A phosphorylated light-chain component of myosin

Perrie, W. T.; Smillie, L. B.; Perry, S. V.

doi:10.1042/bj1280105p

Cited by 39 publications

(16 citation statements)

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“…Therefore, it is of great interest to understand how myosin and actin function in fibroblasts. Recent work by Perrie et al (17) and by Stull et al (18) has shown that contractile proteins may be phosphorylated. Perrie et al reported a light chain of myosin to be phosphorylated, and Stull et al reported a similar finding with troponin I.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Adelstein

Conti

Johnson

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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Myosin has been isolated from cloned mouse fibroblasts, line L-929. Fibroblast myosin: (i) binds to rabbit muscle actin and is dissociated from it by ATP, (ii) has an ATPase activity that is suppressed by Mg2+ in 0.6 M KCI and is activated by rabbit muscle actin in the presence of Mgg+ in 14 mM KCI, (iii) forms thin bi. polar aggregates in 0.1 M KCI when viewed in the electron microscope, (iv) possesses a heavy chain with the same mobility as muscle myosin (molecular weight 200,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these respects, fibroblast myosin appears to be similar to muscle myosin in structure and function.Since the work of Ishikawa et al. (1) on the presence of actinlike filaments in fibroblasts, the possible existence of a myosin counterpart in these cells has been raised. Recently, Yang and Perdue (2) succeeded in isolating and purifying actin from tertiary cultures of chick embryo cells and found it similar to muscle actin in its molecular properties. Furthermore, the the work of Bray (3) has indicated that cytoplasmic actin can be isolated from several different embryonic sources.cAMP has a marked effect on the motility, adhesion, morphology, and growth of fibroblasts grown in vitro (4-9). It is possible that some of the effects of cAMP could be mediated through one or more of the "contractile" proteins (actin, myosin, and troponin-tropomyosin). To explore this hypothesis, we began by attempting to isolate a myosin-like protein from cloned fibroblasts. Using a modification of a technique developed for the isolation of platelet myosin (10), we isolated myosin from cloned mouse fibroblasts grown in vitro. This protein has been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, ATPase assay with and without muscle actin, actin binding, and electron microscopy. MATERIALS AND METHODSCloned mouse (L-929) fibroblasts were grown either as monolayers or in spinner bottles. The cells were scraped off the sides of the bottles or sedimented from the suspended culture. They were washed three times in ten volumes of 0.9% NaCl-0.3% sodium citrate-5 mM dithiothreitol (S2threitol)-1 mM EDTA, and sedimented each time at 13,000 X g for 15 min.

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Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Adelstein

Conti

Johnson

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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“…According to this hypothesis, membrane protein phosphorylation would be able to regulate the arrangement of cell surface proteins by modulating membrane protein-cytoskeletal interactions. Various proteins that bind to actin have been shown to be phosphorylated (16)(17)(18)(19)(20)(21). Most interesting has been data that show that phosphorylation of nonmuscle and smooth muscle myosin changes the activity of actomyosin ATPase (24) and that phosphorylation of spectrin changes its interaction with actin in vtitro (25).…”

Section: Discussionmentioning

confidence: 99%

“…Glycophorin A, which has a transmembrane arrangement similar to that proposed for HLA-A and -B antigens, is phosphorylated in its intracellular carboxy terminal region, though only to a small extent (15). Most interesting are the observations that a number of proteins that interact with actin, including myosin (16), troponin (17,18), tropomyosin (19), filamin (20), and spectrin (21), are phosphorylated. In skeletal muscle, myosin phosphorylation fluctuates with muscle contraction (22,23), and in smooth muscle and nonmuscle cells, myosin phosphorylation alters actomyosin ATPase (24).…”

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confidence: 99%

Phosphorylation in vivo and in vitro of human histocompatibility antigens (HLA-A and HLA-B) in the carboxy-terminal intracellular domain.

Pober¹,

Guild²,

Strominger³

1978

Proc. Natl. Acad. Sci. U.S.A.

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HLA-A and -B antigens are phosphorylated in transformed lymphoblastoid cells and peripheral blood lymphocytes, both incubated with 32Pi. The phosphate group is attached to HLA-A and -B heavy chain (p44) as identified by immunoprecipitation with anti -microglobulin IgG, sodium dodecyl sulfate/polyacrlamide gel electrophoresis, isoelectric focusing, and susceptibility to limited proteolysis by papain and trypsin. The site(s) of phosphorylation is identified as a serine residue(s) located in the hyophilic carboxy terminus of the p44 chain. HLA antigens are a so phosphorylated in isolated membranes from transformed lymphoblastoid cells that are incubated with ['yzP IAThThe phosphorylation of the carboxy terminus of HLA-A and -B antigens im vivo is good evidence that this portion of the molecule is intracellular. Furthermore, this modification suggests a general way in which interactions between membrane proteins and cytoskeletal elements may be regulated.The means by which cells arrange their surface proteins is a fundamental problem in cell biology and is believed to involve interactions between membrane proteins and cytoplasmic elements (1, 2). Histocompatibility antigens are cell surface proteins whose movement in the plasma membrane appears to be coordinated with the movement of intracellular cytoskeletal elements (3, 4), suggesting an association. More is known about the structure of human histocompatibility antigens (HLA-A and -B) than about the structure of any other plasma membrane protein of a nucleated cell, so these molecules offer the best opportunity to study the structural basis of putative membrane protein-cytoplasmic protein interactions. Recently evidence has been presented for a direct association between mouse histocompatibility antigens (H2-K and -D) and actin (5). Similar associations involving HLA-A and -B antigens may also occur.HLA-A and -B antigens, when purified in detergent solutions, are composed of a 44,000-dalton glycoprotein heavy chain (p44) and a 12,000-dalton light chain (p12), j32-microglobulin. A complex between the amino terminus of the p44 chain and the p12 chain forms an extracellular domain that may be liberated by papain from the cell surface as a p34,12 soluble antigen (6). The carboxy terminus of the p44 chain contains both a hydrophilic stretch of 32 amino acid residues and a penultimate sequence of 25 amino acid residues that is markedly hydrophobic (7,8). It has been proposed that the hydrophobic sequence of HLA-A and -B spans the lipid bilayer of the membrane and that the hydrophilic carboxy-terminal sequence is located intracellularly (7,8). A simlar structure has been demonstrated for human glycophorin A (9, 10). This intracellular carboxy-terminal domain of HLA-A and -B would thus be able to interact with cytoplasmic elements. Walsh and Crumpton (11) have presented evidence that HLA-A and -B antigens do span the plasma membrane, but they did not identify the intracellular portion of the chain.Recently, Robb et-al. (8) have determined the amino acid sequence ...

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“…Alkaline urea polyacrylamide gels were used to measure the TnC content of the myofibrils as previously described (Blanchard et al, 1984). The gels were run according to a modified method of Perrie et al (1973) and contained 7.5% acrylamide (0.8% bis-acrylamide), 8 M urea, 25 mM Tris (pH 8.3), and 192 mM glycine. Gel samples were dialyzed against 1 liter of 8 mM urea and 25 mM Tris (pH 8.3) for 5 hours.…”

Section: Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH.

Blanchard¹,

Solaro²

1984

Circulation Research

168

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SUMMARY. The aim of experiments described here was to test whether deactivation of cardiac myofibrils in acidic pH is associated with decreases in amounts of calcium bound to myofilament troponin. We determined the amounts of myofibrillar bound calcium attributable to troponin, from measurements of calcium binding to myofibrils and to myosin and from determination of the troponin C content of the myofibrillar preparations (0.40 nmol troponin C/mg protein). In measurements done at 2 mvi free magnesium, 2 imi (magnesium-adenosine triphosphate, ionic strength 0.12, 22°C, the pCa 50 (-log of the half maximally activating molar free calcium) for myofibrillar magnesium-adenosine triphosphatase activity was 5.87 at pH 7.0, 5.49 at pH 6.5, and 5.04 at pH 6.2. This change in calcium sensitivity of myofibrillar magnesium-adenosine triphosphatase activity was present whether or not ethyleneglycol-bis(/3-aminoethyl ether)-N,N'-tetraacetic acid, was used to buffer the free calcium and whether or not myofibrillar troponin I had been phosphorylated by cyclic adenosine 3',5'-monophosphate-dependent protein kinase. However, the change in pCa 50 of myofibrillar adenosine triphosphatase activity induced by acidic pH, was greater when free magnesium was reduced from 2.0 to 0.05 HIM, and less when free magnesium was increased from 2.0 mM to 10 and 15 mM. The change in pCa 50 with acidic pH was less if the ionic strength was reduced from 0.12 to 0.035 M. The magnesium-adenosine triphosphatase activity of troponin/tropomyosin-free myofibrils was independent of pCa and unaffected by a reduction of pH from 7.0 to 6.5. The affinity of myofibrillar troponin C for calcium decreased as pH was reduced from 7.0 to 6.5 and to 6.2 with and without ethyleneglycolbis(/?-aminoethyl ether)-N,7V'-tetraacetic acid, and in a manner predicted from the effect of acidic pH on pCa 50 for myofibrillar activation. Our results are consistent with the idea that at least part of the mechanism responsible for deactivation of the adenosine triphosphatase activity of cardiac myofilaments in acidic pH is a reduction in the affinity of myofibrillar troponin C for calcium. (Circ Res 55: 382-391, 1984)

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A phosphorylated light-chain component of myosin

Cited by 39 publications

References 4 publications

Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Phosphorylation in vivo and in vitro of human histocompatibility antigens (HLA-A and HLA-B) in the carboxy-terminal intracellular domain.

Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH.

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