A study of protein phosphorylation in shape change and Ca++-dependent serotonin release by blood platelets

Bennett, William F.; Belville, J S; Lynch, Gary

doi:10.1016/0092-8674(79)90214-9

Cited by 45 publications

(16 citation statements)

References 21 publications

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“…It now appears likely that stimulus-induced shape change is the factor that elicits phosphorylation, rather than some direct action of these agents on the biochemical machinery of the platelet . It should be noted that previous experiments have shown that phosphorylation of the 20,000 and 40,000 Mr proteins had components dependent on and independent of external divalent cation levels (2,16). In the present study, only the calcium-dependent aspect of the reaction appeared to be present.…”

Section: Discussioncontrasting

confidence: 34%

“…This conclusion is also supported by recent experiments using the phosphodiesterase inhibitor papaverine . This drug completely and selectively blocks phosphorylation of the 20,000 and 40,000 Mr peptides, while only slightly influencing the shape change reaction (2). In the same study it was shown that robust phosphorylation of the 20,000 and 40,000 Mr proteins can be obtained in the absence of detectable serotonin release.…”

Section: Discussionmentioning

confidence: 57%

“…In Situ Protein Phosphorylation Assay in situ protein phosphorylation wasassessed essentially as previously described (2). Platelet suspensions in medium B were incubated with ['ZP]orthophosphate at I mCi/ml for I h at 25°C .…”

Section: Preparation Of Plateletsmentioning

confidence: 99%

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Low-temperature induction of calcium-dependent protein phosphorylation in blood platelets.

Bennett¹,

Lynch²

1980

The Journal of Cell Biology

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Exposure to low temperature causes platelets to change shape in a manner similar to the shape change that precedes secretagogue-induced serotonin release. Previous studies have shown that two proteins, of approximately 20,000 and approximately 40,000 Mr, become phosphorylated before secretion. We have investigated whether low temperature can induce phosphorylation of these proteins and/or serotonin secretion. The data indicate that low-temperature-induced shape change has no requirement for extracellular calcium, whereas phosphorylation of the two proteins and subsequent serotonin release both have strong calcium requirements. Because cold treatment is thought to influence platelet shape through an effect on microtubules, the events in the shape change-release sequence would seem to be ordered as follows: microtubule disassembly leads to shape change leads to protein phosphorylation leads to secretion.

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Section: Discussioncontrasting

confidence: 34%

Section: Discussionmentioning

confidence: 57%

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Low-temperature induction of calcium-dependent protein phosphorylation in blood platelets.

Bennett¹,

Lynch²

1980

The Journal of Cell Biology

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“…This activated form of the kinase is presumably involved in the phosphorylation of a number of intracellular proteins, including components of the cytoskeletal network. Phosphorylation, particularly of40-and 20-kDa polypeptides, is frequently associated with the activation of platelets (9,10,(30)(31)(32)(33) and neutrophils (11), including cells activated by PMA (9)(10)(11)33). The identification of myosin light chain as the 20-kDa polypeptide phosphorylated by protein kinase C is supported by the observation that myosin light chains purified from chicken gizzard muscle are phosphorylated by this kinase (34).…”

Section: Discussionmentioning

confidence: 63%

Phosphorylation by protein kinase C of a 20-kDa cytoskeletal polypeptide enhances its susceptibility to digestion by calpain.

Pontremoli¹,

Melloni²,

Michetti³

et al. 1987

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

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Incubation of the cytoskeletal fraction from human neutrophils with the proteolytically activated form of protein kinase C results in the phosphorylation of several components, including a 20-kDa polypeptide, probably consisting of myosin light chains. The 20-kDa polypeptide is also specifically phosphorylated by activated protein kinase C in a solubilized 20-kDa/80-kDa complex that was obtained after sonication of the insoluble cytoskeletal fraction. Phosphorylation of this polypeptide, in either the insoluble cytoskeletal fraction or the soluble 20-kDa/80-kDa complex, greatly enhances its susceptibility to digestion by the Ca2+-requiring proteinase (calpain, EC 3.4.22.17) of human neutrophils. Thus, signals that activate calpain by mobilizing intracellular calcium would lead to proteolytic activation of protein kinase C, phosphorylation of cytoskeletal proteins, and remodeling of the cytoskeleton by proteolysis of at least one cytoskeletal component.

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“…In previous experiments we found that a high dose of thrombin could overcome the effects of the inhibitors on the incorporation of 32p, into phosphatidic acid and a 40-kDa protein without overcoming the effects of the inhibitors on the phosphorylation of a 20-kDa protein or the secretion of serotonin (8). Other laboratories have also found that different platelet responses show different sensitivities to cAMP (15,16).…”

Section: Discussionmentioning

confidence: 99%

Agents that elevate the concentration of cAMP in platelets inhibit the formation of a NaDodSO4-resistant complex between thrombin and a 40-kDa protein.

Lerea¹,

Glomset²

1987

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

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We studied the influence of prostaglandin El and theophylline on the ability of rabbit or human platelets to form NaDodSO4-resistant complexes between 1251-labeled thrombin and a platelet protein of -40 kDa. Control platelets formed two types of these complexes, one that sedimented with the platelets and one that was found in the suspension medium. There were 30-40 sedimentable complexes per platelet and about three times this number of soluble complexes. Pretreatment of rabbit or human platelets with prostaglandin El and theophylline decreased the formation of both types of complex by as much as 60-80%. The pretreatment was particularly effective when low doses of thrombin were used. When added to control platelets, such doses of thrombin caused the formation of sedimentable complexes and the concomitant formation of inositol trisphosphate. However, when added to platelets that had been pretreated with prostaglandin El and theophylline, low doses of thrombin had little or no effect on either reaction.Thrombin, a serine protease, is a potent activator of several platelet responses, including the very early hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol (1-3). It presumably stimulates these responses by interacting with a receptor on the platelet surface, which then mediates stimulus transduction. However, the precise identity of the receptor remains to be established, and how thrombin interacts with the receptor to promote stimulus transduction is unknown. What is known is that thrombin can bind to several different platelet proteins, including a glycoprotein of 170 kDa, referred to as GP1b (4), and a protein of about 40 kDa, which appears to be a protease nexin (5). It is also known that thrombin can hydrolyze at least one platelet protein, a glycoprotein referred to as GP V (6), and that thrombin that has been inactivated by covalent modification binds to platelets without stimulating them (7). This suggests, but does not prove, that thrombin first binds to platelets and then activates them by a mechanism that involves a proteolytic cleavage reaction.We recently demonstrated that treatment of rabbit or human platelets with prostaglandin E1 (PGE1) and theophylline or other agents that elevate the concentration of cAMP decreases the ability of these platelets to bind thrombin (8). This raised the possibility that these agents might be used to identify cAMP-sensitive components of a functional thrombin receptor complex. To explore this possibility we decided to test the ability of PGE1 and theophylline to inhibit the binding of thrombin to known platelet proteins and began by focusing attention on the protein of 40 kDa. This protein seemed of special interest because it is the only platelet protein that has been identified so far that binds thrombin only when the latter is proteolytically active (9). It forms a NaDodSO4-resistant complex with thrombin that may be an intermediate in a proteolytic cleavage reaction because the complex resembles one tha...

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A study of protein phosphorylation in shape change and Ca++-dependent serotonin release by blood platelets

Cited by 45 publications

References 21 publications

Low-temperature induction of calcium-dependent protein phosphorylation in blood platelets.

Low-temperature induction of calcium-dependent protein phosphorylation in blood platelets.

Phosphorylation by protein kinase C of a 20-kDa cytoskeletal polypeptide enhances its susceptibility to digestion by calpain.

Agents that elevate the concentration of cAMP in platelets inhibit the formation of a NaDodSO4-resistant complex between thrombin and a 40-kDa protein.

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