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. 2009 Jul;29(14):3929-40.
doi: 10.1128/MCB.00427-09. Epub 2009 May 11.

The Ability of GAP1IP4BP to Function as a Rap1 GTPase-activating Protein (GAP) Requires Its Ras GAP-related Domain and an Arginine Finger Rather Than an Asparagine Thumb

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The Ability of GAP1IP4BP to Function as a Rap1 GTPase-activating Protein (GAP) Requires Its Ras GAP-related Domain and an Arginine Finger Rather Than an Asparagine Thumb

Sabine Kupzig et al. Mol Cell Biol. .
Free PMC article

Abstract

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP-related domain (RasGRD), surrounded by amino-terminal C2 domains and a carboxy-terminal PH/Btk domain, these proteins, with the notable exception of GAP1(m), possess an unexpected arginine finger-dependent GAP activity on the Ras-related protein Rap1 (S. Kupzig, D. Deaconescu, D. Bouyoucef, S. A. Walker, Q. Liu, C. L. Polte, O. Daumke, T. Ishizaki, P. J. Lockyer, A. Wittinghofer, and P. J. Cullen, J. Biol. Chem. 281:9891-9900, 2006). Here, we have examined the mechanism through which GAP1(IP4BP) can function as a Rap1 GAP. We show that deletion of domains on either side of the RasGRD, while not affecting Ras GAP activity, do dramatically perturb Rap1 GAP activity. By utilizing GAP1(IP4BP)/GAP1(m) chimeras, we establish that although the C2 and PH/Btk domains are required to stabilize the RasGRD, it is this domain which contains the catalytic machinery required for Rap1 GAP activity. Finally, a key residue in Rap1-specific GAPs is a catalytic asparagine, the so-called asparagine thumb. By generating a molecular model describing the predicted Rap1-binding site in the RasGRD of GAP1(IP4BP), we show that mutagenesis of individual asparagine or glutamine residues that lie in close proximity to the predicted binding site has no detectable effect on the in vivo Rap1 GAP activity of GAP1(IP4BP). In contrast, we present evidence consistent with a model in which the RasGRD of GAP1(IP4BP) functions to stabilize the switch II region of Rap1, allowing stabilization of the transition state during GTP hydrolysis initiated by the arginine finger.

Figures

FIG. 1.
FIG. 1.
Disruption of any aspect of the domain structure perturbs the ability of GAP1IP4BP to function as a Rap GAP in vivo. (A) Schematic diagram of the GAP1IP4BP deletion constructs used in this study. (i) CHO-T cells were transiently cotransfected with 2.5 μg H-Ras (B) or 2.5 μg HA-tagged Rap1A (C) and 1 μg of the relevant GAP1IP4BP expression vector. Compared to control cells, the amount of Ras- and Rap1-GTP is significantly decreased in cells expressing wild-type GAP1IP4BP. This decrease in Ras-GTP only requires a functional GRD (cf. GAP1IP4BP with GAP1IP4BP ΔGRD/PH/C-tail; panel B). By contrast, Rap GAP activity of GAP1IP4BP requires the presence of all domains (panel C). Ras-GTP (B, part ii) and Rap1-GTP (C, part ii) levels from CHO-T cells are expressed as percentages of the pulldown level in control cells (average of six separate experiments ± the standard error of the mean).
FIG. 2.
FIG. 2.
Exchange of the PH and Btk domains between GAP1IP4BP and GAP1m has no effect on the Ras and Rap GAP activities of either hybrid protein in vivo. (A) Schematic diagram of the PH/Btk domain exchange constructs used in this study. (B, part i) CHO-T cells were transiently cotransfected with 2.5 μg Ras and 1 μg of the relevant GAP1IP4BP or GAP1m expression vector. Compared to that in control cells, the amount of Ras-GTP is significantly decreased in cells expressing wild-type GAP1IP4BP and GAP1m, as well as in cells expressing the GAP1IP4BP and GAP1m PH/Btk domain hybrid proteins. (C, part i) CHO-T cells were transiently cotransfected with 2.5 μg HA-tagged Rap1A and 1 μg of the relevant GAP1IP4BP or GAP1m expression vector. Compared to that in control cells, the amount of Rap1-GTP is only significantly decreased in cells expressing wild-type GAP1IP4BP and in cells expressing GAP1IP4BP with the PH/Btk domain from GAP1m. Wild-type GAP1m and GAP1m with the PH/Btk domain from GAP1IP4BP do not display Rap1 GAP activity. Ras-GTP (B, part ii) and Rap1-GTP (C, part ii) levels from CHO-T cells are expressed as percentages of the pulldown level in control cells (average of six separate experiments ± the standard error of the mean).
FIG. 3.
FIG. 3.
Exchange of the C2A or C2B domains between GAP1IP4BP and GAP1m has no effect on the Ras and Rap GAP activities of either hybrid protein in vivo. (A) Schematic diagram of the C2A and C2B domain exchange constructs used in this study. (B, part i) CHO-T cells were transiently cotransfected with 2.5 μg Ras and 1 μg of the relevant GAP1IP4BP or GAP1m expression vector. Compared to that in control cells, the amount of Ras-GTP is significantly decreased in cells expressing wild-type GAP1IP4BP and GAP1m, as well as in cells expressing the GAP1IP4BP and GAP1m C2 domain hybrid proteins. (C, part i) CHO-T cells were transiently cotransfected with 2.5 μg HA-tagged Rap1A and 1 μg of the relevant GAP1IP4BP or GAP1m expression vector. Compared to that in control cells, the amount of Rap1-GTP is only significantly decreased in cells expressing wild-type GAP1IP4BP and in cells expressing GAP1IP4BP with the C2A or C2B domain from GAP1m. Wild-type GAP1m and the hybrid forms of GAP1m do not display Rap1 GAP activity. Ras-GTP (B, part ii) and Rap1-GTP (C, part ii) levels from CHO-T cells are expressed as percentages of the pulldown level in control cells (average of six separate experiments ± the standard error of the mean).
FIG. 4.
FIG. 4.
Exchange of the GRDs between GAP1IP4BP and GAP1m transfers the Rap1 GAP activity from GAP1IP4BP to GAP1m in vivo. (A) Schematic diagram of the GRD exchange constructs used in this study. (B, part i) CHO-T cells were transiently cotransfected with 2.5 μg Ras and 1 μg of the relevant GAP1IP4BP or GAP1m expression vector. Compared to that in control cells, the amount of Ras-GTP is significantly decreased in cells expressing wild-type GAP1IP4BP and GAP1m, as well as in cells expressing the GAP1IP4BP and GAP1m GRD hybrid proteins. (C, part i) CHO-T cells were transiently cotransfected with 2.5 μg HA-tagged Rap1A and 1 μg of the relevant GAP1IP4BP or GAP1m expression vector. Compared to that in control cells, the amount of Rap1-GTP is significantly decreased in cells expressing wild-type GAP1IP4BP and in cells expressing GAP1m with the GRD from GAP1IP4BP. Wild-type GAP1m and GAP1IP4BP with the GRD from GAP1m do not appear to have Rap1 GAP activity. Ras-GTP (B, part ii) and Rap1-GTP (C, part ii) levels from CHO-T cells are expressed as percentages of the pulldown level in control cells (average of six separate experiments ± the standard error of the mean).
FIG. 5.
FIG. 5.
Molecular modeling of the RasGRD of GAP1IP4BP. (A) The sequence alignment used for modeling of GAP1IP4BP and GAP1m on p120GAP (GRD 1WER) and TAPP1 (PH domain 1EAZ). The secondary structure of the templates is indicated as follows: helix, wavy overlining; sheet, zigzag overlining. Residue identities are in red, and similarities are in magenta. The asparagine and glutamine residues in GAP1IP4BP that were mutated to alanine are underlined. (B) Structure of the Ras/p120GAP complex (1WQ1). The p120GAP GRD is shown as a light blue ribbon with the residues beyond L991 shown in yellow. Ras is shown as a green ribbon, GDP is shown as pink sticks, and R789 is shown as orange sticks. (C) Model of the Ras/GAP1IP4BP complex. The GAP1IP4BP GRD (modeled on p120GAP) is shown as a cyan ribbon with the residues beyond E573 shown in dark blue and modeled as a PH domain. Ras is shown as a green ribbon, GDP is shown as pink sticks, and R371 is shown as orange sticks.
FIG. 6.
FIG. 6.
Identifying potential asparagine thumbs of GAP1IP4BP. (A) GAP1IP4BP model showing the asparagine residues (red sticks) mutated to alanine. The protein is shown as a light blue (GRD) and dark blue (PH domain) ribbon with R371 as orange sticks. (B) GAP1IP4BP model showing the glutamine residues (pink sticks) mutated to alanine. The protein is shown as a light blue (GRD) and dark blue (PH domain) ribbon with R371 as orange sticks. (C) Ribbon representations of the GAP1IP4BP (light blue)-and-Ras (green) complex overlaid on the GAP1m (yellow)-and-Rap (red) complex. Residues V515 (GAP1m) and P489 (GAP1IP4BP) are displayed as sticks to show their close proximity to GTPase residue 64 on the switch II loop (Ras, Y; Rap, F). The view is a rotation of approximately 180° on the vertical axis with respect to panels A and B. R371 in GAP1IP4BP (orange sticks) and the Ras GTP (pink sticks) are shown for reference.
FIG. 7.
FIG. 7.
Mutation of asparagine residues within the GRD of GAP1IP4BP has no effect on Ras and Rap1 GAP activities in vivo. (i) CHO-T cells were transiently cotransfected with 2.5 μg H-Ras (A) or 2.5 μg HA-tagged Rap1A (B) and 1 μg of the relevant GAP1IP4BP expression vector. Compared to that in control cells, the amount of Ras- and Rap1-GTP is significantly decreased in cells expressing wild-type GAP1IP4BP, as well as in the cell lines expressing the asparagine point mutations. Ras-GTP (A, part ii) and Rap1-GTP (B, part ii) levels from CHO-T cells are expressed as percentages of the pulldown level in control cells (average of four separate experiments ± the standard error of the mean).
FIG. 8.
FIG. 8.
Mutation of glutamine residues within the GRD of GAP1IP4BP also has no effect on Ras and Rap1 GAP activities in vivo. (i) CHO-T cells were transiently cotransfected with 2.5 μg H-Ras (A) or 2.5 μg HA-tagged Rap1A (B) and 1 μg of the relevant GAP1IP4BP expression vector. Compared to that in control cells, the amount of Ras- and Rap1-GTP is significantly decreased in cells expressing wild-type GAP1IP4BP, as well as in the cell lines expressing the glutamine point mutations. Ras-GTP (A, part ii) and Rap1-GTP (B, part ii) levels from CHO-T cells are expressed as percentages of the pulldown level in control cells (average of four separate experiments ± the standard error of the mean).
FIG. 9.
FIG. 9.
Mutation of proline 489 to valine largely abolishes Rap1 GAP activity in GAP1IP4BP but has no effect on Ras GAP activity in vivo. (A, part i) CHO-T cells were transiently cotransfected with 2.5 μg H-Ras and 1 μg of the GAP1IP4BP, GAP1m, GAP1IP4BP (P489V), and GAP1m (V515P) expression vectors. Compared to that in control cells, the amount of Ras-GTP is significantly decreased in cells expressing wild-type GAP1IP4BP and GAP1m, as well as in cells expressing GAP1IP4BP (P489V) and GAP1m (V515P). (B, part i) CHO-T cells were transiently cotransfected with 2.5 μg Rap1 and 1 μg of the GAP1IP4BP, GAP1m, GAP1IP4BP (P489V), and GAP1m (V515P) expression vectors. Compared to that in control cells, the amount of Rap1-GTP is only significantly decreased in cells expressing wild-type GAP1IP4BP. Wild-type GAP1m, GAP1IP4BP (P489V), and GAP1m (V515P) do not appear to have Rap1 GAP activity. Ras-GTP (A, part ii) and Rap1-GTP (B, part ii) levels from CHO-T cells are expressed as percentages of the pulldown level in control cells (average of four separate experiments ± the standard error of the mean).

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