Protein kinase C (PKC) isoforms are serine threonine kinases that are activated upon agonist stimulation and regulate several functions. In the previous grant period, we demonstrated the functional role of nPKC isoforms in platelets and the molecular interactions of PKC? with SHIP1 and Lyn to regulate differential granule release. We have strong preliminary data indicating that PKC? is phosphorylated on Y155 and Y311, but these two tyrosines are differentially phosphorylated upon stimulation of Collagen or Thrombin receptors. We have generated knock-in mice for PKC? Y155F mutation and our preliminary data point to the regulation of GPVI-mediated functional events by PKC? through the Y155 residue. We also have strong preliminary data suggesting that Y311 might regulate cPLA2 activation and thromboxane generation. Finally, we have identified a negative regulatory role for PKC? in megakaryopoiesis and platelet production. Hence we will focus our efforts on the molecular mechanisms of regulation of platelet function and megakaryopoiesis by PKC? in this grant period. Our overall hypothesis is that PKC? plays a crucial role in platelet function and megakaryopoiesis through tyrosine phosphorylation of Y155 and Y311 residues. We propose that these phosphorylated tyrosines form nodal points for signal transduction through interaction with other signaling molecules thus regulating functional responses. 1) We hypothesize that diverse signaling pathways downstream of G protein-coupled receptors and tyrosine kinase-linked receptors phosphorylate different tyrosine residues on PKC? and these differential phosphorylations modify the functional role of this isoform. We hypothesize that PKC? Y155 phosphorylation regulates dense granule release through association with SHIP1 and Lyn, whereas PKC? Y311 phosphorylation regulates cPLA2 activation and thromboxane generation. We will investigate the functional role of phosphorylation of Y155 and Y311 on PKC? in platelets using PKC? knock-in mice with the specific mutation Y155F or Y311F. We hypothesize that phosphorylated Y155 and Y311 on PKC? initiate different signaling pathways than other tyrosine residues on PKC? through association of different signaling molecules and we will test this hypothesis by biochemical/proteomic approaches. 2) We hypothesize that PKC? plays a distinct role in megakaryocyte differentiation and platelet production. We propose to test this hypothesis using bone marrow megakaryocytes from mice lacking PKC? as well as from Y155F and Y311F knock-in mice. The studies proposed in this application will enhance our understanding of PKC? signaling networks and their regulation of functional responses in platelets and the production of platelets. These studies will then help us identify targets to trea thrombotic disorders as well as thrombocytopenia.

Public Health Relevance

Platelet activation is critical for hemostasis and can lead to thrombotic events. The receptor targeted by successful drugs to treat heart attacks and stroke may be involved in some inflammatory diseases. An in-depth understanding of the role of this receptor in these disease states will aid in identifying novel targets of antithrombotic therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL093231-05
Application #
8760798
Study Section
Special Emphasis Panel (ZRG1-VH-B (02))
Program Officer
Sarkar, Rita
Project Start
2008-07-01
Project End
2018-06-30
Budget Start
2014-09-08
Budget End
2015-06-30
Support Year
5
Fiscal Year
2014
Total Cost
$463,131
Indirect Cost
$166,252
Name
Temple University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol A et al. (2015) C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets. Biochem Pharmacol 93:163-70
Bhavanasi, Dheeraj; Kostyak, John C; Swindle, John et al. (2015) CGX1037 is a novel PKC isoform delta selective inhibitor in platelets. Platelets 26:9-Feb
Liverani, Elisabetta; Kilpatrick, Laurie E; Tsygankov, Alexander Y et al. (2014) The role of P2Y?? receptor and activated platelets during inflammation. Curr Drug Targets 15:720-8
Liverani, Elisabetta; Rico, Mario C; Yaratha, Laxmikausthubha et al. (2014) LPS-induced systemic inflammation is more severe in P2Y12 null mice. J Leukoc Biol 95:313-23
Dangelmaier, C; Manne, B K; Liverani, E et al. (2014) PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses. Thromb Haemost 111:508-17
Kostyak, John C; Bhavanasi, Dheeraj; Liverani, Elisabeta et al. (2014) Protein kinase C ? deficiency enhances megakaryopoiesis and recovery from thrombocytopenia. Arterioscler Thromb Vasc Biol 34:2579-85
Liverani, Elisabetta; Rico, Mario C; Garcia, Analia E et al. (2013) Prasugrel metabolites inhibit neutrophil functions. J Pharmacol Exp Ther 344:231-43
Buitrago, Lorena; Bhavanasi, Dheeraj; Dangelmaier, Carol et al. (2013) Tyrosine phosphorylation on spleen tyrosine kinase (Syk) is differentially regulated in human and murine platelets by protein kinase C isoforms. J Biol Chem 288:29160-9
Kim, Soochong; Cipolla, Lina; Guidetti, Gianni et al. (2013) Distinct role of Pyk2 in mediating thromboxane generation downstream of both G12/13 and integrin *IIb*3 in platelets. J Biol Chem 288:18194-203
Manne, Bhanu Kanth; Getz, Todd M; Hughes, Craig E et al. (2013) Fucoidan is a novel platelet agonist for the C-type lectin-like receptor 2 (CLEC-2). J Biol Chem 288:7717-26

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