Myosin phosphorylation in intact platelets.

Daniel, James L.; Molish, Ilean R.; Holmsen, Holm

doi:10.1016/s0021-9258(19)68991-x

Cited by 142 publications

(12 citation statements)

References 33 publications

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“…Myosin phosphorylation is closely correlated with shape change induced by thrombin, ADP, or cold (Daniel et al, 1981(Daniel et al, , 1984Nachmias et al, 1985). In the present experiments this correlation was only sustained if the platelets were stimulated within 20 s after being exposed to PR6.…”

Section: Discussionsupporting

confidence: 50%

“…Additional Tris was added to bring the samples to alkaline pH. The method was that of Daniel et al (1981).…”

Section: Gel Analysismentioning

confidence: 99%

“…Recently it has been shown that when platelets are treated with the agonists ADP or thrombin, phosphorylation of myosin regulatory light chain occurs rapidly (Daniel et al, 1981;Fox and Phillips, 1982), and shape change occurs in correlation with the phosphorylation (Daniel et al, 1984). When platelets are chilled, shape change occurs much more slowly, but again it is in correlation with the phosphorylation of the regulatory light chain (Nachmias et al, 1985).…”

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confidence: 99%

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Lowering pH in blood platelets dissociates myosin phosphorylation from shape change and myosin association with the cytoskeleton.

Nachmias

Yoshida

Glennon

1987

The Journal of Cell Biology

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Abstract. Platelet shape change induced by ADP is relatively independent of external pH over the range 6-7. If the chloride ion in the buffer is replaced by weak acids, however, shape change is rapidly and reversibly inhibited as a function of lowered pH (92% at pH 6.0). This inhibition is correlated with lowered internal pH caused by the weak acids, as measured by the 5,5-dimethyloxazolidine 2,4-dione technique. Shape change was 50% inhibited at internal pH 6.4 when 50 mM NaCI was replaced by propionate (PR). When platelets were stimulated with ADP 10-20 s after addition of PR to a final pH of 6 (PR6), both myosin light chain (MLC) phosphorylation and myosin and actin association with the cytoskeleton were reduced in correlation with the inhibition of shape change. But when ADP was added 30 s after PR6, the MLC phosphorylation was essentially the same in PR or in chloride, although shape change and myosin and actin association with the cytoskeleton remained inhibited. This was shown to be due mainly to endogenous phosphorylation of MLC. On return to neutral pH, platelets in PR immediately changed shape and myosin and actin became associated with the cytoskeleton. Twodimensional tryptic peptides of MLC showed two major spots after PR6 treatment, indicating that both the MLC kinase site and the protein kinase C sites were phosphorylated.The results show that increased internal pH is not required for shape change, although it may affect the rate. In PR6, as after phorbol esters, MLC phosphorylation can be uncoupled from shape change. The association of myosin and actin with the cytoskeleton is closely correlated with shape change, suggesting that shape change requires the active interaction of these contractile proteins.

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Section: Discussionsupporting

confidence: 50%

“…Additional Tris was added to bring the samples to alkaline pH. The method was that of Daniel et al (1981).…”

Section: Gel Analysismentioning

confidence: 99%

mentioning

confidence: 99%

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Lowering pH in blood platelets dissociates myosin phosphorylation from shape change and myosin association with the cytoskeleton.

Nachmias

Yoshida

Glennon

1987

The Journal of Cell Biology

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“…Phosphorylation of the myosin light chains is thought to be one of the primary steps in the activation of contractile events in many types of cells, including blood platelets (Adelstein, 1982;Scholey et al, 1980). Stimulusdependent phosphorylation of myosin in platelets is correlated with its polymerization and association with actin filaments (Lebowitz & Cooke, 1979;Daniel et al, 1981;Cox et al, 1984). Myosin light-chain kinase is found in platelets and is activated in vitro by calcium and calmodulin (Scholey et al, 1980), and in intact platelets, calcium ionophore stimulates the phosphorylation of myosin light chains, Mr 20000 (Kaibuchi et al, 1983).…”

Section: Introductionmentioning

confidence: 99%

Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets

Hallam¹,

Daniel²,

Kendrick‐Jones³

et al. 1985

Biochemical Journal

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Human platelets were prepared and loaded with the fluorescent Ca2+ indicator quin2. The relation between cytoplasmic free calcium concentration, [Ca2+]i, and the extent of the phosphorylation of myosin light chains of Mr 20 000 could then be examined. When the calcium ionophore ionomycin is used to stimulate platelets, little phosphorylation is seen until [Ca2+]i exceeds 400 nM; half-maximal response occurs at 600 nM with a full response at about 1 microM-[Ca2+]i. Under optimal conditions, physiological stimuli such as platelet-activating factor and thrombin can increase [Ca2+]i to sufficiently high levels [Rink, Smith & Tsien (1982) FEBS Lett. 148, 21-26; Hallam, Sanchez & Rink (1984) Biochem. J. 218, 819-827] that Ca2+ ions could be the trigger for the myosin phosphorylation evoked by these agonists. However, in this paper we show that, in the absence of external calcium, platelet-activating factor and thrombin can stimulate myosin phosphorylation while [Ca2+]i remains at levels which are well below those needed when the calcium ionophore is the stimulus. This observation suggests that myosin light chain phosphorylation may be controlled by an additional pathway.

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“…Furthermore, this phosphorylation is coupled to the initiation of contraction (Hartshorne and Siemankowski, 1981). Phosphorylation of non-muscle myosin RMLC has been implicated in the regulation of platelet aggregation (Daniel et al, 1981(Daniel et al, , 1984, and capping in lymphocytes (Bourguignon et al, 1981). In Dictyostelium discoideum, myosin filament assembly is regulated by phosphorylation of the heavy chain (Kuczmarski and Spudich, 1980;Kuczmarski et al, 1987;Egelhoff et al, 1993).…”

mentioning

confidence: 99%

Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development.

Chen

Ostrow

Tafuri

et al. 1994

The Journal of Cell Biology

101

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Abstract. Conventional myosin has two different light chains bound to the neck region of the molecule. It has been suggested that the light chains contribute to myosin function by providing structural support to the neck region, therefore amplifying the conformational changes in the head following ATP hydrolysis (Raymerit et al., 1993). The regulatory light chain is also believed to be important in regulating the actinactivated ATPase and myosin motor function as assayed by an in vitro motility assay (Griffith et al., 1987). Despite extensive in vitro biochemical study, little is known regarding RMLC function and its regulatory role in vivo. To better understand the importance and contribution of RMLC in vivo, we engineered Dictyostelium cell lines with a disrupted RMLC gene. Homologous recombination between the introduced gene disruption vector and the chromosomal RMLC locus (mlcR) resulted in disruption of the RMLC-coding region, leading to cells devoid of both the RMLC transcript and the 18-kD RMLC polypeptide. RMLC-deficient cells failed to divide in suspension, becoming large and multinucleate, and could not complete development following starvation. These results, similar to those from myosin heavy chain mutants (DeLozanne et al., 1987;Manstein et al., 1989), suggest the RMLC subunit is required for normal cytokinesis and cell motility. In contrast to the myosin heavy chain mutants, however, the mlcR cells are able to cap cell surface receptors following concanavilin A treatment. By immunofluorescence microscopy, RMLC null cells exhibited myosin localization patterns different from that of wild-type cells. The myosin localization in RMLC null cells also varied depending upon whether the cells were cultured in suspension or on a solid substrate. In vitro, purified RMLC-myosin assembled to form thick filaments comparable to wildtype myosin, but the filaments then exhibited abnormal disassembly properties. These results indicate that in vivo RMLC is necessary for myosin function.

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Myosin phosphorylation in intact platelets.

Cited by 142 publications

References 33 publications

Lowering pH in blood platelets dissociates myosin phosphorylation from shape change and myosin association with the cytoskeleton.

Lowering pH in blood platelets dissociates myosin phosphorylation from shape change and myosin association with the cytoskeleton.

Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets

Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development.

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