Abstract: Activated protein C (APC) exerts endothelial cytoprotective actions that require protease-activated receptor 1 (PAR1), whereas thrombin acting via PAR1 causes endothelial disruptive, proinflammatory actions. APC's activities, but not thrombin's, require PAR1 located in caveolae. PAR1 is a biased 7-transmembrane receptor because G proteins mediate thrombin's signaling, whereas β-arrestin 2 mediates APC's signaling. Here we elucidate novel mechanisms for APC's initiation of signaling. Biochemical studies of APC's protease specificity showed that APC cleaved PAR1 sequences at both Arg41 and Arg46. That PAR1 cleavage at Arg46 can occur on cells was supported by APC's cleavage of N-terminal-SEAP-tagged R41Q-PAR1 but not R41Q/R46Q-PAR1 mutants transfected into cells and by anti-PAR1 epitope mapping of APC-treated endothelial cells. A synthetic peptide composing PAR1 residues 47-66, TR47, stimulated protective signaling in endothelial cells as reflected in Akt and glycogen synthase kinase 3β phosphorylation, Ras-related C3 botulinum toxin substrate 1 activation, and barrier stabilization effects. In mice, the TR47 peptide reduced VEGF-induced vascular leakage. These in vitro and in vivo data imply that the novel PAR1 N-terminus beginning at residue Asn47, which is generated by APC cleavage at Arg46, mediates APC's cytoprotective signaling and that this unique APC-generated N-terminal peptide tail is a novel biased agonist for PAR1
Abstract: Type II phosphatidylinositol (PtdIns) 4-kinases produce PtdIns 4-phosphate, an early key signaling molecule in phosphatidylinositol cycle, which is indispensable for T cell activation. Type II PtdIns 4-kinase alpha and beta have similar biochemical properties. To distinguish these isoforms Epigallocatechin gallate (EGCG) has been evaluated as a specific inhibitor. EGCG is the major active catechin in green tea having anti-inflammatory, antiatherogenic and cancer chemopreventive properties. The precise mechanism of actions and molecular targets of EGCG in early signaling cascades are not well understood. In the present study, we have shown that EGCG inhibits type II PtdIns 4-kinases (α and β isoforms) and PtdIns 3-kinase activity in vitro. EGCG directly bind to both alpha and beta isoforms of type II PtdIns 4-kinases with a Kd of 2.62 μM and 1.02 μM, respectively. Type II PtdIns 4-kinase-EGCG complex have different binding pattern at its excited state. Both isoforms showed significant change in helicity upon binding with EGCG. EGCG modulates its effect by interacting with ATP binding pocket; the residues likely to be involved in EGCG binding were predicted by Autodock. Our findings suggest that EGCG inhibits two isoforms and could be a key to regulate T cell activation.
Abstract: Phosphorylated derivatives of phosphatidylinositol (PtdIns) are key components of many signaling cascades. Many isoforms of PtdIns kinases, PtdIns phosphate kinases and phosphatases use these lipids in amazing networks of signaling cascades that are yet to be understood fully. PtdIns 4-kinase(s) phosphorylates PtdIns at the 4th -OH position of inositol head group and are classified in to type II and III PtdIns 4-kinases. While type III PtdIns 4-kinases are implicated in vesicular trafficking, type II PtdIns 4-kinases are suggested to play a role in cell signaling, cytoskeletal rearrangements, cell motility and in microbial pathogenicity. This paper reviews the role of type II PtdIns 4-kinases in cell signaling cascades in health and disease.
Abstract: Phosphatidylinositol lipid signaling cascades are integral part of TCR-CD3 signaling. The mechanisms by which phosphatidylinositol kinases are coupled to TCR-CD3 complex remain elusive. Here we report an association of type II PtdIns 4-kinase with TCR-CD3 zeta chain upon cross-linking. Mapping studies have revealed that the C-terminal ITAM is critical for docking of the enzyme on the zeta chain. The association is shown to be tyrosyl phosphorylation dependent as mutation of Y-151 and Y-142 on the C-terminal ITAM disrupts interaction of the two proteins. Identification of the associated type II PtdIns 4-kinase revealed that the beta isoform of the enzyme interacts with the zeta chain in vivo.
Abstract: T cells show rapid reorganization of cytoskeleton in response to antigenic stimulation. The molecular mechanisms by which TCR-CD3 regulates actin cytoskeleton are not well defined. Here we show that a type II PtdIns 4-kinase associates with cytoskeletal fraction in splenic lymphocytes in response to Con A. Protein tyrosyl phosphorylation of type II PtdIns 4-kinase appears to be the mechanism for its association with cytoskeleton. Over-lay blots suggest that the enzyme binds to TCR-CD3 zeta chain in the cytoskeletal fraction. Anti-TCR-CD3 zeta antibodies competitively inhibit PtdIns 4-kinase association with TCR-CD3 zeta chain. Immunodepletion of TCR-CD3 zeta decreases PtdIns 4-kinase activity in the cytoskeletal fraction with a concomitant increase in PtdIns 4-kinase activity in anti-TCR-CD3 zeta immunoprecipitates. We propose that the association of type II PtdIns 4-kinase with TCR-CD3 zeta chain may bring the enzyme into close proximity of actin and a possible regulation of actin polymerization through localized production of PtdIns4P and PtdIns(4,5)P2.
