Further characterization and thiophosphorylation of smooth muscle myosin

Malik, Mazhar N.; Fenko, Michael D.; Howard, Rudolph G.; Wı́sniewski, Henryk M.

doi:10.1016/0003-9861(82)90256-9

Cited by 10 publications

(5 citation statements)

References 30 publications

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“…Electrophoresis in denaturing conditions (SDS soft gels) enabled the identification of two [20,29,55,67,130,175,223] isoforms in vascular and nonvascular SM, whose apparent molecular mass is around 200 kD. More over, differences in the distribution of SM myosin can be shown in arterial SM tissue (i.e., human pulmonary artery seems to contain an extra myosin component; table 3) [178].…”

Section: Myhc Isoforms In Sm Tissuesmentioning

confidence: 99%

Myosin Isoform Expression in Smooth Muscle Cells during Physiological and Pathological Vascular Remodeling

et al. 1994

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There is substantial evidence indicating that the study of cytoskeletal and cytocontractile protein composition in vascular smooth muscle cells (SMCs) can be valuable in tracing structural changes during vascular remodeling. Recent nucleic acid and protein investigations suggest that myosin can be used as a new specific marker for the identification of SMC phenotypes in some pathological conditions affecting the vascular wall. In view of this new information, it would seem timely to review the structural bases of myosin isoform expression in the vascular smooth muscle system as well as the factors involved in its regulation. A puzzling feature has arisen in recent studies on this topic: the presence of non-muscle myosin variants in SMCs during physiological and pathological vascular remodeling. In the response to injury caused by mechanical, chemical and hormonal factors in animals, characterized by proliferation and migration of vascular SMCs from the media to the intima, there is a partial or complete recapitulation of a myosin isoform pattern pertinent to developing vascular smooth muscle tissue. Analysis of myosin isoform content in the vascular wall also demonstrates that: (1) changes in SMC composition may occur independent of medial SMC migration into intima, and (2) the presence of fetal-type SMCs in the neointima is not necessarily related to specific positional changes of medial SMCs.

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Section: Myhc Isoforms In Sm Tissuesmentioning

confidence: 99%

Myosin Isoform Expression in Smooth Muscle Cells during Physiological and Pathological Vascular Remodeling

et al. 1994

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“…To rule out the possibility that the differential synapsin I phosphorylation was due to a differential phosphatase activity, perhaps to phosphatase inactivation resulting from protein phosphorylation, [35S]thio-ATP was used to label synapsin 1 by the same methods described above. Previous studies have demonstrated that proteins phosphorylated with [35S]thio-ATP are much more resistant to dephosphorylation by phosphatases than proteins labeled with [ Y -~~P I A T P (Cassel and Glaser, 1982;Malik et al, 1982). Results from experiments with 35S-thiophosphorylated synapsin I were in close agreement with those just described: i.e., the labeling of synapsin I by phosphorylated SJs was much greater than that observed for unphosphorylated SJs at low calmodulin concentrations with the differential effect disappearing at high calmodulin concentrations (data not shown).…”

Section: Effect Of Phosphorylation On Cam-kinase I1 Activitymentioning

confidence: 99%

Autophosphorylation of Calmodulin‐Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity

Shields

Vernon

Kelly

1984

Journal of Neurochemistry

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Previous studies have purified from brain a Ca2+/calmodulin-dependent protein kinase II (designated CaM-kinase II) that phosphorylates synapsin I, a synaptic vesicle-associated phosphoprotein. CaM-kinase II is composed of a major Mr 50K polypeptide and a minor Mr 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin-dependent manner. Recent studies have demonstrated that the 50K component of CaM-kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM-kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [alpha-32P]8-azido-ATP was used to demonstrate that the 60K and 50K polypeptides of SJ-associated CaM-kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin-dependent manner. Experiments using 32P-labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I-calmodulin binding sites. These results suggested that the autophosphorylation of CaM-kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I-calmodulin overlay techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM-kinase II polypeptides bound more calmodulin (50-70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM-kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaM-kinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM-kinase II. The CaM-kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300-1,000 nM). These findings show that the degree of autophosphorylation of CaM-kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.

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“…We found that addition of V i caused a very slow inhibition of active force. However, during the long contractions (>30 min) required to study this phenomenon the force of the muscles was not stable and decayed gradually, probably because of slow dephosphorylation of the thiophosphorylated myosin light chains [14,24]. In order to enable analysis of slow changes in active force, we used the phosphatase inhibitors OA or Microcystin LR.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of force and shortening in smooth muscle by vanadate

Jaworowski

Öztürk

Arner

1999

Pfl�gers Archiv European Journal of Physiology

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We have investigated the effects of vanadate (Vi) on force generation by, and shortening of, chemically skinned smooth muscle preparations from guinea-pig taenia coli at 22 degrees C. A method, using phosphatase inhibitors, was introduced to obtain stable, long-lasting contractions in thiophosphorylated preparations. Vi (10-1000 microM) dose-dependently inhibited active force, to about 20% of its maximum level. At a higher temperature (30 degrees C), the rate of inhibition was faster but the extent of inhibition was less. The rate of contraction following photolytic release of ATP to fibres in rigor was not affected by Vi (30 microM). The maximal shortening velocity (Vmax) was inhibited in a similar manner as active force by Vi (30 microM). In conclusion, the results suggest that Vi interacts with a force-generating actomyosin-ADP (AMADP) state reached after phosphate release. The rate of inhibition of smooth muscle contraction was markedly lower than in skeletal muscle, suggesting differences either in properties of the Vi-bound states or, more likely, in the concentration of AMADP states capable of binding Vi. This suggests that the long duty cycle in smooth muscle is not associated with a higher relative population of AMADP states reached immediately after Pi release, but rather by an increase in the population of subsequent force-generating cross-bridge states. The Vi-bound cross-bridges introduce an internal load to shortening, possibly acting in a similar manner as cross-bridge states introduced at low levels of activation.

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Further characterization and thiophosphorylation of smooth muscle myosin

Cited by 10 publications

References 30 publications

Myosin Isoform Expression in Smooth Muscle Cells during Physiological and Pathological Vascular Remodeling

Myosin Isoform Expression in Smooth Muscle Cells during Physiological and Pathological Vascular Remodeling

Autophosphorylation of Calmodulin‐Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity

Inhibition of force and shortening in smooth muscle by vanadate

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