Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.6.4.1 (myosin ATPase)
1,140 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In smooth muscle and specific nonmuscle cells the phosphorylation of the regulatory myosin light chains by myosin light chain kinase (MLCK) is an obligatory step in actin-induced activation of myosin ATPase and subsequent contractile events. We have previously demonstrated that CaM phosphorylated by casein kinase II fails to activate bovine platelet MLCK (Sacks et al. (1992) Biochem. J. 283, 21-24). While myosin light chains are perceived as the only known substrate for MLCK phosphorylation activity, we now show that MLCK phosphorylates CaM. This phosphorylation of CaM is dependent upon the presence of basic peptides such as poly-L-arginine (optimal basic peptide/CaM ratio = 0.08) and is stimulated by saturating [Ca2+] (K0.5 = 16 microM). CaM phosphorylation was inhibited by KT5926, a specific MLCK inhibitor, with a dose-dependency identical to that for inhibition of myosin light chain phosphorylation. Native and MLCK-phosphorylated CaM were indistinguishable in activating MLCK to phosphorylate myosin light chains. Interestingly, MLCK in which the CaM-binding site has been removed is able to phosphorylate CaM in a Ca(2+)-independent manner, suggesting that two CaM molecules bind to intact MLCK simultaneously, one on the inhibitory (pseudosubstrate) domain and one at the catalytic site. CaM phosphorylation by MLCK occurred exclusively on Thr 29 (90%) and Thr 26 (10%) in the first Ca(2+)-binding pocket. In summary, CaM phosphorylation by MLCK differs from CaM phosphorylation catalyzed by other kinases (i.e., the insulin receptor or casein kinase II) in both basic peptide and Ca2+ requirements as well as in the sites of phosphorylation. Further investigations of this model may provide insight into the mechanisms of MLCK activation and substrate recognition.
...
PMID:Phosphorylation of calmodulin in the first calcium-binding pocket by myosin light chain kinase. 880 14

We have designed a series of recombinant peptides derived from the C-terminus of human caldesmon (amino acids 663-793, domain 4) to determine the structural basis of the multiple-sited caldesmon-actin-tropomyosin interaction. All the recombinant peptides are able to bind to actin and inhibit actin-activated myosin ATPase activity; 1 mol of peptide is bound per actin for >90% inhibition. However, equivalent inhibition of actin-tropomyosin activation of myosin ATPase requires less than one peptide per seven actin to be bound. We have found two sequences, H2 (amino acids 683-767) and H2+12 (amino acids 683-779), from the center of domain 4 which potentiate actin-tropomyosin filament activity; i.e., their effect is opposite to caldesmon. Maximum potentiation correlates with one H2 or H2+12 bound per four actin. This effect is completely dependent upon the presence of tropomyosin on the actin filament. H2 and H2+12 also increase actin-tropomyosin filament velocity in the in vitro motility assay. If the H2 sequence is extended by 20 amino acids at the N-terminal end to the N-terminus of domain 4, the peptide becomes an inhibitor. If H2 is extended by 19 amino acids at its C terminus, it becomes a tropomyosin-dependent inhibitor, and with a further extension of 7 amino acids to reach the C-terminus of human caldesmon (H2+26), inhibition is more potent. We conclude that three regions in domain 4 of caldesmon contribute to tropomyosin-dependent inhibition of actomyosin ATPase: a central segment [747-767 (690-710 in the chicken sequence)], which is essential but not sufficient for tropomyosin-dependent inhibition of actomyosin ATPase; and two actin binding segments N-terminal and C-terminal to this segment, 663-682 (606-625) and 770-793 (713-737). If only the central segment is present (H2, H2+12), the actin-tropomyosin-caldesmon peptide complex is not inhibitory, and its properties resemble actin-tropomyosin-caldesmon-Ca2+ x calmodulin.
...
PMID:The inhibitory complex of smooth muscle caldesmon with actin and tropomyosin involves three interacting segments of the C-terminal domain 4. 915 31

We have used isotope-edited nuclear magnetic resonance spectroscopy, binding studies, and ATPase activity assays to investigate the interaction with F-actin of the 10 kDa C-terminal 658C fragment of chicken gizzard caldesmon and two site-directed mutants of this fragment. Simultaneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competition experiments showed that both 658C contacts with actin are displaced by substoichiometric concentrations of the short inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon. Substitution of caldesmon serine-702 by aspartic acid within the spacer region linking the two actin contacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site. Differential binding affinity of the two sites was achieved by replacement of the sequence Glu691-Trp-Leu-Thr-Lys-Thr696 by Pro-Gly-His-Tyr-Asn-Asn. Consistent with these data, the concentration of this Cg1 mutant required to achieve 50% inhibition of actin-tropomyosin-activated myosin ATPase was 4-fold greater than found for the 658C fragment. Although calmodulin binding to Cg1 was observed, calmodulin proved ineffective in relieving the inhibition induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the inhibitory activity possessed by different regions of the C-terminus of caldesmon.
...
PMID:Structure-activity studies of the regulatory interaction of the 10 kilodalton C-terminal fragment of caldesmon with actin and the effect of mutation of caldesmon residues 691-696. 948 78

We have investigated the functional properties of a mutant (Cg1) derived from the C-terminal 99 amino acids of chicken caldesmon, 658-756 (658C) where the sequence 691glu-trp-leu-thr-lys-thr696 is changed to pro-gly-his-tyr-asn-asn. Cg1 bound Ca2+-calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+-calmodulin with the Trp-722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+-calmodulin is not able to reverse the Cg1-induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin-tropomyosin activation of myosin ATPase. The results are interpreted in terms of multisite attachment of actin and Ca2+-calmodulin to overlapping sequences in caldesmon domain 4b.
...
PMID:Characterisation of the effects of mutation of the caldesmon sequence 691glu-trp-leu-thr-lys-thr696 to pro-gly-his-tyr-asn-asn on caldesmon-calmodulin interaction. 950 48

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.
...
PMID:Protein kinase C--catalyzed calponin phosphorylation in swine carotid arterial homogenate. 969 7

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.
...
PMID:Myosin light chain kinase: functional domains and structural motifs. 988 70

We have previously shown that p21-activated kinase, PAK, induces Ca(2+)-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J. E., Arrell, D. K., Foster, D. B., Strauss, J. D., Heinonen, T. Y., Furmaniak-Kazmierczak, E., Cote, G. P., and Mak, A. S. (1998) J. Biol. Chem. 273, 23433-23439). In this study, we provide biochemical evidence implicating a role for PAK in Ca(2+)-independent contraction of smooth muscle via phosphorylation of caldesmon. Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon. PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation. Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin. We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca(2+) sensitivity of smooth muscle contraction.
...
PMID:Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction. 1063 98

Calponin, an F-actin-associated protein implicated in the regulation of smooth muscle contraction, is known to be phosphorylated in vitro by protein kinase C (PKC) and Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II). Unphosphorylated calponin binds to F-actin and inhibits the actin-activated myosin ATPase activity; these properties are lost on phosphorylation. In the present study, we found that Rho-kinase phosphorylated basic calponin stoichiometrically in vitro. We identified the sites of phosphorylation of calponin by Rho-kinase as Thr-170, Ser-175, Thr-180, Thr-184, and Thr-259, and prepared antibodies that specifically recognized calponin phosphorylated at Thr-170 and Thr-184. We showed that the phosphorylation of calponin by Rho-kinase inhibited the binding of calponin to F-actin. Taken together, these results suggest that calponin is a substrate of Rho-kinase and that Rho-kinase regulates the interaction of calponin with F-actin.
...
PMID:Identification of calponin as a novel substrate of Rho-kinase. 1087 72

Airway smooth muscle (ASM) cells in culture stiffen when exposed to contractile agonists. Such cell stiffening may reflect activation of the contractile apparatus as well as polymerization of cytoskeletal biopolymers. Here we have assessed the relative contribution of these mechanisms in cultured ASM cells stimulated with serotonin (5-hydroxytryptamine; 5-HT) in the presence or absence of drugs that inhibit either myosin-based contraction or polymerization of filamentous (F) actin. Magnetic twisting cytometry was used to measure cell stiffness, and associated changes in structural organization of actin cytoskeleton were evaluated by confocal microscopy. We found that 5-HT increased cell stiffness in a dose-dependent fashion and also elicited rapid formation of F-actin as marked by increased intensity of FITC-phalloidin staining in these cells. A calmodulin antagonist (W-7), a myosin light chain kinase inhibitor (ML-7) and a myosin ATPase inhibitor (BDM) each ablated the stiffening response but not the F-actin polymerization induced by 5-HT. Agents that inhibited the formation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, and Y-27632) attenuated both baseline stiffness and the extent of cell stiffening in response to 5-HT. Together, these data suggest that agonist-evoked stiffening of cultured ASM cells requires actin polymerization as well as myosin activation and that neither actin polymerization nor myosin activation by itself is sufficient to account for the cell stiffening response.
...
PMID:Stiffness changes in cultured airway smooth muscle cells. 1217 36

To explore possible mechanisms involving the thin filament-linked regulation of contraction in living smooth muscles, we studied the effects of a synthetic peptide of rabbit cardiac troponin I [residues 136-147] (TnIp), which is a minimal sequence required to inhibit striated muscle acto-tropomyosin-myosin ATPase activity, on the mechanical properties of beta-escin skinned preparations of taenia caeci from guinea pig. TnIp reversibly suppressed the Ca(2+)-activated force without significant effects on the Ca(2+) sensitivity and on the phosphorylation level of myosin regulatory light chain (MLC(20)). TnIp also reversibly suppressed the Ca(2+)/calmodulin-independent contraction induced by 30mM Mg(2+). An analogue of TnIp, which lost inhibiting action on acto-tropomyosin-myosin ATPase activity, affected neither Ca(2+)-activated nor 30mM Mg(2+)-induced contraction. These results indicate that TnIp suppresses the force generation in smooth muscle by directly interfering with cross-bridge formation rather than inhibiting the Ca(2+)/calmodulin-dependent thick and thin filament activating processes.
...
PMID:Troponin I inhibitory peptide suppresses the force generation in smooth muscle by directly interfering with cross-bridge formation. 1285 45


<< Previous 1 2 3 4 5 Next >>

---