca2+ entry. mtCaMKII was necessary and sufficient for platelet-derived growth factor-induced mitochondrial Ca2+ uptake. This effect was dependent on MCU. mtCaMKII and MCU inhibition abrogated VSMC migration and mitochondrial translocation to the leading edge. Overexpression of wild-type MCU, but not MCU S92A, mutant in MCU-/- VSMC rescued migration and mitochondrial mobility. Inhibition of microtubule, but not of actin assembly, blocked mitochondrial mobility. The outer mitochondrial membrane GTPase Miro-1 promotes mitochondrial mobility via microtubule transport but arrests it in subcellular domains of high Ca2+ concentrations. In Miro-1-/- VSMC, mitochondrial mobility and VSMC migration were abolished, and overexpression of mtCaMKII or a CaMKII inhibitory peptide in mitochondria (mtCaMKIIN) had no effect. Consistently, inhibition of mtCaMKII increased and prolonged cytosolic Ca2+ transients. mtCaMKII inhibition diminished phosphorylation of focal adhesion kinase and myosin light chain, leading to reduced focal adhesion turnover and cytoskeletal remodeling. In a transgenic model of selective mitochondrial CaMKII inhibition in VSMC, neointimal hyperplasia was significantly reduced after vascular injury. CONCLUSIONS:These findings identify mitochondrial CaMKII as a key regulator of mitochondrial Ca2+ uptake via MCU, thereby controlling mitochondrial translocation and VSMC migration after vascular injury.

Project description:During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII), are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic models of postsynaptic signal transduction during learning.

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Characterization of Ca2+/calmodulin-dependent protein kinase II from smooth muscle.

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