Limits...
A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Karunanithi S, Xiong T, Uhm M, Leto D, Sun J, Chen XW, Saltiel AR - Mol. Biol. Cell (2014)

Bottom Line: Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2.Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10.Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.

Show MeSH

Related in: MedlinePlus

Rab10 activates RalA through Rlf. (A) Rab10 activates RalA through a GEF. Wild-type and F39L mutant of RalA were expressed with or without HA-Rab10 in Cos-1 cells. Pull-down assay was performed with GST-RalBP1 beads, and samples were run on SDS–PAGE and Western blotted. (B) Stealth siRNA knockdown of Rlf in 293T cells. Two days posttransfection, cells were lysed, and RalA pull down with GST-RalBP1 beads was performed. Levels of indicated proteins in total cell lysates and pull down are shown. (C) Images from experiment in B were quantified by ImageJ and plotted. *p = 0.05. (D) Cos-1 cells were transfected with FLAG-RalA together with Myc-Rab10 (QL), HA-Rlf, or both. At 24 h later, cells were lysed and subjected to pull-down assay using GST-Sec5-RBD beads. Lysates and pull-down samples were run on SDS–PAGE and immunoblotted.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230594&req=5

Figure 3: Rab10 activates RalA through Rlf. (A) Rab10 activates RalA through a GEF. Wild-type and F39L mutant of RalA were expressed with or without HA-Rab10 in Cos-1 cells. Pull-down assay was performed with GST-RalBP1 beads, and samples were run on SDS–PAGE and Western blotted. (B) Stealth siRNA knockdown of Rlf in 293T cells. Two days posttransfection, cells were lysed, and RalA pull down with GST-RalBP1 beads was performed. Levels of indicated proteins in total cell lysates and pull down are shown. (C) Images from experiment in B were quantified by ImageJ and plotted. *p = 0.05. (D) Cos-1 cells were transfected with FLAG-RalA together with Myc-Rab10 (QL), HA-Rlf, or both. At 24 h later, cells were lysed and subjected to pull-down assay using GST-Sec5-RBD beads. Lysates and pull-down samples were run on SDS–PAGE and immunoblotted.

Mentions: The activation of RalA by Rab10 could be due to inactivation of the RalGAP complex, activation of a RalGEF, or both. RGC1/2 is inactivated upon insulin stimulation through Akt phosphorylation of the catalytic subunit RGC2 (Chen et al., 2011b). Rab10 overexpression did not accelerate or affect the phosphorylation status of RGC2 (unpublished data). Moreover, a point mutation in RalA (F39L/FL) with increased nucleotide dissociation rates that quickly cycles between GDP and GTP (Reinstein et al., 1991; Lim et al., 2005), and thus does not require a GEF for activation, was not influenced by overexpression of Rab10 (QL), unlike what was observed for RalA (wild type [WT]; Figure 3A).


A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Karunanithi S, Xiong T, Uhm M, Leto D, Sun J, Chen XW, Saltiel AR - Mol. Biol. Cell (2014)

Rab10 activates RalA through Rlf. (A) Rab10 activates RalA through a GEF. Wild-type and F39L mutant of RalA were expressed with or without HA-Rab10 in Cos-1 cells. Pull-down assay was performed with GST-RalBP1 beads, and samples were run on SDS–PAGE and Western blotted. (B) Stealth siRNA knockdown of Rlf in 293T cells. Two days posttransfection, cells were lysed, and RalA pull down with GST-RalBP1 beads was performed. Levels of indicated proteins in total cell lysates and pull down are shown. (C) Images from experiment in B were quantified by ImageJ and plotted. *p = 0.05. (D) Cos-1 cells were transfected with FLAG-RalA together with Myc-Rab10 (QL), HA-Rlf, or both. At 24 h later, cells were lysed and subjected to pull-down assay using GST-Sec5-RBD beads. Lysates and pull-down samples were run on SDS–PAGE and immunoblotted.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230594&req=5

Figure 3: Rab10 activates RalA through Rlf. (A) Rab10 activates RalA through a GEF. Wild-type and F39L mutant of RalA were expressed with or without HA-Rab10 in Cos-1 cells. Pull-down assay was performed with GST-RalBP1 beads, and samples were run on SDS–PAGE and Western blotted. (B) Stealth siRNA knockdown of Rlf in 293T cells. Two days posttransfection, cells were lysed, and RalA pull down with GST-RalBP1 beads was performed. Levels of indicated proteins in total cell lysates and pull down are shown. (C) Images from experiment in B were quantified by ImageJ and plotted. *p = 0.05. (D) Cos-1 cells were transfected with FLAG-RalA together with Myc-Rab10 (QL), HA-Rlf, or both. At 24 h later, cells were lysed and subjected to pull-down assay using GST-Sec5-RBD beads. Lysates and pull-down samples were run on SDS–PAGE and immunoblotted.
Mentions: The activation of RalA by Rab10 could be due to inactivation of the RalGAP complex, activation of a RalGEF, or both. RGC1/2 is inactivated upon insulin stimulation through Akt phosphorylation of the catalytic subunit RGC2 (Chen et al., 2011b). Rab10 overexpression did not accelerate or affect the phosphorylation status of RGC2 (unpublished data). Moreover, a point mutation in RalA (F39L/FL) with increased nucleotide dissociation rates that quickly cycles between GDP and GTP (Reinstein et al., 1991; Lim et al., 2005), and thus does not require a GEF for activation, was not influenced by overexpression of Rab10 (QL), unlike what was observed for RalA (wild type [WT]; Figure 3A).

Bottom Line: Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2.Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10.Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.

Show MeSH
Related in: MedlinePlus