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A Study was conducted on recombinant HIV-1 Tat and its interaction with TAR RNA. Results showed that recombinant Tat's structure contains a 15 to 20% alpha-helix. Tat was also found to bind to a 63-nucleotide target TAR RNA. The source of free energy of association was traced to nonelectrostatic interactions. Protein-RNA cross-linking also showed that the Tat protein and TAR RNA came into direct contact.

Abstract: Cyclooxygenase-2 (COX-2) is rapidly induced by angiotensin (Ang)-II in the non-tumorigenic, rat intestinal epithelial cell line, IEC-18, through its G-protein coupled receptor, AT1R. Here, we investigate the ability of Ang II to regulate transcription of the COX-2 promoter and a Gal4-CREB heterologous promoter in IEC-18 cells. Ang II and EGF induced similar levels of transcription from the COX-2 and Gal4-CREB promoters. Over-expression of constitutively active Gα proteins q, 11, 12 and 13, showed induction by GαqQ209L and by Gα11Q209L for both the COX-2 and Gal4-CREB promoters. Co-expression of RGS 2, 3 or 4 but not the RGS domain of p115RhoGEF inhibited Ang II-dependent induction of the COX-2 and Gal4-CREB promoters. Expression of constitutively active MKK6 EE but not MKK3 EE induced the COX-2 and Gal4-CREB promoters via p38MAPK. SB202190 but not PD98059 inhibited induction of the COX-2 promoter by over-expression of the constitutively active PAK1T423E. Expression of the kinase-inactive PAK1K299R inhibited both Ang II-dependent induction of the COX-2 promoter and induction of the COX-2 and Gal4-CREB promoters by GαqQ209L. These data demonstrate that in IEC-18 cells, Ang II-dependent activation of the COX-2 promoter is mediated primarily through Gq/11 signaling via a PAK/MKK6/p38β/CREB signaling cascade. [Copyright &y& Elsevier]

Cyclooxygenase (COX)-2 derived prostaglandins (PGs) play a major role in intestinal inflammation and colorectal carcinogenesis. Because COX-2 is the rate-limiting step in the production of PGs, mechanisms that regulate COX-2 expression control PG production in the cell. Using the non-tumorigenic, rat intestinal epithelial cell, IEC-18, we demonstrate that co-activation of endogenously expressed AT1 receptor and EGFR resulted in synergistic expression of COX-2 mRNA and protein involving transcriptional and post-transcriptional mechanisms. Ang II and EGF induced transient phosphorylation of ERK, p38MAPK and CREB. Co-stimulation with Ang II and EGF prolonged phosphorylation of ERK, p38MAPK, and CREB. The p38MAPK selective inhibitor, SB202190, but not the MEK selective inhibitor, PD98059, or the EGFR kinase inhibitor, AG1478, inhibited Ang II-dependent COX-2 expression and CREB phosphorylation. EGF-dependent COX-2 expression and CREB phosphorylation were inhibited by SB202190, PD98059, and AG1478. Inhibition of CREB expression using two separate RNAi methods blocked COX-2 expression by Ang II and EGF. Expression of a dominant negative CREB mutant inhibited Ang II- and EGF-dependent induction of the COX-2 promoter. Ang II induced luciferase expression in cells transfected with the CRE-luc reporter vector and cells co-transfected with Gal4-luc reporter vector and a Gal4-CREB expression vector. Chromatin immunoprecipitation assays demonstrated CREB binding to the proximal rat COX-2 promoter region containing a CRE cis-acting element. These results indicate that co-stimulation with Ang II and EGF synergistically induced COX-2 expression in these intestinal epithelial cells through p38MAPK mediated signaling cascades that converge onto CREB. J. Cell. Physiol. 214:96–109, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Cyclooxygenase (COX)-2 expression in intestinal epithelial cells is associated with colorectal carcinogenesis. COX-2 expression is induced by numerous growth factors and gastrointestinal hormones through multiple protein kinase cascades. Here, the role of mitogen activated protein kinases (MAPKs) and small GTPases in COX-2 expression was investigated. Anisomycin and sorbitol induced COX-2 expression in non-transformed, intestinal epithelial IEC-18 cells. Both anisomycin and sorbitol activated p38MAPK followed by phosphorylation of CREB. SB202190 and PD169316 but neither PD98059 nor U0126 blocked COX-2 expression and CREB phosphorylation by anisomycin or sorbitol. Clostridium difficile toxin B inhibition of small GTPases did not affect anisomycin-induced COX-2 mRNA expression or phosphorylation of p38MAPK and CREB but did inhibit sorbitol-dependent COX-2 expression and phosphorylation of p38MAPK and CREB. Angiotensin (Ang) II-dependent induction of COX-2 mRNA and induced phosphorylation of p38MAPK and CREB were inhibited by toxin B. Reduction of CREB protein in cells transfected with CREB siRNAs inhibited anisomycin-induced COX-2 expression. These results indicate that activation of p38MAPK signaling is sufficient for COX-2 expression in IEC-18 cells. Ang II and sorbitol require small GTPase activity for COX-2 expression via p38MAPK while anisomycin-induced COX-2 expression by p38MAPK does not require small GTPases. This places small GTPase activity down-stream of the AT1 receptor and hyperosmotic stress and up-stream of p38MAPK and CREB. [Copyright &y& Elsevier]

Tests the hypothesis that lysophosphatidic acid induces protein kinase D (PKD) through multiple G protein signal transduction pathways. Experimental procedures; Attenuation of PKD activity; Point of convergence in the active of G protein pathways.

Cyclooxygenase (COX)-2 derived prostaglandins (PGs) play a major role in intestinal inflammation and colorectal carcinogenesis. Because COX-2 is the rate-limiting step in the production of PGs, mechanisms that regulate COX-2 expression control PG production in the cell. Using the non-tumorigenic, rat intestinal epithelial cell, IEC-18, we demonstrate that co-activation of endogenously expressed AT(1) receptor and EGFR resulted in synergistic expression of COX-2 mRNA and protein involving transcriptional and post-transcriptional mechanisms. Ang II and EGF induced transient phosphorylation of ERK, p38(MAPK) and CREB. Co-stimulation with Ang II and EGF prolonged phosphorylation of ERK, p38(MAPK), and CREB. The p38(MAPK) selective inhibitor, SB202190, but not the MEK selective inhibitor, PD98059, or the EGFR kinase inhibitor, AG1478, inhibited Ang II-dependent COX-2 expression and CREB phosphorylation. EGF-dependent COX-2 expression and CREB phosphorylation were inhibited by SB202190, PD98059, and AG1478. Inhibition of CREB expression using two separate RNAi methods blocked COX-2 expression by Ang II and EGF. Expression of a dominant negative CREB mutant inhibited Ang II- and EGF-dependent induction of the COX-2 promoter. Ang II induced luciferase expression in cells transfected with the CRE-luc reporter vector and cells co-transfected with Gal4-luc reporter vector and a Gal4-CREB expression vector. Chromatin immunoprecipitation assays demonstrated CREB binding to the proximal rat COX-2 promoter region containing a CRE cis-acting element. These results indicate that co-stimulation with Ang II and EGF synergistically induced COX-2 expression in these intestinal epithelial cells through p38(MAPK) mediated signaling cascades that converge onto CREB., ((c) 2007 Wiley-Liss, Inc.)

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Galpha(q/11) (Galpha(q/11) KO cells) or Galpha(12/13) (Galpha(12/13) KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Galpha(12/13) KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Galpha(12/13) KO cells in autophosphorylation activity) achieved at 3 microm. In contrast, LPA did not induce any significant increase in PKD activity in Galpha(q/11) KO cells. However, LPA induced a significantly increased PKD activity when Galpha(q/11) KO cells were transfected with Galpha(q). LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Galpha(12/13) KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that G(q) is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to G(q), LPA requires additional G-protein pathways to elicit a maximal response with G(i) playing a critical role in Galpha(12/13) KO cells. We conclude that LPA induces PKD activation through G(q), G(i), and G(12) and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.