Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets

1991 ◽  
Vol 178 (3) ◽  
pp. 878-883 ◽  
Author(s):  
Maryse Crabos ◽  
Roland Imber ◽  
Thomas Woodtli ◽  
Doriano Fabbro ◽  
Paul Erne
Keyword(s):  
Phorbol Ester ◽  
Human Platelets ◽  
Pkc Isoforms

scholarly journals Effect of tumour-promoting phorbol ester, thrombin and vasopressin on translocation of three distinct protein kinase C isoforms in human platelets and regulation by calcium

1992 ◽  
Vol 288 (3) ◽  
pp. 891-896 ◽  
Author(s):  
M Crabos ◽  
D Fabbro ◽  
S Stabel ◽  
P Erne
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Human Platelets ◽  
Second Phase ◽  
Percentage Increase ◽  
Control Value ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Alpha Beta

Protein kinase C (PKC) acts in synergy with Ca2+ mobilization for the activation of platelets. Three different PKC subtypes that specifically react with antibodies to alpha- beta- and zeta-PKC have been detected in human platelets. We have compared the subcellular redistribution of these isoforms in platelets after exposure to the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and to two physiological agonists, thrombin and vasopressin. In the presence of PMA, beta-PKC is most rapidly translocated to membranes, followed by zeta-PKC and alpha-PKC [membrane contents of 39 +/- 6, 31 +/- 4 and 24 +/- 4% (means +/- S.E.M.) respectively after 2 min incubation]. In contrast, both thrombin and vasopressin induced a biphasic translocation of PKC isoforms. For both agonists, the first phase of translocation occurred within 1 min and was identical for the three isoforms. However, during the second phase, the translocation of zeta-PKC by thrombin and vasopressin differed [membrane contents (mean +/- S.E.M.) of 24 +/- 3 and 46 +/- 4% respectively after 10 min]. These results suggest a differential activation of zeta-PKC by vasopressin and thrombin. PMA-induced translocation of alpha-PKC was decreased from 278 +/- 27 to 198 +/- 24 (mean +/- S.E.M., P = 0.02; percentage increase over control value) in the presence of 1 mM-EDTA, whereas chelation of intracellular Ca2+ by Quin2-AM does not influence this response. These results suggest that the PMA-induced translocation of alpha-PKC depends on the presence of 1 mM concentration of extracellular Ca2+. In addition, the chelation of either extracellular or intracellular Ca2+ inhibited both vasopressin- and thrombin-induced translocation of all three isoforms, suggesting that Ca2+ is an important requirement for the translocation of alpha-, beta- and zeta-PKC by physiological agonists. In conclusion, the translocation of PKC varies between different isoforms and between different agonists.

Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3541-3541
Author(s):  
Swaminathan Murugappan ◽  
Haripriya Shankar ◽  
Satya Kunapuli
Keyword(s):  
Tyrosine Kinase ◽  
Intracellular Calcium ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Glycoprotein Vi ◽  
Pkc Isoforms

Abstract Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.

scholarly journals Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets

1996 ◽  
Vol 318 (1) ◽  
pp. 207-212 ◽  
Author(s):  
Angelika G. BÖRSCH-HAUBOLD ◽  
Ruth M. KRAMER ◽  
Steve P WATSON
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Substrate Inhibition ◽  
Human Platelets ◽  
Mitogen Activated Protein Kinase ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2´-amino-3´-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol ester-stimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 µM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST–MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.

Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion

1997 ◽  
Vol 272 (1) ◽  
pp. C263-C269 ◽  
Author(s):  
D. Zoukhri ◽  
R. R. Hodges ◽  
C. Sergheraert ◽  
A. Toker ◽  
D. A. Dartt
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Secretion ◽  
Phorbol Ester ◽  
Lacrimal Gland ◽  
Pkc Delta ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha ◽  
Kinase A

In the present study, we have synthesized and N-myristoylated peptides derived from the pseudosubstrate sequences of protein kinase C (PKC)-alpha, -delta, and -epsilon [Myr-PKC-alpha-(15-28), Myr-PKC-delta-(142-153), and Myr-PKC-epsilon-(149-164)], three isoforms present in rat lacrimal gland, and a peptide derived from the sequence of the endogenous inhibitor of protein kinase A [Myr-PKI-(17-25)]. Lacrimal gland acini were preincubated for 60 min with the myristoylated peptides (10(-10) to 3 x 10(-7) M), then protein secretion was stimulated with a phorbol ester, phorbol 12,13-dibutyrate (10(-6) M); vasoactive intestinal peptide (10(-8) M); a cholinergic agonist, carbachol (10(-5) M); or an alpha 1-adrenergic agonist, phenylephrine (10(-4) M), for 20 min. In intact lacrimal gland acini, Myr-PKC-alpha-(15-28) inhibited phorbol 12,13-dibutyrate-induced protein secretion. This effect was not reproduced by the acetylated peptide or by the myristoylated PKI, which inhibited vasoactive intestinal peptide-induced protein secretion, a response mediated by protein kinase A. Carbachol-induced protein secretion was inhibited by all three peptides. In contrast, phenylephrine-induced protein secretion was inhibited only by Myr-PKC-epsilon-(149-164), whereas Myr-PKC-alpha-(15-28) and Myr-PKC-delta-(142-153) had a stimulatory effect. None of these myristoylated peptides affected the calcium increase evoked by cholinergic or alpha 1-adrenergic agonists. We concluded that phorbol ester- and receptor-induced protein secretion involve different PKC isoforms in lacrimal gland.

Synergistic functions of phorbol ester and calcium in serotonin release from human platelets

1983 ◽  
Vol 112 (2) ◽  
pp. 778-786 ◽  
Author(s):  
Junji Yamanishi ◽  
Yoshimi Takai ◽  
Kozo Kaibuchi ◽  
Kimihiko Sano ◽  
Monique Castagna ◽  
...  
Keyword(s):  
Phorbol Ester ◽  
Human Platelets ◽  
Serotonin Release

scholarly journals Activation of Na+/H+ exchange in human platelets stimulated by thrombin and a phorbol ester

1987 ◽  
Vol 241 (1) ◽  
pp. 301-303 ◽  
Author(s):  
W Siffert ◽  
G Siffert ◽  
P Scheid
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fluorescent Probe ◽  
Phorbol Ester ◽  
Human Platelets ◽  
Cytoplasmic Ph ◽  
Kinase C ◽  
Stimulation Of

We have investigated changes in cytoplasmic pH (pHi) in activated human platelets, using the fluorescent probe 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. Stimulation of platelets by thrombin or 12-O-tetradecanoylphorbol 13-acetate increased pHi by about 0.11 pH unit above the resting value. This increase in pHi depended on the presence of external Na+ and was inhibited by ethylisopropylamiloride. The data suggest that protein kinase C mediates Na+/H+ exchange in human platelets.

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