Biomedical subjects

C Vasudevan

Publications and source records attributed to C Vasudevan.

7 recordsLinked to original sources

PubMed2000

The activation of phospholipase D by endothelin-1, angiotensin II, and platelet-derived growth factor in vascular smooth muscle A10 cells is mediated by small G proteins of the ADP-ribosylation factor family.

We show here that A10 cells express the phospholipase D (PLD) isoforms PLD1b and PLD2. The activation of PLD in these cells by angiotensin II (AngII), endothelin-1 (ET-1), and platelet-derived growth factor (PDGF) was found to be sensitive to inhibitors of the activation of ADP-ribosylation factor (ARF) but not to blockers of Rho protein function. PDGF, AngII, and ET-1 induced the binding of ARF proteins to cell membranes in a permeabilized cell assay. Cells permeabilized and depleted of ARF were no longer sensitive to stimulation with AngII, ET-1, or PDGF, but the addition of recombinant myristoylated human ARF1 restored agonist-dependent PLD activity. Expression of dominant negative ARF mutants blocked receptor-dependent activation of PLD. PLD activity was also potently stimulated by treatment with phorbol esters, but this activity was only partially inhibited by brefeldin A or by the overexpression of ARF dominant negative mutants. Transient expression of catalytically inactive mutants of PLD2, but not PLD1, inhibited significantly PDGF- and AngII-dependent PLD activity. We conclude: 1) the activation of PLD by cell surface receptors occurs primarily by an ARF-dependent mechanism in A10 cells, whereas the activation of PLD by protein kinase C-dependent pathways is only partially dependent on the regulation of ARF proteins; and 2) cell surface receptors, such as AngII and PDGF, signal primarily via PLD2 in A10 cells.

ADP Ribose Transferases↗

PubMed1999

The KDEL receptor regulates a GTPase-activating protein for ADP-ribosylation factor 1 by interacting with its non-catalytic domain.

ADP-ribosylation factor 1 (ARF1) is a key regulator of transport in the secretory system. Like all small GTPases, deactivation of ARF1 requires a GTPase-activating protein (GAP) that promotes hydrolysis of GTP to GDP on ARF1. Structure-function analysis of a GAP for ARF1 revealed that its activity in vivo requires not only a domain that catalyzes hydrolysis of GTP on ARF1 but also a non-catalytic domain. In this study, we show that the non-catalytic domain of GAP is required for its recruitment from cytosol to membranes and that this domain mediates the interaction of GAP with the transmembrane KDEL receptor. Blocking its interaction with the KDEL receptor leaves the GAP cytosolic and prevents the deactivation in vivo of Golgi-localized ARF1. Thus, these findings suggest that the KDEL receptor plays a critical role in the function of GAP by regulating its recruitment from cytosol to membranes, where it can then act on its membrane-restricted target, the GTP-bound form of ARF1.

ADP-Ribosylation Factor 1↗

PubMed1999

Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway.

The primary known function of phospholipase D (PLD) is to generate phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine. However, the functional role of PA is not well understood. We report here evidence that links the activation of PLD by insulin and the subsequent generation of PA to the activation of the Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A (BFA), an inhibitor of the activation of ADP-ribosylation factor proteins, inhibited insulin-dependent production of PA and MAPK phosphorylation. The addition of PA reversed the inhibition of MAPK activation by BFA. Overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocked insulin-dependent activation of PLD and phosphorylation of MAPK. Real time imaging analysis showed that insulin induced Raf-1 translocation to cell membranes by a process that was inhibited by BFA. PA addition reversed the effects of BFA on Raf-1 translocation. However, PA did not activate Raf-1 in vitro or in vivo, suggesting that the primary function of PA is to enhance the recruitment of Raf-1 to the plasma membrane where other factors may activate it. Finally, we found that the recruitment of Raf-1 to the plasma membrane was transient, but Raf-1 remained bound to endocytic vesicles.

Animals↗

PubMed1998

The distribution and translocation of the G protein ADP-ribosylation factor 1 in live cells is determined by its GTPase activity.

ADP-ribosylation factors (ARF) are small G proteins that play key roles in vesicular transport processes. We have studied the distribution of ARF1 in live cells using chimeras of ARF1 mutants (wild type (wt) ARF1; Q71L-ARF1 (reduced GTPase); T31N (low affinity for GTP); and (Delta)Nwt (deletion of amino acids 2-18)) with green fluorescent protein (GFP). Confocal microscopy studies showed that the wt and Q71L proteins were localized in the Golgi and cytoplasm. The (Delta)Nwt and the T31N mutants were exclusively cytoplasmic. The behavior of the wt and Q71L proteins was studied in detail. About 15% of wt-ARF1-GFP was bound to the Golgi. Bound wt-ARF1-GFP dissociated rapidly after addition of Brefeldin A (BFA). This process did not appear to be a consequence of BFA-induced disappearance of the Golgi. Photobleaching recovery showed that essentially all the ARF-GFP was mobile, although it diffused very slowly. In contrast, about 40-50% of the Q71L mutant was found in the Golgi, and its rate of dissociation in the presence of BFA was slow and biphasic. Q71L-ARF1-GFP diffused more slowly than the wt. We conclude that ARF1 proteins exist in a dynamic equilibrium between Golgi-bound and cytosolic pools, and that the translocation of ARF in live cells requires the hydrolysis of GTP by the Golgi-bound protein.

ADP-Ribosylation Factor 1↗

PubMed1997

The elevation of cellular phosphatidic acid levels caused by polyomavirus transformation can be disassociated from the activation of phospholipase D.

Middle T (mT), the oncogene of murine polyomavirus, causes transformation of rat fibroblasts by activating a number of signal transducing pathways usually used by polypeptide growth factors and their receptors. Here, we report data regarding the activation of signal transducing pathways involving phospholipase D (PL-D). The hydrolysis of phospholipids by PL-D produces phosphatidic acid (PA), a compound with multiple biological effects. The PA content of cells expressing wild-type mT, introduced via a number of different methods, is approximately 50% higher than their untransformed counterparts. This increase in cellular PA content is associated with an approximately 65% increase in PL-D activity in cells expressing wild-type mT. We have also examined the effects of a number of site-directed mutants of mT, on both cellular PA levels and on PL-D activity. Mutants that do not produce mT (Py808A) or that produce a truncated, nonmembrane bound mT (Py1387T) have PA levels similar to that of control cells. Cells expressing the 322YF mutant of mT (which abolishes interaction of mT with phospholipase C gamma1) show increases in both PA levels and PL-D activity that are similar to those seen with wild-type mT. Expression of mutants that abolish the interaction of mT with either shc or with phosphatidylinositol 3-kinase (250YS and 315YF, respectively) cause an increase in PL-D activity comparable to that seen with wild-type mT. However, the PA content of cells expressing these mutants is not elevated. These results suggest that mT causes activation of cellular PL-D, but this activation alone is not sufficient to cause an increase in cellular PA content. Therefore, wild-type mT must affect another, as yet unknown, step in PA metabolism.

Animals↗

PubMed1997

ARF proteins mediate insulin-dependent activation of phospholipase D.

BACKGROUND: ADP-ribosylation factors (ARFs) have been shown to activate phospholipase D (PLD), an enzyme modulated by extracellular signals, including several growth factors and, in particular, insulin. We have tested the hypothesis that ARF proteins are involved specifically in insulin-induced activation of PLD. RESULTS: We found that in membranes obtained from HIRcB cells, a cell line derived from Rat-1 fibroblasts that overexpresses normal human insulin receptors, binding of the GTP analogue GTPgammaS to purified bovine or recombinant ARF was enhanced in the presence of insulin. Membranes obtained from cells that overexpressed a mutated, nonfunctional insulin receptor failed to stimulate ARF activation. Insulin promoted the association of ARF proteins with membranes in the presence of GTPgammaS in permeabilized cells. Insulin activated PLD in permeabilized HIRcB cells by a process that required GTPgammaS and ARF. Azido-gamma[32P]-GTP labelling of immunoprecipitated receptors revealed the presence of a unique 19 kD band; ARF proteins are approximately this size, and analysis using specific monoclonal antibodies demonstrated that ARF proteins coimmunoprecipitated with the insulin receptor. Coimmunoprecipitation of ARF with the receptor was inhibited by guanine nucleotides and stimulated by insulin. No evidence of the coprecipitation of ARF with mutant receptors could be obtained using azido-gamma[32P]-GTP or anti-ARF antibodies. CONCLUSIONS: The activation of ARF proteins is stimulated by insulin and this process plays an important role in insulin-mediated regulation of PLD.

ADP-Ribosylation Factors↗

PubMed1990

Artificial intelligence for medical decision making.

Artificial intelligence techniques find extensive applications in medical decision making and other aspects of health care. A number of successful expert systems have been developed in various disciplines of medicine. This paper gives an overview of expert system techniques, describes some practical systems, and discusses the relevance of such systems in clinical diagnosis and management of diseases.

Decision Making, Computer-Assisted↗