[Abstract unavailable

Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2-to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/ PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/ threonine rich C-terminal region impaired both RAC-PKa basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKa activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.The RAC-PKs (for related to PKA and C protein kinases; also known as PKB/Akt) represent a subfamily of second messenger-regulated serine/threonine protein kinases (1). Two human genes have been identified, termed RACa and -Pf that are 90% homologous (2-4). Both genes appear to be widely expressed in human tissues, implying that they play an important role in cell signaling. Mouse RACa (c-akt) is the cellular homologue of the viral oncogene v-akt, generated by fusion of the Gag protein from the AKT8 retrovirus to the N terminus of mouse RAC-PKa, giving rise to a 105-kDa phosphoprotein that is myristilated at its N terminus (5, 6). The mouse protein is mainly cytosolic (90%), whereas the oncoprotein is apparently equally distributed between the plasma membrane, nucleus, and cytoplasm (6). Human RACf3 was found to be amplified in 10% of human ovarian carcinomas (4), suggesting the involvement of the RAC-PK subfamily members in regulation of cell growth. The Drosophila homologue (DRAC) shows 75% homology to the human isoforms and is ubiquitously expressed throughout the Drosophila life cycle (7,8).All characterized members of the RAC-PK subfamily have a similar domain structure: an N-terminal pleckstrin homologyThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.(PH) domain, a centrally located catalytic do...

Background-Arginase competes with endothelial nitric oxide synthase (eNOS) for the substrate L-arginine and decreases NO production. This study investigated regulatory mechanisms of arginase activity in endothelial cells and its role in atherosclerosis. Methods and Results-In human endothelial cells isolated from umbilical veins, thrombin concentration-and timedependently stimulated arginase enzymatic activity, reaching a 1.9-fold increase (PϽ0.001) at 1 U/mL for 24 hours. The effect of thrombin was prevented by C3 exoenzyme or the HMG-CoA reductase inhibitor fluvastatin, which inhibit RhoA, or by the ROCK inhibitors Y-27632 and HA-1077. Adenoviral expression of constitutively active RhoA or ROCK mutants enhanced arginase activity (Ϸ3-fold, PϽ0.001), and the effect of active RhoA mutant was inhibited by the ROCK inhibitors. Neither thrombin nor the active RhoA/ROCK mutants affected arginase II protein level, the only isozyme detectable in the cells. Moreover, a significantly higher arginase II activity (1.5-fold, not the protein level) and RhoA protein level (4-fold) were observed in atherosclerotic aortas of apoE Ϫ/Ϫ compared with wild-type mice. Interestingly, L-arginine (1 mmol/L), despite a significantly higher eNOS expression in aortas of apoE Ϫ/Ϫ mice, evoked a more pronounced contraction, which was reverted to a greater vasodilation by the arginase inhibitor L-norvaline (20 mmol/L) compared with the wild-type animals (nϭ5, PϽ0.001). Conclusions-Thrombin

BackgroundMacrophage‐mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg‐II) in macrophage function remains elusive. This study characterizes the role of Arg‐II in macrophage inflammatory responses and its impact on obesity‐linked type II diabetes mellitus and atherosclerosis.Methods and ResultsIn human monocytes, silencing Arg‐II decreases the monocytes’ adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low‐density lipoprotein or lipopolysaccharides, as evaluated by real‐time quantitative reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg‐II–deficient (Arg‐II−/−) mice express lower levels of lipopolysaccharide‐induced proinflammatory mediators than do macrophages of wild‐type mice. Importantly, reintroducing Arg‐II cDNA into Arg‐II−/− macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N‐acetylcysteine prevents the Arg‐II–mediated inflammatory responses. Moreover, high‐fat diet–induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg‐II−/− mice. Accordingly, Arg‐II−/− mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)–deficient mice with Arg‐II deficiency (ApoE−/−Arg‐II−/−) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE−/−Arg‐II−/− than ApoE−/−Arg‐II+/+ monocytes infiltrate into the plaque of ApoE−/−Arg‐II+/+ mice. Conversely, recipient ApoE−/−Arg‐II−/− mice accumulate fewer donor monocytes than do recipient ApoE−/−Arg‐II+/+ animals.ConclusionsArg‐II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg‐II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.)

It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However