Differential Roles of the PKC Novel Isoforms, PKCδ and PKCε, in Mouse and Human Platelets

Pears, Catherine J.; Thornber, Kelly; Auger, Jocelyn M.; Hughes, Craig E.; Grygielska, Beata; Protty, Majd; Pearce, Andrew C.; Watson, Steve P.

doi:10.1371/journal.pone.0003793

Cited by 40 publications

(75 citation statements)

References 38 publications

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“…However, Pears et al 45 and Buensuceso et al 46 failed to identify PKCε in human platelets. Thus, in order to resolve the issue, we subjected human and murine platelet lysates to Western Blot analysis using custom made antibody against PKCε (Gift from Dr. Messing, Gallo Centre, San Francisco).…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Bynagari-Settipalli

Lakhani

Jin

et al. 2012

ATVB

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Objective— Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. Methods and Results— A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA 2 phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca 2+ i ) mobilization, 2 signaling molecules, upstream of cPLA 2 activation. Thus, PKCs inhibit cPLA 2 activation by inhibiting ERK and Ca 2+ i mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKCε null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε knockout and wild-type was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in an FeCl 3 -induced arterial injury model and shorter bleeding times in tail-bleeding experiments. Conclusion— We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

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Section: Discussionmentioning

confidence: 99%

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Bynagari-Settipalli

Lakhani

Jin

et al. 2012

ATVB

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“…Further, the presence of a threonine group at position Y ϩ 1 or Y ϩ 2 is of also of interest given previous observations in mast cells and mouse platelets that phosphorylation of a conserved threonine at position Y ϩ 1 in 1 of the 2 YxxL sequences of the FcR␥-chain by a novel isoform of protein kinase C (␦ or ⑀) is required for maximal binding and activation of Syk. [27][28][29] Based on these alignments, experiments were designed to investigate the role of the YxxL, and the conserved serine and threonine residues in the cytoplasmic tail of CLEC-2 through a combination of peptide pull-down studies using human CLEC-2 For personal use only. on March 28, 2019. by guest www.bloodjournal.org From cytosolic peptides and human mutant CLEC-2 transfected into chicken DT40 B cells.…”

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confidence: 99%

CLEC-2 activates Syk through dimerization

Hughes

Pollitt

Mori

et al. 2010

Blood

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147

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The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C ␥2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x 6-12 Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor. IntroductionThe C-type lectin receptor CLEC-2 is expressed on platelets and on a subpopulation of other hematopoietic cells, including mouse neutrophils and dendritic cells. 1-3 CLEC-2 is a receptor for the snake venom toxin rhodocytin 4 and the transmembrane protein podoplanin, 5,6 which is expressed on the leading edge of tumor cells and on kidney podocytes, lung type 1 alveolar cells, and lymphatic endothelium. In addition, recent evidence suggests that activated platelets express or release a ligand for CLEC-2 that supports platelet aggregation at arteriolar rates of flow. 7 Mice pretreated with a specific antibody to CLEC-2 exhibit a selective loss of the C-type lectin receptor and impaired platelet activation on collagen at high shear in vitro or in vivo. 7 Cross-linking of CLEC-2 by rhodocytin, podoplanin, or specific antibodies elicits powerful platelet aggregation and secretion. 4,6 CLEC-2 signals through Src-and Syk-dependent tyrosine kinases, leading to phosphorylation of a series of adapter and effector proteins that culminate in activation of phospholipase-C ␥2 (PLC␥2) and platelet activation. 4 This mechanism of platelet activation resembles that used by the immunoglobulin collagen receptor, glycoprotein VI (GPVI), which is constitutively associated with the FcR␥ chain at the platelet surface. Cross-linking of GPVI by collagen or specific agonists, such as the snake venom toxin convulxin or antibodies, leads to Src kinase-dependent phosphorylation of 2 conserved tyrosines in the FcR␥-chain immunoreceptor tyrosine-based activation motif (ITAM). 8,9 ITAMs are present in a variety of hematopoietic receptors, including T-and B-cell antigen receptors and th...

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“…In fact, it is known that in human normal platelets, PKC epsilon is nearly absent (25,26). This kinetic of expression can be altered during malignant megakaryopoiesis, as we showed that PKCepsilon is over-expressed in PMF MKs accounting for enhanced proliferation and impaired differentiation (13,14).…”

Section: Mf Platelets Are Characterized By Higher Levels Of Pkcepsilonmentioning

confidence: 53%

Platelet expression of PKCepsilon oncoprotein in myelofibrosis is associated with disease severity and thrombotic risk

Masselli¹,

Carubbi²,

Pozzi³

et al. 2017

Ann. Transl. Med.

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Background: Myelofibrosis (MF) is the most aggressive Philadelphia-negative chronic myeloproliferative neoplasm (MPN) with high morbidity and mortality due to thrombo-hemorrhagic complications and leukemic transformation. MF is characterized by profound alterations of megakaryocytopoiesis, with consequent abnormalities in platelet number and function. We recently showed that the overexpression of the oncoprotein PKCepsilon plays a key role in the aberrant differentiation of MF megakaryocyte clone and that its levels correlate with disease burden. Moreover, our group previously demonstrated that PKCepsilon is over-expressed in platelets from patients with acute myocardial infarction (MI) and accounts for their increased reactivity. On these bases, we investigated here the activation state and PKCepsilon expression of MF platelets, testing potential correlations with thrombotic risk and disease aggressiveness.Methods: Platelets were isolated from peripheral blood samples of MF patients and healthy donors (HDs).Patients were stratified according to the IPSS/DIPSS risk category and history of cardiovascular events.Platelet activation was assessed by flow cytometry. PKCepsilon mRNA and protein levels were determined by real time-PCR and western blot.Results: MF platelets circulate in an activated status and display significantly higher levels of PKCepsilon compared to HDs. In MF patients, PKCepsilon platelet levels were associated with high-risk disease as well as with a positive history of major cardiovascular events.Conclusions: PKCepsilon is configuring as the common denominator of neoplastic transformation and thrombus formation in MF. Overall, our data pinpoint PKCepsilon as a potential novel biomarker of disease aggressiveness and thrombotic risk in this hematologic neoplasm.

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Differential Roles of the PKC Novel Isoforms, PKCδ and PKCε, in Mouse and Human Platelets

Cited by 40 publications

References 38 publications

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

CLEC-2 activates Syk through dimerization

Platelet expression of PKCepsilon oncoprotein in myelofibrosis is associated with disease severity and thrombotic risk

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