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Insulin-stimulated Phosphorylation of a Rab GTPase-activating Protein Regulates GLUT4 Translocation*

Open AccessPublished:March 11, 2003DOI:https://doi.org/10.1074/jbc.C300063200
      Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.
      GAP
      GTPase-activating protein
      GFP
      green fluorescence protein
      MS/MS
      tandem mass spectrometry
      PBS
      phosphate-buffered saline
      HA
      hemagglutinin
      Insulin rapidly stimulates glucose transport into fat and muscle cells by causing the insertion of additional glucose transporters of the GLUT4 isotype into the plasma membrane, in a process referred to as GLUT4 translocation. The overall process consists of generation of the specialized vesicles containing GLUT4 from the endosomal system, the movement of these vesicles from the perinuclear region to the plasma membrane, and the fusion of the vesicles with the plasma membrane (
      • Zeigerer A.
      • Lampson M.A.
      • Karylowski O.
      • Sabatini D.D.
      • Adesnik M.
      • Ren M.
      • McGraw T.E.
      ). The steps in this process that insulin accelerates, and the complete signaling pathways from the insulin receptor that lead to their acceleration, have not yet been fully defined. One partial insulin signaling pathway that has been established to be required for GLUT4 translocation is the pathway that proceeds from the receptor through tyrosine phosphorylation of the insulin receptor substrates to activation of phosphatidylinositol 3-kinase and generation of phosphatidylinositol 3,4,5-trisphosphate. The latter leads to the activation of the protein kinase Akt and also protein kinase C λ/ζ, and there is evidence that GLUT4 translocation requires the activation of one or both of these kinases (reviewed in Refs.
      • Czech M.P.
      • Corvera S.
      and
      • Khan A.H.
      • Pessin J.E.
      ). However, although a substrate linking either kinase to GLUT4 translocation has been sought for several years, hitherto none has been clearly identified.
      Recently, we reported the discovery of a new substrate for insulin-activated Akt in 3T3-L1 adipocytes, which was designated AS160 for Akt subtrate of 160 kDa (
      • Kane S.
      • Sano H.
      • Liu S.C.H.
      • Asara J.M.
      • Lane W.S.
      • Garner C.W.
      • Lienhard G.E.
      ). The most prominent feature of AS160 is the presence of a GTPase activating domain for a Rab. Since Rabs are small G proteins that play critical roles in vesicle formation, movement, and fusion (
      • Zerial M.
      • McBride H.
      ), we investigated the role of AS160 in GLUT4 translocation. Our present results strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and also that an active GAP1 domain in AS160 is required for AS160 control of GLUT4 translocation. This study thus identifies AS160 as a new and likely key connection between the phosphatidylinositol 3-kinase insulin signaling pathway and the vesicle trafficking machinery in GLUT4 translocation.

      DISCUSSION

      Our results show that AS160 undergoes a marked increase in phosphorylation at five sites in response to insulin. Since each site lies within the consensus sequence for Akt phosphorylation, and since we have shown previously that Akt phosphorylates the Thr642site (
      • Kane S.
      • Sano H.
      • Liu S.C.H.
      • Asara J.M.
      • Lane W.S.
      • Garner C.W.
      • Lienhard G.E.
      ), it seems likely that activated Akt is responsible for phosphorylation at these sites, although it remains possible that one or more other insulin-activated kinases, such as protein kinase C λ/ζ, also participates.
      Expression of AS160 mutated at two or more of its phosphorylation sites markedly inhibited insulin-stimulated GLUT4 translocation, whereas equivalent expression of wild type AS160 had no effect. This result strongly indicates that phosphorylation of AS160 is necessary for GLUT4 translocation to occur. Expression of AS160 with combined mutations in the phosphorylation sites and the Rab GAP domain largely reversed the inhibition given by AS160 mutated at the phosphorylation sites alone. This result demonstrates that the inhibitory effect of AS160 mutated at its phosphorylation sites requires a functional Rab GAP domain. A hypothesis that explains these findings is the following: insulin-stimulated translocation of GLUT4 requires a Rab in its active GTP form; in the unstimulated state this Rab is maintained in its inactive GDP form by the GAP domain of AS160; phosphorylation of AS160 inhibits its GAP activity toward this Rab, through an effect either on GAP function and/or on localization of the protein; as a consequence the GTP form of the Rab increases, and the Rab-dependent step(s) in GLUT4 translocation proceed.
      If the above hypothesis is correct, it might be thought that AS160 with an inactive GAP domain would trigger translocation in the absence of insulin, whereas in fact expression of the GAP mutant of AS160 did not have this effect. One possibility is that extent of expression of the GAP mutant was not sufficient for it to block the action of the endogenous AS160. A second possible explanation is that generation of the GTP form of a Rab is only one of several signals required for GLUT4 translocation. There is considerable evidence that a second signaling pathway involving activation of the Rho-type G protein TC10 and the rearrangement of cortical actin is required for GLUT4 translocation (
      • Khan A.H.
      • Pessin J.E.
      ). This pathway is independent of Akt activation. In addition, there is suggestive evidence that Akt phosphorylation of the syntaxin 4-interacting protein Synip may be required for GLUT4 translocation (
      • Min J.
      • Okada S.
      • Kanzaki M.
      • Elemendorf J.S.
      • Coker K.J.
      • Ceresa B.P.
      • Syu L.-J.
      • Noda Y.
      • Saltiel A.R.
      • Pessin J.E.
      ,
      • Yamada E.
      • Okada S.
      • Saito T.
      • Sato M.
      • Ohshima K.
      • Pessin J.E.
      • Mori M.
      ).
      This study indicates that AS160 is a key component linking the phosphatidylinositol 3-kinase insulin signaling pathway to the vesicle trafficking machinery in GLUT4 translocation. In the future it will be important to identify the Rab(s) on which AS160 acts and to determine whether phosphorylation of AS160 directly inhibits its GAP activity. Rab4 and Rab11 are present in the intracellular vesicles that contain GLUT4 (reviewed in Ref.
      • Cormont M.
      • Le Marchand-Brustel Y.
      ). Consequently these two Rabs are candidates to be substrates for the GAP function of AS160. We are currently attempting to determine whether AS160 exhibits GAP activity toward either of these Rabs or toward another one of the ∼60 Rabs present in mammals (
      • Zerial M.
      • McBride H.
      ).

      Acknowledgments

      We are deeply indebted to Renee Robinson for technical assistance with the mass spectrometry measurements, to Dr. Samuel Cushman for the HA-GLUT4-GFP plasmid, to Dr. Michael Czech and his associates for the guidance in the electroporation method, to Dr. Timothy McGraw for advice about the fluorescence microscopy, and to Dr. William Wickner for the use of the fluorescence microscope. We are especially grateful to Dr. Alexey Merz for his guidance in the fluorescence microscopy.

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