The GPI-anchored superoxide dismutase SodC is essential for regulating basal Ras activity and for chemotaxis of Dictyostelium discoideum

Abstract

A genetic screen for Dictyostelium mutant displaying high level of constitutive phosphatidylinositol (3,4,5)-trisphosphate led to the finding that the glycosylphosphatidylinositol (GPI)-anchored superoxide dismutase SodC regulates small GTPase Ras. Cells that lack SodC exhibited constitutively high levels of active Ras, more membrane localization of GFP-PHcrac, and defects in chemoattractant sensing, cell polarization and motility. These defects of SodC-lacking cells were partially restored by expression of wild-type SodC but not by the catalytically inactive mutant SodC (H245R, H247Q). Furthermore, an inhibition of PI3K activity in SodC-deficient cells by LY294002 only partially restored chemoattractant sensing and cell polarization, consistent with the fact that SodC-deficient cells have aberrantly high level of active Ras, which functions upstream of PI3K. A higher level of active GFP-RasG was observed in SodC-deficient cells, which significantly decreased upon incubation of SodC-deficient cells with the superoxide scavenger XTT. Having constitutively high levels of active Ras proteins and more membrane localization of GFP-PHcrac, SodC-deficient cells exhibited severe defects in chemoattractant sensing, cell polarization and motility.

Mechanism of Superoxide Mediated Regulation of Particle Uptake and Exocytosis by a GPI-anchored Superoxide Dismutase C in Dictyostelium

“…Considering that SodC is regulating RasG, which in turn controls PI3K activation and thus macropinocytosis, the aberrant hyperactivation of RasG in sodC -cells is likely the cause for the decreased particle uptake. It is, however, puzzling that PI3K inhibition did not improve particle uptake of sodC -cells, considering that LY294002 treated sodC -cells displayed excellent chemotaxis (Veeranki et al, 2008). It is thus likely that there exist additional defects that hamper particle uptake behavior of This study, for the first time, demonstrated that proper metabolism of reactive oxygen species Superoxide is critical for efficient particle uptake, proper trafficking of the internalized particles, and exocytosis in Dictyostelium cells.…”

“…Cells that lack Superoxide Dismutase C, experience oxidative stress as a result of modestly increased intracellular superoxide level (~18% higher compared to wild type), high basal RasG activity and defects in chemoattractant sensing, cell polarization and motility (Veeranki et al, 2008).…”

“…In our laboratory we have previously generated Dictyostelium discoideum cells lacking one of the superoxide dismutases, SodC, in order to understand the role of superoxide in biological processes such as chemotaxis and particle uptake. SodC is a glycosylphosphatidylinositol (GPI)-anchored enzyme that is located in the outer leaflet of the plasma membrane and is involved in the regulation of intracellular level of superoxide in Dictyostelium cells (Veeranki et al, 2008).…”

“…The goal of the study was to determine if EC-SOD localize to the nucleus, but the endosomal EC-SOD proteins did not translocate to the nucleus and so the function of endosomal EC-SOD is not known. Dictyostelium cells also contain multiple types of SOD protein including the extracellular one, SodC (Veeranki, et al, 2008). Dictyostelium SodC localizes on the plasma membrane (Veeranki et al, 2008), but the follow up study showed that cells lacking SodC (sodC Earlier studies on sodC -cells disclosed its role in directional cell migration through regulating redox sensitive small GTPase RasG (Veeranki et al, 2008).…”

Two Adaptation Mechanisms Regulate Cellular Migration in Dictyostelium discouideum

“…Secondly, GTP bound Ras proteins were purified from cells lysates using pulldown assays as previously described by Veeranki, S. et al 2008. The activated forms of RasD and RasG were purified using recombinant GST-Ras binding domain (RBD) from mammalian Raf1 (GST-RBDRaf1) and the activated form of RasC was purified using recombinant GST-Ras Binding Domain of Byr2 from Schizosaccharomyces pombe (GST-RBDByr2).…”