New isoforms of Ca2+/calmodulin-dependent protein kinase II in smooth muscle.
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ABSTRACT:
SUBMITTER: Zhou ZL
PROVIDER: S-EPMC1138298 | biostudies-other | 1994 Apr
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC1135925 | biostudies-other
Project description:Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions.
| S-EPMC4750416 | biostudies-literature
Project description:Ca(2+)/calmodulin-dependent protein kinase kinase ? (CaMKK?) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKK? phosphorylates Thr(172) in the AMPK? subunit more efficiently than CaMKK?, with a lower Km (?2 ?m) for AMPK, whereas the CaMKI? phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKK?/CaMKK? chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKK?/Ile(322) in CaMKK? confer, at least in part, a distinct recognition of AMPK but not of CaMKI?.
| S-EPMC4919462 | biostudies-literature
Project description:Ca(v)1 (L-type) channels and calmodulin-dependent protein kinase II (CaMKII) are key regulators of Ca(2+) signaling in neurons. CaMKII directly potentiates the activity of Ca(v)1.2 and Ca(v)1.3 channels, but the underlying molecular mechanisms are incompletely understood. Here, we report that the CaMKII-associated protein densin is required for Ca(2+)-dependent facilitation of Ca(v)1.3 channels. While neither CaMKII nor densin independently affects Ca(v)1.3 properties in transfected HEK293T cells, the two together augment Ca(v)1.3 Ca(2+) currents during repetitive, but not sustained, depolarizing stimuli. Facilitation requires Ca(2+), CaMKII activation, and its association with densin, as well as densin binding to the Ca(v)1.3 alpha(1) subunit C-terminal domain. Ca(v)1.3 channels and densin are targeted to dendritic spines in neurons and form a complex with CaMKII in the brain. Our results demonstrate a novel mechanism for Ca(2+)-dependent facilitation that may intensify postsynaptic Ca(2+) signals during high-frequency stimulation.
| S-EPMC2990970 | biostudies-literature
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.
| S-EPMC5970052 | biostudies-literature
Project description:Calcium/calmodulin-dependent protein kinase II (CaMKII) is highly concentrated in the brain where its activation by the Ca2+ sensor CaM, multivalent structure, and complex autoregulatory features make it an ideal translator of Ca2+ signals created by different patterns of neuronal activity. We provide direct evidence that graded levels of kinase activity and extent of T287 (T286? isoform) autophosphorylation drive changes in catalytic output and substrate selectivity. The catalytic domains of CaMKII phosphorylate purified PSDs much more effectively when tethered together in the holoenzyme versus individual subunits. Using multisubstrate SPOT arrays, high-affinity substrates are preferentially phosphorylated with limited subunit activity per holoenzyme, whereas multiple subunits or maximal subunit activation is required for intermediate- and low-affinity, weak substrates, respectively. Using a monomeric form of CaMKII to control T287 autophosphorylation, we demonstrate that increased Ca2+/CaM-dependent activity for all substrates tested, with the extent of weak, low-affinity substrate phosphorylation governed by the extent of T287 autophosphorylation. Our data suggest T287 autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.
| S-EPMC5748111 | biostudies-literature
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.
| S-EPMC2820514 | biostudies-literature
Project description:AimsCalmodulin (CaM) regulates Na+ channel gating through binding to an IQ-like motif in the C-terminus. Ca2+/CaM-dependent protein kinase II (CaMKII) regulates Ca2+ handling, and chronic overactivity of CaMKII is associated with left ventricular hypertrophy and dysfunction and lethal arrhythmias. However, the acute effects of Ca2+/CaM and CaMKII on cardiac Na+ channels are not fully understood.Methods and resultsPurified Na(V)1.5-glutathione-S-transferase fusion peptides were phosphorylated in vitro by CaMKII predominantly on the I-II linker. Whole-cell voltage-clamp was used to measure Na+ current (I(Na)) in isolated guinea-pig ventricular myocytes in the absence or presence of CaM or CaMKII in the pipette solution. CaMKII shifted the voltage dependence of Na+ channel availability by approximately +5 mV, hastened recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased persistent (late) current, but did not change I(Na) decay. These CaMKII-induced changes of Na+ channel gating were completely abolished by a specific CaMKII inhibitor, autocamtide-2-related inhibitory peptide (AIP). Ca2+/CaM alone reproduced the CaMKII-induced changes of I(Na) availability and the fraction of channels undergoing slow inactivation, but did not alter recovery from inactivation or the magnitude of the late current. Furthermore, the CaM-induced changes were also completely abolished by AIP. On the other hand, cAMP-dependent protein kinase A inhibitors did not abolish the CaM/CaMKII-induced alterations of I(Na) function.ConclusionCa2+/CaM and CaMKII have distinct effects on the inactivation phenotype of cardiac Na+ channels. The differences are consistent with CaM-independent effects of CaMKII on cardiac Na+ channel gating.
| S-EPMC2802203 | biostudies-literature
Project description:Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, we designed mutant CaMs that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the "closed" Ca2+-free conformation. We have measured their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation. We show that CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. Our results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways.
| S-EPMC1599897 | biostudies-literature
Project description:Catecholamines increase heart rate by augmenting the cAMP-responsive hyperpolarization-activated cyclic nucleotide-gated channel 4 pacemaker current (I(f)) and by promoting inward Na(+)/Ca(2+) exchanger current (I(NCX)) by a "Ca(2+) clock" mechanism in sinoatrial nodal cells (SANCs). The importance, identity, and function of signals that connect I(f) and Ca(2+) clock mechanisms are uncertain and controversial, but the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to ?-adrenergic receptor (?-AR) stimulation. The aim of this study was to measure the contribution of the Ca(2+) clock and CaMKII to cardiac pacing independent of ?-AR agonist stimulation.We used the L-type Ca(2+) channel agonist Bay K8644 (BayK) to activate the SANC Ca(2+) clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from wild-type control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs but increased L-type Ca(2+) current (I(Ca)) equally in all SANCs, indicating that increasing I(Ca) was insufficient and suggesting that CaMKII activation was required for heart rate increases by BayK. BayK did not increase I(f) or protein kinase A-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca(2+) by BayK did not augment cAMP/protein kinase A signaling at these targets. Late-diastolic intracellular Ca(2+) release and I(NCX) were significantly reduced in AC3-I SANCs, and the response to BayK was eliminated by ryanodine in all groups.The Ca(2+) clock is capable of supporting physiological fight-or-flight responses, independent of ?-AR stimulation or I(f) increases. Complete Ca(2+) clock and ?-AR stimulation responses require CaMKII.
| S-EPMC3116039 | biostudies-literature