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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.

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Related in: MedlinePlus

Rab10 is an in vivo target of AS160. (A–C) COS-1 cell lysates overexpressing FLAG-AS160 was incubated with immobilized GST or GST-Rab4, 5, 10, 11, or RalA bound to GDP, GTPγS, or GDP/AIFx as indicated. Pull downs were subjected to SDS–PAGE and Western blot with anti-FLAG antibody. GST proteins were visualized with Ponceau S. (D) COS-1 cells were transiently transfected with HA-Rab10 WT, QL, or TN constructs as indicated, and cell lysates were incubated with immobilized GST-Rim1-RBD. Pull downs were resolved on SDS–PAGE and visualized by Western blot with anti-HA antibody. (E) Quantification of Rab10 activity in D from three independent experiments. Rab10 activity was quantified by normalizing the amount of pull down with the total cell lysate. The value of the activity of wild-type Rab10 was then set as 1 arbitrary unit. *p < 0.05. (F) 3T3-L1 adipocytes were transfected with FLAG-AS160 and HA-Rab10 (QL), and the activity of Rab10 was determined by GST-Rim1-RBD pull-down assay as described previously. (G) COS-1 cells were transiently transfected with HA-Rab10 alone or with FLAG-AS160 4A and/or Myr-Akt constructs as indicated. Cell lysates were subjected to GST-Rim1-RBD pull-down assay as described previously.
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Figure 1: Rab10 is an in vivo target of AS160. (A–C) COS-1 cell lysates overexpressing FLAG-AS160 was incubated with immobilized GST or GST-Rab4, 5, 10, 11, or RalA bound to GDP, GTPγS, or GDP/AIFx as indicated. Pull downs were subjected to SDS–PAGE and Western blot with anti-FLAG antibody. GST proteins were visualized with Ponceau S. (D) COS-1 cells were transiently transfected with HA-Rab10 WT, QL, or TN constructs as indicated, and cell lysates were incubated with immobilized GST-Rim1-RBD. Pull downs were resolved on SDS–PAGE and visualized by Western blot with anti-HA antibody. (E) Quantification of Rab10 activity in D from three independent experiments. Rab10 activity was quantified by normalizing the amount of pull down with the total cell lysate. The value of the activity of wild-type Rab10 was then set as 1 arbitrary unit. *p < 0.05. (F) 3T3-L1 adipocytes were transfected with FLAG-AS160 and HA-Rab10 (QL), and the activity of Rab10 was determined by GST-Rim1-RBD pull-down assay as described previously. (G) COS-1 cells were transiently transfected with HA-Rab10 alone or with FLAG-AS160 4A and/or Myr-Akt constructs as indicated. Cell lysates were subjected to GST-Rim1-RBD pull-down assay as described previously.

Mentions: Previous studies demonstrated that GAP proteins preferentially interact with their cognate small GTPases during the transition state of GTP hydrolysis (Scheffzek et al., 1997; Bos et al., 2007). To identify a cellular target of AS160, we loaded glutathione S-transferase (GST)–Rab proteins with guano­sine diphosphate (GDP)/AlF4 to mimic this transition state, incubating the fusion proteins with lysates from COS-1 cells overexpressing FLAG-AS160. AS160 specifically interacted with GST-Rab10 bound to GDP/AIFx but not with GST-Rab10 bound to GDP or GTPγS (Figure 1A), indicating that the interaction between AS160 and Rab10 is transition state dependent. Meanwhile, AS160 did not interact, or interacted only modestly, with other GDP/AIFx-loaded small GTPases, including the Ras subfamily member RalA and the Rab subfamily members Rab4, Rab5, and Rab11 (Figure 1, B and C). These results suggest that the transition state–dependent binding between AS160 and Rab10 is specific and characteristic of a GAP-substrate GTPase interaction, consistent with the notion that AS160 possesses GAP activity toward Rab10 in vitro.


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 is an in vivo target of AS160. (A–C) COS-1 cell lysates overexpressing FLAG-AS160 was incubated with immobilized GST or GST-Rab4, 5, 10, 11, or RalA bound to GDP, GTPγS, or GDP/AIFx as indicated. Pull downs were subjected to SDS–PAGE and Western blot with anti-FLAG antibody. GST proteins were visualized with Ponceau S. (D) COS-1 cells were transiently transfected with HA-Rab10 WT, QL, or TN constructs as indicated, and cell lysates were incubated with immobilized GST-Rim1-RBD. Pull downs were resolved on SDS–PAGE and visualized by Western blot with anti-HA antibody. (E) Quantification of Rab10 activity in D from three independent experiments. Rab10 activity was quantified by normalizing the amount of pull down with the total cell lysate. The value of the activity of wild-type Rab10 was then set as 1 arbitrary unit. *p < 0.05. (F) 3T3-L1 adipocytes were transfected with FLAG-AS160 and HA-Rab10 (QL), and the activity of Rab10 was determined by GST-Rim1-RBD pull-down assay as described previously. (G) COS-1 cells were transiently transfected with HA-Rab10 alone or with FLAG-AS160 4A and/or Myr-Akt constructs as indicated. Cell lysates were subjected to GST-Rim1-RBD pull-down assay as described previously.
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Related In: Results  -  Collection

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Figure 1: Rab10 is an in vivo target of AS160. (A–C) COS-1 cell lysates overexpressing FLAG-AS160 was incubated with immobilized GST or GST-Rab4, 5, 10, 11, or RalA bound to GDP, GTPγS, or GDP/AIFx as indicated. Pull downs were subjected to SDS–PAGE and Western blot with anti-FLAG antibody. GST proteins were visualized with Ponceau S. (D) COS-1 cells were transiently transfected with HA-Rab10 WT, QL, or TN constructs as indicated, and cell lysates were incubated with immobilized GST-Rim1-RBD. Pull downs were resolved on SDS–PAGE and visualized by Western blot with anti-HA antibody. (E) Quantification of Rab10 activity in D from three independent experiments. Rab10 activity was quantified by normalizing the amount of pull down with the total cell lysate. The value of the activity of wild-type Rab10 was then set as 1 arbitrary unit. *p < 0.05. (F) 3T3-L1 adipocytes were transfected with FLAG-AS160 and HA-Rab10 (QL), and the activity of Rab10 was determined by GST-Rim1-RBD pull-down assay as described previously. (G) COS-1 cells were transiently transfected with HA-Rab10 alone or with FLAG-AS160 4A and/or Myr-Akt constructs as indicated. Cell lysates were subjected to GST-Rim1-RBD pull-down assay as described previously.
Mentions: Previous studies demonstrated that GAP proteins preferentially interact with their cognate small GTPases during the transition state of GTP hydrolysis (Scheffzek et al., 1997; Bos et al., 2007). To identify a cellular target of AS160, we loaded glutathione S-transferase (GST)–Rab proteins with guano­sine diphosphate (GDP)/AlF4 to mimic this transition state, incubating the fusion proteins with lysates from COS-1 cells overexpressing FLAG-AS160. AS160 specifically interacted with GST-Rab10 bound to GDP/AIFx but not with GST-Rab10 bound to GDP or GTPγS (Figure 1A), indicating that the interaction between AS160 and Rab10 is transition state dependent. Meanwhile, AS160 did not interact, or interacted only modestly, with other GDP/AIFx-loaded small GTPases, including the Ras subfamily member RalA and the Rab subfamily members Rab4, Rab5, and Rab11 (Figure 1, B and C). These results suggest that the transition state–dependent binding between AS160 and Rab10 is specific and characteristic of a GAP-substrate GTPase interaction, consistent with the notion that AS160 possesses GAP activity toward Rab10 in vitro.

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