Identification of Protein Kinase Cα as an Essential, but Not Sufficient, Cytosolic Factor for Ca2+-induced α- and Dense-core Granule Secretion in Platelets

Yoshioka, Akira; Shirakawa, Ryutaro; Nishioka, Hiroaki; Tabuchi, Arata; Higashi, Tomohito; Ozaki, H.; Yamamoto, Akira; Kita, Toru; Horiuchi, Hisanori

doi:10.1074/jbc.m102933200

Cited by 45 publications

(61 citation statements)

References 46 publications

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“…Washed human platelets from healthy donors were prepared (24), resuspended in ice-cold Buffer A (50 mM Hepes/KOH, pH 7.2, 78 mM KCl, 4 mM MgCl 2 , 0.2 mM CaCl 2 , 2 mM EGTA, 1 mM dithiothreitol, and the calculated free [Ca 2ϩ ] was ϳ20 nM (25)) containing 4 mg/ml bovine serum albumin and 20 ng/ml prostaglandin E 1 and kept at 4°C. The platelets were incubated with 0.6 g/ml SLO at 4°C for 10 min and washed once to remove unbound SLO (19,20,26,27), The treated platelets were resuspended in ice-cold Buffer A containing 4 mg/ml bovine serum albumin at a density of 5 ϫ 10 8 /ml, quantified with a Coulter Counter, and incubated at 30°C for 5 min to make holes in their plasma membrane (19,20,26,27). The permeabilized platelets were kept on ice for 15-30 min with 2 mg of proteins/ml human platelet cytosol (or rat brain cytosol), an ATP-regenerating system (19,20,26,27), and tested substances.…”

Section: Methodsmentioning

confidence: 99%

“…For the granule secretion, we have recently demonstrated the direct involvement of PKC (19). We have established a semi-intact secretion assay (19,20) where the secretion does not occur upon stimulation without adding exogenous cytosol, indicating the existence of cytosolic essential factors. We purified an essential factor for the secretion and identified it to be PKC␣ (19).…”

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confidence: 99%

“…In this method, we permeabilized only the plasma membrane judging from observations that the intracellular membrane structures of the platelets appeared to be intact morphologically (30) and that vWF stored in ␣-granules did not leak out (19,20). Because the condition used here with 0.6 g/ml SLO induced leakage of 80% cytosolic lactate dehydrogenase from platelets (19,20), it was presumed that most of ATP and cytosol were also lost by diffusion through the pores in the plasma membrane. Therefore, we exogenously added ATP and cytosol in the assay to reconstitute the aggregation.…”

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confidence: 99%

“…We have established a semi-intact secretion assay (19,20) where the secretion does not occur upon stimulation without adding exogenous cytosol, indicating the existence of cytosolic essential factors. We purified an essential factor for the secretion and identified it to be PKC␣ (19). On the other hand, it has been so far difficult to demonstrate the involvement of PKC in platelet aggregation without using small compounds of PKC inhibitors or phorbol esters.…”

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confidence: 99%

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Direct Demonstration of Involvement of Protein Kinase Cα in the Ca2+-induced Platelet Aggregation

Tabuchi¹,

Yoshioka²,

Higashi³

et al. 2003

Journal of Biological Chemistry

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Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin ␣ IIb ␤ 3 . However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca 2؉ -induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of ␣ IIb ␤ 3 , suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKC␣ and ␤ from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKC␣. Furthermore, the addition of purified PKC␣ in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKC␣ is involved in the regulation of Ca 2؉ -induced platelet aggregation.Platelets play critical roles in hemostasis and thrombosis through aggregation following activation of integrin ␣ IIb ␤ 3 (1-3). Although ␣ IIb ␤ 3 of platelets in the resting stage does not bind fibrinogen or von Willebrand factors (vWF), 1 the activation of platelets results in conformation changes, which allow ␣ IIb ␤ 3 to bind these ligands (1-3). When fibrinogen or vWF binds to ␣ IIb ␤ 3 , the ligand-occupied integrin signals downstream to stabilize platelet aggregation through reorganization of the actin cytoskeletal network and the release of bioactive substances stored in the granules (1-3). Thus, the process of platelet activation and aggregation consists of a series of orchestrated responses (1-3). However, the molecular mechanisms that underlie this process remain unclear because it is difficult to use molecular biology and biochemistry in platelets that do not synthesize new proteins. To overcome this difficulty, semi-intact assays using permeabilized platelets have been established for the study of granule secretion (4 -8). Although some semi-intact aggregation assays have now been developed, it has been difficult to demonstrate a cytosol dependence in these experiments (8).Protein kinase C (PKC) family members are important signaling molecules regulating many cellular functions (9, 10). Among the family members, conventional PKCs (cPKC), which include PKC␣, ␤I, ␤II, and ␥...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Direct Demonstration of Involvement of Protein Kinase Cα in the Ca2+-induced Platelet Aggregation

Tabuchi¹,

Yoshioka²,

Higashi³

et al. 2003

Journal of Biological Chemistry

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“…PKC isoforms are sequentially activated by calcium and diacylglycerol signals (Newton, 1997;Oancea & Meyer, 1998). PKC-a has been observed as an activator of secretion, for example, Ca 2+ -induced secretion of a-and dense-core granules in platelets (Yoshioka et al, 2001), or secretion of amyloid precursor protein (Benussi et al, 1998). However, in the case of rabbit gastric parietal cells, our data indicate an inhibitory function of PKC-a during cholinergically induced acid secretion.…”

Section: Introductionmentioning

confidence: 58%

Protein kinase C‐α attenuates cholinergically stimulated gastric acid secretion of rabbit parietal cells

Fährmann

Kaufhold

Pfeiffer³

et al. 2003

British J Pharmacology

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1 The phorbolester 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C (PKC), inhibits cholinergic stimulation of gastric acid secretion. We observed that this effect strongly correlated with the inhibition of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) activity in rabbit parietal cells. 2 The aim of this study was to specify the function of PKC-a in cholinergically stimulated H + secretion. PKC-a represents the only calcium-dependent PKC isoenzyme that has been detected in rabbit parietal cells. 3 Go¨6976, an inhibitor of calcium-dependent PKC, concentration-dependently antagonized the inhibitory effect of TPA, and, therefore, revealed the action of PKC-a on carbachol-induced acid secretion in rabbit parietal cells. 4 TPA exerted no additive inhibition of carbachol-stimulated acid secretion if acid secretion was partially inhibited by the potent CaMKII inhibitor 1-[N,O-bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62). 5 Since both kinase modulators, TPA and KN-62, affected no divergent signal transduction pathways in the parietal cell, an in vitro model has been used to study if PKC directly targets CaMKII. CaMKII purified from parietal cell-containing gastric mucosa of pig, was transphosphorylated by purified cPKC containing PKC-a up to 1.8 mol P i per mol CaMKII in vitro. The autonomy site of CaMKII was not transphosphorylated by PKC. 6 The phosphotransferase activity of the purified CaMKII was in vitro inhibited after transphosphorylation by PKC if calmodulin was absent during transphosphorylation. Attenuation of CaMKII activity by PKC showed strong similarity to the downregulation of CaMKII by basal autophosphorylation. 7 Our results suggest that PKC-a and CaMKII are closely functionally linked in a cholinergically induced signalling pathway in rabbit parietal cells. We assume that in cholinergically stimulated parietal cells PKC-a transinhibits CaMKII activity, resulting in an attenuation of acid secretion.

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Cardiovascular and Hemostatic Disorders: SOCE and Ca2+ Handling in Platelet Dysfunction

López

Salido

Rosado

2017

Store-Operated Ca²⁺ Entry (SOCE) Pathways

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Among the Ca entry mechanisms in platelets, store-operated Ca entry (SOCE) plays a prominent role as it is necessary to achieve full activation of platelet functions and replenish intracellular Ca stores. In platelets, as in other non-excitable cells, SOCE has been reported to involve the activation of plasma membrane channels by the ER Ca sensor STIM1. Despite electrophysiological studies are not possible in human platelets, indirect analyses have revealed that the Ca-permeable channels involve Orai1 and, most likely, TRPC1 subunits. A relevant role for the latter has not been found in mouse platelets. There is a body of evidence revealing a number of abnormalities in SOCE or in its molecular regulators that result in qualitative platelet disorders and, as a consequence, altered platelet responsiveness upon stimulation with multiple physiological agonists. Platelet SOCE abnormalities include STIM1 and Orai1 mutations. This chapter summarizes the current knowledge in this field, as well as the disorders associated to platelet SOCE dysfunction.

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Identification of Protein Kinase Cα as an Essential, but Not Sufficient, Cytosolic Factor for Ca2+-induced α- and Dense-core Granule Secretion in Platelets

Cited by 45 publications

References 46 publications

Direct Demonstration of Involvement of Protein Kinase Cα in the Ca2+-induced Platelet Aggregation

Direct Demonstration of Involvement of Protein Kinase Cα in the Ca2+-induced Platelet Aggregation

Protein kinase C‐α attenuates cholinergically stimulated gastric acid secretion of rabbit parietal cells

Cardiovascular and Hemostatic Disorders: SOCE and Ca2+ Handling in Platelet Dysfunction

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