Platelet phosphorylase kinase activity and its regulation by the calcium-dependent regulatory protein, calmodulin

Gergely, Pál; Castle, Alan G.; Crawford, N.

doi:10.1016/0005-2744(80)90277-6

Cited by 14 publications

(4 citation statements)

References 18 publications

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“…We have been unable to purify and positively identify the protein kinase responsible, since it seems to resist extensive solubilization . Co-incubation of the PSD preparation with phosphorylase b, glycogen synthase, as well as with myosin light chains from canine skeletal muscle, substrates known to require a calmodulin-dependent protein kinase (12,14,15,18,24,30,41,53,56,59,60,62,64), did not induce any phosphorylation in the substrate proteins used. Histone II-AS and phosvitin were also not phosphorylated by the PSD kinase.…”

Section: Resultsmentioning

confidence: 92%

“…A further examination of the possible function of the calmodulin in the PSD was prompted by the demonstration by several groups of the existence of calmodulin-dependent protein kinases . Calmodu-lin has been shown to be a subunit of the Ca"-dependent myosin light chain kinase (14,15,30,41,59,60,62,64) as well as activating muscle (12,53) and platelet (24) phosphorylase kinase, glycogen synthase kinase, (18,56), and the phosphorylation of histones (58). It has also been implicated in the phosphorylation of brain cytosol (63) and of synaptic vesicle (13) proteins .…”

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confidence: 99%

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Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Grab

Carlin

Siekevitz

1981

The Journal of Cell Biology

129

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Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity . As seen by autoradiographic analysis of Coomassie Blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [y-32 P]ATP and Mg t+ alone. Addition of Ca t+ alone to the native PSD preparation had little or no effect on phosphorylation . However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 Mr. Similar results were also obtained in sonicated or freeze-thawed native PSD preparations by addition of Ca t+ alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions . The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulinactivatable phosphorylation, but not of the Mgt'-dependent activity, by the Ca" chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca 21 . In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [Y-32 P]ATP, Mgt ' and Ca 21, one could not induce a Ca t+ -stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins . Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 Mr PSD protein is the one that is phosphorylated and not the 51,000 Mr component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 Mr protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg t+ -dependent kinase(s) . Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins l a and lb, the cyclic AMP-dependent protein kinase, which definitely does phosphorylate proteins l a and Ib and is present in the PSD, does not phosphorylate the 51,000 and 62,000 Mr proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins .In the previous paper we have shown that the postsynaptic density (PSD) contains a cryptic calmodulin-activatable cyclic nucleotide phosphodiesterase activity (29). A further examination of the possible function of the calmodulin in the PSD was prompted by the demonstration by several groups of the existence of calmodulin-dependent protein kinases . Calmodulin has been shown to be a subunit of the Ca"-dependent myosin light chain kinase (14,15,30,41,59,60,62,64) as well as activating muscle (12, 53) and plat...

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Section: Resultsmentioning

confidence: 92%

mentioning

confidence: 99%

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Grab

Carlin

Siekevitz

1981

The Journal of Cell Biology

129

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“…The Ca2+/calmodulin complex activates a number of platelet enzymes including myosin light chain kinase (Hathaway & Adelstein, 1979), phosphorylase kinase (Gergely et al, 1980), and a phosphoprotein phosphatase (Tallant & Wallace, 1985). In addition, under in vitro conditions, calmodulin increases the rate of Mg2+-induced polymerization of platelet actin and alters the structural characteristics of the resulting actin filaments (Piazza & Wallace, 1985) which suggests that calmodulin may also be involved in the regulation of platelet microfilament structure. Many calmodulin-regulated enzymes are irreversibly activated in a calmodulin-independent manner by limited proteolysis (Lin & Cheung, 1980;Kincaid et al, 1985;Depaoli-Roach et al, 1979;Higgli et al, 1981;Walsh et al, 1982;Manalan & Klee, 1983; Tallant & Cheung, 1984; Meijer & Guerrier, 1982;Keller et al, 1980).…”

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confidence: 99%

Human platelet calmodulin-binding proteins: identification and calcium-dependent proteolysis upon platelet activation

Rw¹,

Ea²,

Mc³

1987

Biochemistry

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Calmodulin-binding proteins have been identified in human platelets by using Western blotting techniques and 125I-calmodulin. Ten distinct proteins of 245, 225, 175, 150, 90, 82 (2), 60, and 41 (2) kilodaltons (kDa) bound 125I-calmodulin in a Ca2+-dependent manner; the binding was blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA), trifluoperazine, and nonradiolabeled calmodulin. Proteins of 225 and 90 kDa were labeled by antisera against myosin light chain kinase; 60- and 82-kDa proteins were labeled by antisera against the calmodulin-dependent phosphatase and caldesmon, respectively. The remaining calmodulin-binding proteins have not been identified. Calmodulin-binding proteins were degraded upon addition of Ca2+ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin, or N-ethylmaleimide which suggests that the degradation was due to a Ca2+-dependent protease. Activation of intact platelets by thrombin, adenosine 5'-diphosphate, and collagen under conditions which promote platelet aggregation (i.e., stirring with extracellular Ca2+) also resulted in limited proteolysis of calmodulin-binding proteins including those labeled with antisera against myosin light chain kinase and the calmodulin-dependent phosphatase. Activation by the Ca2+ ionophores A23187 and ionomycin also promoted degradation of the calmodulin-binding proteins in the presence of extracellular Ca2+; however, degradation in response to the ionophores did not require stirring of the platelet suspension to promote aggregation. Many Ca2+/calmodulin-regulated enzymes are irreversibly activated in vitro by limited proteolysis. Our data indicate that limited proteolysis of Ca2+/calmodulin-regulated enzymes also occurs in the intact platelet and suggest that the proteolysis is triggered by an influx of extracellular Ca2+ associated with platelet aggregation.

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“…Many of these biochemical reactions are triggered by an increase in the intraplatelet concentration of free calcium (2,3). Calmodulin, one of the primary Ca2l-receptor proteins in the platelet (4)(5)(6), is converted into an active conformation upon binding Ca2+ (7,8); the Ca2+-calmodulin complex activates at least three platelet enzymes-myosin light chain kinase (9), phosphorylase kinase (10), and a calmodulin-dependent phosphatase (11)-all of which may play important roles in the platelet reaction sequence. In addition, a number of other calmodulin-binding proteins have been identified in the platelet that may be additional calmodulin-regulated enzymes or structural proteins (12)(13)(14).…”

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confidence: 99%

Calmodulin accelerates the rate of polymerization of human platelet actin and alters the structural characteristics of actin filaments.

Piazza¹,

Wallace²

1985

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

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Calmodulin stimulated the rate of Mg2+-induced polymerization of human platelet actin. The stimulatory effect was due to an increase in the nucleation phase of the reaction; there was no effect on the steady-state viscosity. The calmodulin antagonist trifluoperazine blocked the stimulatory effect of calmodulin. Addition of EGTA to the reaction mixture also stimulated the rate of actin polymerization; however, the effect of calmodulin on actin polymerization is not due to Ca2+ chelation, as is presumed to be the case for EGTA. Electron microscopy revealed structural differences in the filaments prepared in the presence of calmodulin as compared to those prepared with trifluoperazine. In the presence of calmodulin, the filaments were thicker, suggesting that they consisted of multiple actin polymers. In addition, numerous projections were present perpendicular to the filaments, as well as localized areas of filament bundling. It was not possible to demonstrate a direct interaction between calmodulin and actin, which raises the possibility that the calmodulin effect may be indirect through a calmodulin-binding protein or calmodulin-dependent enzyme. Regardless of whether calmodulin is acting directly or indirectly, these results provide evidence that calmodulin may play a regulatory role in either the polymerization of actin or in determining the structural characteristics of actin filaments.

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Platelet phosphorylase kinase activity and its regulation by the calcium-dependent regulatory protein, calmodulin

Cited by 14 publications

References 18 publications

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Human platelet calmodulin-binding proteins: identification and calcium-dependent proteolysis upon platelet activation

Calmodulin accelerates the rate of polymerization of human platelet actin and alters the structural characteristics of actin filaments.

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