scholarly journals Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

Blood ◽  
2011 ◽  
Vol 118 (2) ◽  
pp. 416-424 ◽  
Author(s):  
Olga Konopatskaya ◽  
Sharon A. Matthews ◽  
Matthew T. Harper ◽  
Karen Gilio ◽  
Judith M. E. M. Cosemans ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Thrombus Formation ◽  
Dense Granule ◽  
Molecular Pathway ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

SHIP-1 Associates with Tyrosine Phosphorylated Protein Kinase C-Delta at 155 Residue.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3004-3004
Author(s):  
Ramya Chari ◽  
Dheeraj Bhavanasi ◽  
James Daniel ◽  
Satya P. Kunapuli
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Conflicts Of Interest ◽  
Dense Granule ◽  
Protein Kinase C Delta ◽  
Tyrosine Residues ◽  
Kinase C ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Abstract 3004 Poster Board II-981 Protein Kinase C-delta (PKCδ) is a novel PKC isoform that differentially regulates platelet dense granule secretion. PKCδ positively regulates Protease activated receptor (PAR)-mediated dense granule secretion, whereas it negatively regulates glycoproteinVI (GPVI)-mediated dense granule secretion in platelets. PKCδ, a serine/threonine kinase is phosphorylated on its tyrosine residues. There are nine potential tyrosine phosphorylation sites in the regulatory domain of PKCδ. Phosphorylation at different tyrosine residues regulates its substrate specificity. We have previously shown that the association of PKCδ with Lyn and SHIP-1 negatively regulates GPVI-mediated dense granule secretion. However, the event leading to the association between PKCδ and SHIP-1 is not known. We hypothesize that the differential tyrosine phosphorylation of PKCδ downstream of PARs or GPVI receptors result in the preferential association with SHIP-1. In the current study, we show that PKCδ is phosphorylated at tyrosine residues Y332, Y523, Y525 and Y565 upon PAR or GPVI stimulation. Y311 residue is predominantly phosphorylated upon stimulation of PARs, whereas Y155 residue is preferentially phosphorylated upon GPVI stimulation. PAR-mediated Y311 phosphorylation peaks at later timepoint, whereas GPVI-mediated Y155 phosphorylation peaks at an early timepoint. correlating with dense granule secretion. Furthermore, we show that agarose-conjugated Y155 phosphorylated PKCδ peptide associates with SHIP-1 upon GPVI stimulation, and not PARs. These data suggest that the phosphorylation of PKCδ at distinct tyrosine residues differentially regulate its association with SHIP-1. Therefore, we conclude that the GPVI-mediated phosphorylation of PKCδ at 155 is required for its association with SHIP-1. This study is supported by pre-doctoral fellowship to Ramya Chari from American Heart Association, Pennsylvania-Delaware affiliate. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Submaximal Inhibition of Protein Kinase C Restores ADP-induced Dense Granule Secretion in Platelets in the Presence of Ca2+

2011 ◽  
Vol 286 (24) ◽  
pp. 21073-21082 ◽  
Author(s):  
Amanda J. Unsworth ◽  
Holly Smith ◽  
Paul Gissen ◽  
Steve P. Watson ◽  
Catherine J. Pears
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dense Granule ◽  
Kinase C ◽  
Dense Granule Secretion ◽  
Granule Secretion

SHIP-1 Differentially Regulates Dense Granule Secretion in Platelets through Its Association with Protein Kinase C δ.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3630-3630
Author(s):  
Ramya Chari ◽  
Soochong Kim ◽  
Swaminathan Murugappan ◽  
James L. Daniel ◽  
Satya P. Kunapuli
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dense Granule ◽  
Protease Activated Receptors ◽  
Kinase C ◽  
Inositol Phosphatases ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Granule Release ◽  
Stimulation Of

Abstract Collagen-induced glycoprotein (GP) VI-mediated and thrombin-induced protease activated receptors (PAR)-mediated activation are important signaling pathways regulating dense granule secretion in platelets. Protein kinase C (PKC) isoforms play a crucial role in platelet secretion and we have previously shown that PKCδ plays a ying-yang role in dense granule release by different agonists (Murugappan et al, J. Biol. Chem. 2004). PKCδ isoform positively regulates PAR-mediated platelet dense granule release, whereas it negatively regulates GPVI-mediated dense granule release. In this study, we investigated the mechanism of such differential regulation by PKCδ downstream of PAR and GPVI pathways. We hypothesize that the differential association of PKCδ with phosphatases downstream of GPVI and PAR receptors differentially regulate dense granule secretion. More specifically, we explored the functional relevance of the interaction of PKCδ with Src homology 2-domain containing Inositol Phosphatases (SHIP), 5′-inositol phosphatases in platelets. In our studies, SHIP-1 was tyrosine phosphorylated by both PARs and GPVI receptors and its phosphorylation followed different activation kinetics. Whereas PAR-mediated SHIP-1 phosphorylation (Y1020) was delayed and occurred as late as 120 seconds, the GPVI-mediated SHIP-1 phosphorylation was rapid, starting as early as 15 seconds and peaked at 60 seconds. Co-immunoprecipitation experiments revealed that SHIP-1, and not SHIP-2, associated with PKCδ upon stimulation of platelets with GPVI agonist, convulxin. However, such association did not occur with the PAR agonists. GPVI-mediated SHIP-1 phosphorylation failed to occur in platelets from mice lacking Lyn kinase suggesting a role for Lyn in regulating SHIP-1 phosphorylation. In murine platelets lacking either Lyn or SHIP-1, dense granule secretion was potentiated by convulxin and not by thrombin. We attribute the phosphorylation and association of SHIP-1 with PKCδ to be critical for the regulation of agonist-induced dense granule secretion in platelets. Based on the above results, we conclude that the preferential association of SHIP-1 with PKCδ upon stimulation of GPVI receptor results in the negative regulation of collagen-induced dense granule release in platelets.

scholarly journals Endometrial cancer cell survival and apoptosis is regulated by protein kinase C α and δ

2006 ◽  
Vol 13 (4) ◽  
pp. 1251-1267 ◽  
Author(s):  
James M Haughian ◽  
Twila A Jackson ◽  
David M Koterwas ◽  
Andrew P Bradford
Keyword(s):  
Cell Death ◽  
Endometrial Cancer ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cancer Cell ◽  
Molecular Mechanisms ◽  
Endometrial Cancer Cell ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Induced Apoptosis

Endometrial cancer is the most common invasive gynecologic malignancy but the molecular mechanisms underlying its onset and progression are poorly understood. Paradoxically, endometrial tumors exhibit increased apoptosis, correlating with disease progression and poor patient prognosis. Endometrial tumors also show altered activity and expression of protein kinase C (PKC) isoforms, implicated in the regulation of programmed cell death; however, PKC modulation of apoptosis in endometrial cancer cells has not been investigated. We detected nine out of ten PKC isoforms in Ishikawa endometrial cancer cell lines, and demonstrated expression of both PKCα and δ in human endometrial tumors. To determine the functional roles of PKCα and δ in apoptosis in endometrial cancer, Ishikawa cells were treated with selective PKC inhibitors or adenoviral constructs encoding wild-type or isoform-specific, dominant-negative mutants. Apoptosis was assessed by DNA fragmentation and caspase-mediated poly-(ADP-ribose)-polymerase cleavage. The inhibition of PKCδ suppressed etoposide-induced apoptosis, while overexpression of PKCδ enhanced it. In contrast, inhibition of PKCα elevated basal levels of apoptosis and potentiated etoposide-induced cell death. Etoposide treatment also selectively activated PKCδ, but resulted in both cytosolic translocation and decreased activity of PKCα. A fraction of PKCδ also underwent caspase-dependent cleavage, in response to etoposide. Our results suggest that changes in apoptosis and PKC expression in endometrial cancer are mechanistically linked, such that PKCδ is required for DNA damage-induced apoptosis, while PKCα mediates a survival response. Thus, PKCα and δ expression and signaling may be important in endometrial tumorigenesis and could serve as potential prognostic indicators and/or novel targets for therapeutic intervention.

Different Requirement of Intracellular Calcium and Protein Kinase C for Arachidonic Acid Release and Serotonin Secretion in Cathepsin G-activated Platelets

1997 ◽  
Vol 78 (02) ◽  
pp. 919-925 ◽  
Author(s):  
Serenella Rotondo ◽  
Virgilio Evangelista ◽  
Stefano Manarini ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Dense Granule ◽  
Cathepsin G ◽  
Platelet Response ◽  
Relative Contribution ◽  
Kinase C ◽  
Serotonin Secretion ◽  
Dense Granule Secretion

SummaryPrevious studies have shown that platelet stimulation with cathepsin G rapidly results in cytoplasmic calcium ([Ca2+]j) increase and activation of protein kinase C (PKC). To elucidate the relationship between these two biochemical events and their relative contribution to the regulation of platelet response to cathepsin G, arachidonic acid (AA) release and serotonin (5HT) secretion were studied. Platelets made Ca2+- depleted and -permeable by treatment with A23187 were compared to intact platelets to better dissociate calcium changes from other receptor- stimulated events. AA release elicited by cathepsin G in intact platelets was prevented by the Ca2+ chelator BAPTA; in Ca2+-depleted, -permeable platelets AA was released in direct response to added Ca2+ and was not increased by simultaneous stimulation with cathepsin G. In intact platelets, PKC inhibition by Ro 31-8220 or PKC induction with PMA either enhanced or reduced, respectively, cathepsin G-induced AA release. Both BAPTA and Ro 31-8220 prevented 5HT secretion from intact platelets; however, in Ca2+-depleted, -permeable platelets, cathepsin G was able to evoke 5HT secretion and p47 phosphorylation independently of [Ca2+]j increase, both effects being hampered by Ro 31-8220. Ca2+ and PKC therefore regulate PLA2 activity and 5HT secretion in cathepsin G-stimulated platelets in a different manner: the former is mainly triggered by [Ca2+]j increase, while PKC represents the major factor in determining dense granule secretion.

scholarly journals Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen

2010 ◽  
Vol 285 (30) ◽  
pp. 23410-23419 ◽  
Author(s):  
Karen Gilio ◽  
Matthew T. Harper ◽  
Judith M. E. M. Cosemans ◽  
Olga Konopatskaya ◽  
Imke C. A. Munnix ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Thrombus Formation ◽  
Functional Divergence ◽  
Platelet Protein ◽  
Kinase C ◽  
Pkc Isoforms

scholarly journals Collagen-induced platelet activation mainly involves the protein kinase C pathway

1990 ◽  
Vol 268 (2) ◽  
pp. 325-331 ◽  
Author(s):  
A Karniguian ◽  
F Grelac ◽  
S Levy-Toledano ◽  
Y J Legrand ◽  
F Rendu
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Membrane Receptors ◽  
Lag Phase ◽  
Response Curves ◽  
Kinase C ◽  
Metabolic Event ◽  
Granule Secretion

This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage.

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