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

Calponin is a basic, approximately 34,000 M(r), smooth muscle-specific protein which is developmentally expressed in up to four isoforms. Calponin binds very strongly to actin in a Ca(2+)-independent manner and is localized to the thin filaments in smooth muscle, where it is present at a stoichiometry of 1 mol calponin/7 mol actin. The interaction of calponin with actin inhibits the actomyosin MgATPase (cross-bridge cycling rate) without affecting myosin phosphorylation. The calponin-actin interaction is blocked and calponin-mediated inhibition of the actomyosin MgATPase is reversed upon phosphorylation of calponin by either PKC or CaM kinase II; these properties are restored upon dephosphorylation of calponin by a type 2A protein phosphatase. Consistent with these in vitro findings, calponin is phosphorylated in intact smooth muscle in response to contractile stimuli. The increasing body of evidence, both in vitro and in vivo, strongly supports calponin phosphorylation-dephosphorylation as a thin filament-linked regulatory system in smooth muscle.
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PMID:Calponin: thin filament-linked regulation of smooth muscle contraction. 813 72

A caldesmon kinase activity was detected in an ATP extract of the myofibril-like pellet from sheep aorta. The enzyme was purified 745-fold and was identified as casein kinase II on the basis of molecular size, substrate specificity, and high sensitivity to heparin inhibition. Casein kinase II phosphorylated isolated caldesmon and caldesmon incorporated into native thin filaments, and transferred about 1 mol of phosphate per mol of caldesmon-h. Ser-73 was the main site phosphorylated by casein kinase II in chicken gizzard caldesmon. Phosphorylation of caldesmon reduced its affinity for smooth muscle myosin but had no effect upon the ability of caldesmon to inhibit the ATPase activity of actomyosin.
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PMID:Identification of casein kinase II as a major endogeneous caldesmon kinase in sheep aorta smooth muscle. 822 19

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.
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PMID:Characterization and confocal imaging of calponin in gastrointestinal smooth muscle. 823 86

1. Myosin-V from vertebrate brain is a novel molecular motor with a myosin-like head domain, a calmodulin-binding neck region and a unique tail domain of unknown function. Previous studies showed brain myosin-V to be a phosphoprotein substrate for Ca2+/calmodulin-dependent protein kinase associated with actomyosin. In the present study we describe the preparation of a specific actin-cytoskeletal fraction which is enriched in brain myosin-V. 2. We show that Ca2+/calmodulin-dependent protein kinase activity is also associated with this preparation and phosphorylates brain myosin-V. 3. Calpain, a Ca(2+)-dependent protease, generates a M(r) 80,000 fragment from the COOH terminal region of brain myosin-V containing most or all of the phosphorylation sites. 4. These results suggest that the unique tail domain of this novel myosin is subject to Ca2+ control via phosphorylation by kinase activity associated with the actin cytoskeleton.
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PMID:Ca(2+)-dependent phosphorylation of the tail domain of myosin-V, a calmodulin-binding myosin in vertebrate brain. 825 35

Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.
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PMID:Calponin phosphorylation in vitro and in intact muscle. 828 82

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.
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PMID:Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase. 839 39

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin- dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.
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PMID:Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis. 928 79

Activation of protein kinases plays an important role in the Ca2+-dependent stimulation of insulin secretion by nutrients. The aim of the present study was to identify kinase substrates with the potential to regulate secretion because these have been poorly defined. Nutrient stimulation of the rat insulinoma RINm5F cell line and rat pancreatic islets resulted in an increase in the threonine phosphorylation of a 200-kDa protein. This was secondary to the gating of voltage-dependent Ca2+ channels because it was reproduced by depolarizing KCl concentrations and blocked by the Ca2+ channel antagonist, verapamil. The peak rises in [Ca2+]i preceded or were coincident with the maximal threonine phosphorylation in response to both glyceraldehyde and KCl. In digitonin-permeabilized RINm5F cells a rise in Ca2+ from 0.1 to 0.15 microM was sufficient to increase phosphorylation. Protein kinase C, protein kinase A, and Ca2+/calmodulin-dependent kinase II did not appear to be responsible for the phosphorylation, yet the Ca2+ dependence of the response suggests possible involvement of other members of the Ca2+/calmodulin-dependent kinase family. The 200-kDa protein was identified as myosin heavy chain by immunoprecipitation with a polyclonal nonmuscle myosin antibody. Phosphopeptide mapping indicated that the site of phosphorylation on myosin heavy chain was the same for both KCl- and glyceraldehyde-stimulated cells. Phosphoamino acid analysis confirmed a low basal phosphothreonine content of myosin heavy chain, which increased 6-fold in response to KCl. A lesser (2-fold) increase in serine phosphorylation was also detected using this technique. Although myosin IIA and IIB were shown to be present in RINm5F cells and rat islets, myosin IIA was the predominant threonine-phosphorylated species, suggesting that the two myosin species might be independently regulated. Our results identify myosin heavy chain as a novel kinase substrate in pancreatic beta-cells and suggest that it might play an important role in the regulation of insulin secretion.
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PMID:Nutrient stimulation results in a rapid Ca2+-dependent threonine phosphorylation of myosin heavy chain in rat pancreatic islets and RINm5F cells. 971 4

Ca(2+)-dependent regulation of [3H]noradrenaline ([3H]NA) uptake through the NA transporter was studied using PC12 cells. Preincubation for 10 min in the presence of 0.3-10 mM ca2+ in Krebs-Ringer (KR) buffer induced marked enhancement of the uptake (at 1 mM Ca2+, 6.6 times greater than that observed in the absence of Ca2+), which reflected both an increase in Vmax and a decrease in K(m) of the uptake process. Preincubation with 1 mM Ca2+ also induced a significant increase in the Bmax and Kd of [3H]desipramine binding. The uptake was still enhanced after washing cells with Ca(2+)-free buffer following preincubation with 1 mM Ca2+. 1-[N, O-bis(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-c hlo rocinnamyl) -N-methylbenzylamine (KN-93) (inhibitors of Ca2+/calmodulin-dependent kinase II), N-(6-aminohexyl)-5-chloro-1-naphthalenesulonamide (W-7) (a calmodulin antagonist), wortmannin (a myosin light chain kinase inhibitor) significantly reduced Ca(2+)-dependent enhancement of the uptake. Mycalolide B (an inhibitor of actin-myosin interaction) also inhibited the enhancement. Although calphostin C (a protein kinase C (PKC) inhibitor) did not affect the enhancement, 12-o-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake. A synthetic peptide with a sequence (KKVIYKFFS579 IRGSLW) contained in the intracellular COOH-terminal domain of a rat NA transporter was phosphorylated by purified brain Ca2+/calmodulin-dependent protein kinase II. These results suggest that Ca(2+)-dependent enhancement of the [3H]NA uptake in PC12 cells are mediated by activation of calmodulin-dependent protein kinases, probably through stimulation of translocation of the NA transporter to the plasma membrane and/or direct phosphorylation of the transporter itself.
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PMID:Ca(2+)-dependent enhancement of [3H]noradrenaline uptake in PC12 cells through calmodulin-dependent kinases. 985 6

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca2+/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca2+/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca2+/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca2+/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca2+/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.
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PMID:Myosin light chain kinase: functional domains and structural motifs. 988 70


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