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.
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.
|Soroush, Fariborz; Tang, Yuan; Guglielmo, Kimberly et al. (2018) Protein Kinase C-Delta (PKC?) Tyrosine Phosphorylation is a Critical Regulator of Neutrophil-Endothelial Cell Interaction in Inflammation. Shock :|
|Janapati, S; Wurtzel, J; Dangelmaier, C et al. (2018) TC21/RRas2 regulates glycoprotein VI-FcR?-mediated platelet activation and thrombus stability. J Thromb Haemost :|
|Badolia, Rachit; Kostyak, John C; Dangelmaier, Carol et al. (2017) Syk Activity Is Dispensable for Platelet GP1b-IX-V Signaling. Int J Mol Sci 18:|
|Badolia, Rachit; Inamdar, Vaishali; Manne, Bhanu Kanth et al. (2017) Gq pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets. J Biol Chem 292:14516-14531|
|Buitrago, Lorena; Manne, Bhanu Kanth; Andre, Pierrette et al. (2016) Identification of novel Syk-independent functional roles of Fc?RIIa in platelet outside-in signaling using transgenic mice expressing human Fc?RIIa. Platelets 27:488-90|
|Liverani, Elisabetta; Rico, Mario C; Tsygankov, Alexander Y et al. (2016) P2Y12 Receptor Modulates Sepsis-Induced Inflammation. Arterioscler Thromb Vasc Biol 36:961-71|
|Reppschläger, Kevin; Gosselin, Jeanne; Dangelmaier, Carol A et al. (2016) TULA-2 Protein Phosphatase Suppresses Activation of Syk through the GPVI Platelet Receptor for Collagen by Dephosphorylating Tyr(P)346, a Regulatory Site of Syk. J Biol Chem 291:22427-22441|
|Inamdar, Vaishali; Patel, Akruti; Manne, Bhanu Kanth et al. (2015) Characterization of UBO-QIC as a G?q inhibitor in platelets. Platelets 26:771-8|
|Badolia, Rachit; Manne, Bhanu Kanth; Dangelmaier, Carol et al. (2015) IPA3 non-specifically enhances phosphorylation of several proteins in human platelets. Platelets 26:501-3|
|Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol et al. (2015) Distinct pathways regulate Syk protein activation downstream of immune tyrosine activation motif (ITAM) and hemITAM receptors in platelets. J Biol Chem 290:11557-68|
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