Mechanism of activation and functional role of protein kinase Ceta in human platelets.
The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.
Bynagari
Yamini S
YS
Departments of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Nagy
Bela
B
Departments of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Tuluc
Florin
F
Departments of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Bhavaraju
Kamala
K
Departments of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Kim
Soochong
S
Departments of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Vijayan
K Vinod
KV
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Kunapuli
Satya P
SP
Departments of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; Pharmacology and the Temple University School of Medicine, Philadelphia, Pennsylvania 19140; Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140. Electronic address: spk@temple.edu.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D039421:Integrin alpha2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010456:Peptides, Cyclic / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D013931:Thromboxanes / Q000096:biosynthesis
C056564:ITGA2B protein, human; D039421:Integrin alpha2; C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; C546382:P2RY1 protein, human; C546383:P2ry1 protein, mouse; D010456:Peptides, Cyclic; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D013931:Thromboxanes; C475455:YM-254890; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C117446:cangrelor; C081975:protein kinase C eta; D011493:Protein Kinase C; D054645:Protein Phosphatase 1; D043802:GTP-Binding Protein alpha Subunits, Gq-G11