Biologically active lipids promote trafficking and membrane association of Rac1 in insulin-secreting INS 832/13 cells

McDonald, Philip; Veluthakal, Rajakrishnan; Kaur, Hitchintan; Kowluru, Anjaneyulu

doi:10.1152/ajpcell.00467.2006

Cited by 33 publications

(31 citation statements)

References 34 publications

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“…In an attempt to identify precise regulatory mechanisms involved in glucose-mediated activation of Rac1 and insulin secretion, we first proposed [22] and subsequently confirmed experimentally [23] that certain biologically-active lipid second messengers [e.g., PA, PIP 2 ] promote dissociation of Rac1 from Rac1/GDI complex to facilitate activation of Rac1 in rodent islets and clonal β-cells. Therein, we also proposed that Arf6 could represent one of the upstream regulators of Rac1 activation by generating relevant lipid second messengers via phospholipase activation to dissociate the Rac1/GDI complex [22, 23].…”

Section: Introductionmentioning

confidence: 99%

Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets

Jayaram

Syed

Kyathanahalli

et al. 2011

Biochemical Pharmacology

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Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. secinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. secinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS.

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Section: Introductionmentioning

confidence: 99%

Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets

Jayaram

Syed

Kyathanahalli

et al. 2011

Biochemical Pharmacology

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“…Rac1 has been shown to control multiple cellular processes, including actin/microtubule cytoskeleton organisation (Colley, 2000), reactive oxygen species (ROS) generation (Bishop and Hall, 2000; Whaley-connell et al , 2007), membrane trafficking (McDonald et al , 2007), hypoxia-stimulated breast cancer cell migration (Han et al , 2008; Vermeulen et al , 2010), cell adhesion and migration (Nobes and Hall, 1999; Kraynov et al , 2000; Ridley, 2001), and cell proliferation (Benitah et al , 2004; Choi et al , 2009). Because Rac1 is also a member of the NADPH oxidase, which generates ROS (Bokoch and Diebold, 2002), it regulates NADPH oxidase complex assembly in most cells (Diebold, et al , 2009), and it has been shown to be a critical determinant of intracellular redox status.…”

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confidence: 99%

Role of SNTA1 in Rac1 activation, modulation of ROS generation, and migratory potential of human breast cancer cells

et al. 2014

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Background:Alpha-1-syntrophin (SNTA1) has been implicated in the activation of Rac1. However, the underlying mechanism has not yet been explored. Here, we show that a novel complex, involving SNTA1, P66shc, and Grb2 proteins, is involved in Rac1 activation.Methods:Co-immunoprecipitation assays were used to show the complex formation, while siRNAs and shRNAs were used to downregulate expression of these proteins. Various Rac1 activation assays and functional assays, such as migration assays, in vitro wound healing assays, cell proliferation assays, and ROS generation assays, were also performed.Results:The results showed a significant increase in activation of Rac1 when SNTA1 and P66shc were overexpressed, whereas depletion of SNTA1 and P66shc expression effectively reduced the levels of active Rac1. The results indicated a significant displacement of Sos1 protein from Grb2 when SNTA1 and P66shc are overexpressed in breast cancer cell lines, resulting in Sos1 predominantly forming a complex with Eps8 and E3b1. In addition, the SNTA1/P66shc-mediated Rac1 activation resulted in an increase in reactive oxygen species (ROS) production and migratory potential in human breast cancer cells.Conclusion:Together, our results present a possible mechanism of Rac1 activation involving SNTA1 and emphasise its role in ROS generation, cell migration, and acquisition of malignancy.

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“…8). We propose that glucose-induced activation of PLD leads to the generation of biologically active lipids, which, in turn, regulate G-protein (e.g., Rac1) function by dissociating Rac1 from its inhibitory GDI [10]. It is also likely that glucose-induced, Arf6-mediated ERK1/2 activation facilitates Rac1 activation as we have demonstrated recently [6,26].…”

Section: Discussionmentioning

confidence: 67%

Phagocytic NADPH Oxidase Links ARNO-Arf6 Signaling Pathway in Glucose-Stimulated Insulin Secretion from the Pancreatic �-Cell

Jayaram

Kowluru

2012

Cell Physiol Biochem

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Background: Recent findings from our laboratory have demonstrated that glucose-stimulated insulin secretion (GSIS) involves interplay between a variety of small G proteins belonging to the Rho (e.g., Cdc42 and Rac1) and ADP-ribosylation factor (e.g., Arf6) subfamilies. Using immunological, pharmacological and molecular biological approaches, we have also identified guanine nucleotide exchange factors (GEFs) for Rac1 (e.g., Tiam1) and Arf6 (e.g., ARNO) in clonal INS-1 832/13 cells, normal rat islets and human islets. As a logical extension to these studies, we investigated, herein, potential downstream signaling steps involved in Arf6/ARNO-mediated GSIS. Methods: Using a selective pharmacological inhibitor of ARNO/Arf6 signaling axis (e.g., secinH3) we assessed regulatory roles for Arf6/ARNO in promoting phospholipase D (PLD), phagocytic NADPH oxidase (Nox2), reactive oxygen species (ROS), extracellular-regulated kinases (ERK 1/2) and cofilin (actin-severing protein] signaling steps in clonal INS-1 832/13 cells. Results: Our data suggested a marked inhibition by secinH3 of glucose-induced PLD activation, ERK1/2 phosphorylation and dephosphorylation of cofilin, suggesting that Arf6/ ARNO signaling mediates PLD, ERK1/2 and cofilin activation in beta-cells. In addition, secinH3 blocked glucose-induced Nox2 activation and associated ROS generation, thus placing Nox downstream to Arf6/ARNO signaling step. Lastly, we also demonstrate a significantly higher cofilin phosphorylation (inactive) in islets derived from type 2 diabetic human donors as well as the Zucker Diabetic Fatty (ZDF) rat, a model for type 2 diabetes. Conclusion: Together, our current findings identify signaling steps downstream to ARNO/Arf6 axis leading to insulin secretion.

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Biologically active lipids promote trafficking and membrane association of Rac1 in insulin-secreting INS 832/13 cells

Abstract: McDonald P, Veluthakal R, Kaur H, Kowluru A. Biologically active lipids promote trafficking and membrane association of Rac1 in insulin-secreting INS 832/13 cells.

Cited by 33 publications

References 34 publications

Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets

Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets

Role of SNTA1 in Rac1 activation, modulation of ROS generation, and migratory potential of human breast cancer cells

Phagocytic NADPH Oxidase Links ARNO-Arf6 Signaling Pathway in Glucose-Stimulated Insulin Secretion from the Pancreatic �-Cell

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