AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain - PubMed (original) (raw)

AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain

Cristinel P Mîinea et al. Biochem J. 2005.

Abstract

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

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Figures

Figure 1

Figure 1. Rab GAP assay with AS160 GST–GAP domain and GST–Rab14

(A) TLC separation of α32P GDP and GTP in assays with Rab14 alone, Rab14 plus the GAP domain, and Rab14 plus the R/K mutant of the GAP domain. (B) The data from (A) are presented as the percentage of the total radioactivity in GDP versus time for Rab14 (●), Rab14 plus the GAP domain (■), and Rab14 plus the R/K mutant GAP domain (▲). For further details, see the Experimental section.

Figure 2

Figure 2. Activity of AS160 GAP domain against the 18 Rab proteins

The GAP assay was carried out as described in the Experimental section and shown in Figure 1. The data show the increase in the percentage of the total radioactivity in GDP between 0 and 15 min for the Rab alone and the Rab plus the AS160 GAP domain. In addition, for any Rab that appeared to be a GAP substrate, as well as most of the others, the assay was also performed with the GAP R/K mutant. In the case of Rab5A, the data are for 3 min, rather than 15 min, due to the high intrinsic GTPase activity of this Rab.

Figure 3

Figure 3. Dependence of the GAP activity upon the concentration of the AS160 GST–GAP domain

The GAP assay was carried out with Rabs 2A, 8A, 10 and 14, as described in the Experimental section and shown in Figure 1. The concentration of the GST–GAP domain in the assay was: none (■), 0.04 μM (◆), 0.13 μM (●) and 0.4 μM (▲).

Figure 4

Figure 4. Rabs in GLUT4 vesicles

GLUT4 vesicles (G4V) and control adsorbates (CV) were prepared from unstimulated (B) and insulin-treated (I) 3T3-L1 adipocytes, as described in the Experimental section. SDS samples of the vesicles were immunoblotted for IRAP, Rabs 2, 8 and 14, and AS160. The 1× loads were derived from 0.15% of a 10-cm diameter plate for IRAP, 2% of a plate for Rabs 2, 8 and 14, and 4% of a plate for AS160. Two repetitions of this experiment gave similar results, except for variable relative amounts of Rab14 in the insulin-treated GLUT4 vesicles (see the text).

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