TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.
In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor.Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A.We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.
Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.
- GTPase-Activating Proteins/genetics/metabolism*
- rab GTP-Binding Proteins/biosynthesis*/genetics
- Cell Polarity
- Gene Expression Regulation
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Figure 4: Rab11a binds to the TBC domain of TBC1D9B. (A) Left, coimmunoprecipitation of flag-tagged TBC1D9B with GFP-Rab11a-wild-type (WT), GTP-locked GFP-Rab11aQ70L (QL), or GDP-locked GFP-Rab11aS25N (SN) coexpressed in HEK cells. Anti-flag antibody was used to recover flag-TBC1D9B, and coimmunopreciptated GFP-Rab11a was detected using an anti-GFP antibody. Controls included use of IgG instead of the flag-tag antibody and expression of GFP-Rab11a alone (first lane). Right, 2% of each lysate was resolved by SDS–PAGE and the indicated proteins detected by Western blot. Bottom, quantification of coimmunoprecipitations. Values were normalized to the total expression of each protein first and then to the values obtained in experiments using the Rab11a-WT lysate. (B) HEK cells were cotransfected with GFP-Rab11a and either flag-TBC1D9B-wild-type (WT) or the flag-TBC1D9B-RYQ/AAA mutant (RYQ). Flag-tagged TBC1D9B was recovered by immunoprecipitation, and the amount of GFP-Rab11a was quantified. Control reactions were performed for cells that only expressed GFP-Rab11a, or in some cases IgG was substituted for the anti-flag antibody. Right, 2% of each lysate was resolved by SDS–PAGE and the indicated proteins detected by Western blot. Bottom, amount of coimmunoprecipitated GFP-Rab11a normalized to the amount recovered from the TBC1D9B-WT lysate. (C) Fragments of TBC1D9B used in pull-down studies. (D) Top, GST-TBC1D9B fragments, labeled according to C, were used to affinity capture GFP-Rab11a in HEK cell lysates. Here #2RYQ denotes the use of fragment #2 with the RYQ/AAA mutations. GST alone was used as a control. Bottom, GST constructs were resolved by SDS–PAGE and proteins blotted with anti-GST antibody. (E, F) Top left, GST-TBC1D9B-#2 fragment was used to affinity capture GFP-Rab11aQ70L (QL) or GFP-Rab11aS25N (SN) from HEK cell lysates. Right, 2% of each lysate was resolved by SDS–PAGE and GFP-Rab11a detected by Western blot using anti-GFP antibody. Bottom left, GST constructs were resolved by SDS–PAGE and proteins detected using an anti-GST antibody. (G) Quantification of data from E and F. Values were normalized to total expression of the protein of interest and then to the values obtained for the GFP-Rab11a-WT pull down. For A, B, and G, data are from at least three independent experiments, and the mean ± SEM is shown. Values significantly different from the group means, as assessed by ANOVA, are indicated (*p < 0.05).
Next, we explored the nature of the interaction between TBC1D9B and Rab11a. In general, GAPs are believed to interact most robustly to Rabs in their GTP-bound state and less so with Rabs in their GDP-bound state (Will and Gallwitz, 2001; Haas et al., 2005; Fuchs et al., 2007; Hannemann et al., 2012). Consistent with this finding, we observed that immunoisolated flag-tagged TBC1D9B coimmunoprecipitated approximately five times more avidly with Rab11a-QL than with Rab11a-WT or Rab11a-SN (Figure 4A). Mutations of the R and/or Q fingers in the TBC domain are reported to alter TBC–protein interaction with Rab-GTP, either enhancing it (Will and Gallwitz, 2001; Haas et al., 2005; Itoh et al., 2006) or impairing it (Itoh et al., 2006). In the case of TBC1D9B, we observed that full-length flag-TBC1D9B-RYQ/AAA coimmunoprecipitated Rab11a-WT in a manner that was greater than for the wild-type flag-TCB1D9B protein (Figure 4B). Thus, our results were similar to those reported for RUTBC-3/RabGAP-5, USP6NL/RN-Tre, and Gyp6p, all of which show increased binding to variants of their cognate TBC GAPs that contain mutations in their active-site residues (Will and Gallwitz, 2001; Haas et al., 2005).