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TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, Jiang Y, Fukuda M, Apodaca G, Yin XM - Mol. Biol. Cell (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.

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TBC1D9B has GAP activity in cellula. (A) Localization of endogenous Rab11a and endogenous Sec15A in cells transduced with adenovirus encoding CFP-TBC1D9B-WT or the inactive mutant (-RYQ/AAA). Top, from the subapical region of the cell; bottom, xz-sections. (B, C) Coefficient of colocalization for the fraction of Rab11a that colocalizes with Sec15A (or vice versa) in MDCK cells transduced with adenovirus encoding CFP-TBC1D9B-WT or -RYQ/AAA (B) or in cells expressing shRNA-1 or shRNA-2 against canine TBC1D9B (C). In B, GFP expression was used as control, and in C, scrambled shRNA was used as control. (D) GST-Sec15A–mediated pull down of activated Rab11a in MDCK cells expressing scrambled shRNA (Control) or shRNA-2 specific for canine TBC1D9B. Left, immunoprecipitate from the pull down was analyzed by Western blot, using anti-GST and anti-Rab11a antibodies. Right, 2% of each lysate was analyzed by Western blotting, using anti-TBC1D9B and anti-Rab11a antibodies. (E) Data from D and F are quantified and normalized to control incubations. (F) GST-Sec15A pull down of activated Rab11a in MDCK cells expressing CFP-TBC1D9B-RYQ/AAA (+RYQ) compared with control cells expressing endogenous TBC1D9B (–). Left, the precipitate from the pull down was analyzed by Western blot, using anti-GST and anti-Rab11a antibodies. Right, 2% of each lysate was analyzed by Western blot, using anti-GFP and anti-Rab11a antibodies. (G) GST-Sec15A pull down of activated Rab11a in MDCK cells expressing CFP-TBC1D9B-WT (+WT) or -RYQ/AAA (+RYQ). CFP-TBC1D9B-WT and -RYQ/AAA were detected using anti-GFP antibody, endogenous Rab11a was detected using anti-Rab11a antibody, and GST-Sec15A was detected using GST antibody. (H) Data from G are quantified. In B, C, E, and H, data were obtained from at least three independent experiments, and mean ± SEM is shown. Values different from the control, assessed by ANOVA, are indicated (*p < 0.05).
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Figure 9: TBC1D9B has GAP activity in cellula. (A) Localization of endogenous Rab11a and endogenous Sec15A in cells transduced with adenovirus encoding CFP-TBC1D9B-WT or the inactive mutant (-RYQ/AAA). Top, from the subapical region of the cell; bottom, xz-sections. (B, C) Coefficient of colocalization for the fraction of Rab11a that colocalizes with Sec15A (or vice versa) in MDCK cells transduced with adenovirus encoding CFP-TBC1D9B-WT or -RYQ/AAA (B) or in cells expressing shRNA-1 or shRNA-2 against canine TBC1D9B (C). In B, GFP expression was used as control, and in C, scrambled shRNA was used as control. (D) GST-Sec15A–mediated pull down of activated Rab11a in MDCK cells expressing scrambled shRNA (Control) or shRNA-2 specific for canine TBC1D9B. Left, immunoprecipitate from the pull down was analyzed by Western blot, using anti-GST and anti-Rab11a antibodies. Right, 2% of each lysate was analyzed by Western blotting, using anti-TBC1D9B and anti-Rab11a antibodies. (E) Data from D and F are quantified and normalized to control incubations. (F) GST-Sec15A pull down of activated Rab11a in MDCK cells expressing CFP-TBC1D9B-RYQ/AAA (+RYQ) compared with control cells expressing endogenous TBC1D9B (–). Left, the precipitate from the pull down was analyzed by Western blot, using anti-GST and anti-Rab11a antibodies. Right, 2% of each lysate was analyzed by Western blot, using anti-GFP and anti-Rab11a antibodies. (G) GST-Sec15A pull down of activated Rab11a in MDCK cells expressing CFP-TBC1D9B-WT (+WT) or -RYQ/AAA (+RYQ). CFP-TBC1D9B-WT and -RYQ/AAA were detected using anti-GFP antibody, endogenous Rab11a was detected using anti-Rab11a antibody, and GST-Sec15A was detected using GST antibody. (H) Data from G are quantified. In B, C, E, and H, data were obtained from at least three independent experiments, and mean ± SEM is shown. Values different from the control, assessed by ANOVA, are indicated (*p < 0.05).

Mentions: We also determined whether TBC1D9B colocalized to a greater extent with the GFP-tagged Rab11aQ70L mutant. Indeed, we observed higher degree of colocalization between endogenous TBC1D9B and GFP-tagged Rab11a-QL than with GFP-tagged Rab11a-WT (coefficient of colocalization of 0.73 ± 0.03 and 0.61 ± 0.02, respectively; Figure 7, E–G). We also determined whether the localization of Rab11a in MDCK cells was altered by expressing CFP-TBC1D9B-WT or CFP-TBC1D9B-RYQ/AAA. However, we found no difference in the Rab11a distribution (Supplemental Figure S5, A and B; see Figure 9A later in this article), which is consistent with previous reports that the Rab11a-SN mutant remains membrane bound in MDCK cells (Wang et al., 2000b). There was also no change in Rab11a colocalization with these TBC1D9B variants (coefficient of colocalization of 0.72 ± 0.06 and 0.70 ± 0.06 for WT and RYQ/AAA, respectively; Supplemental Figure S5C). In contrast, in HEK cells, there was a significant redistribution of Rab11a from recycling endosomes to the cytosol when TBC1D9B-WT, but not when the inactive mutant TBC1D9B-RYQ/AAA, was expressed (Supplemental Figure S2, A and B). This is the expected result for HEK cells if the GDP-bound form of Rab11a increased as a result of expressing TBC1D9B-WT. Finally, when Rab11a expression was depleted using a specific shRNA, endogenous TBC1D9B's normal accumulation in the apical half of the cell was altered, and its distribution became more dispersed throughout the cell (Supplemental Figure S5D).


TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, Jiang Y, Fukuda M, Apodaca G, Yin XM - Mol. Biol. Cell (2014)

TBC1D9B has GAP activity in cellula. (A) Localization of endogenous Rab11a and endogenous Sec15A in cells transduced with adenovirus encoding CFP-TBC1D9B-WT or the inactive mutant (-RYQ/AAA). Top, from the subapical region of the cell; bottom, xz-sections. (B, C) Coefficient of colocalization for the fraction of Rab11a that colocalizes with Sec15A (or vice versa) in MDCK cells transduced with adenovirus encoding CFP-TBC1D9B-WT or -RYQ/AAA (B) or in cells expressing shRNA-1 or shRNA-2 against canine TBC1D9B (C). In B, GFP expression was used as control, and in C, scrambled shRNA was used as control. (D) GST-Sec15A–mediated pull down of activated Rab11a in MDCK cells expressing scrambled shRNA (Control) or shRNA-2 specific for canine TBC1D9B. Left, immunoprecipitate from the pull down was analyzed by Western blot, using anti-GST and anti-Rab11a antibodies. Right, 2% of each lysate was analyzed by Western blotting, using anti-TBC1D9B and anti-Rab11a antibodies. (E) Data from D and F are quantified and normalized to control incubations. (F) GST-Sec15A pull down of activated Rab11a in MDCK cells expressing CFP-TBC1D9B-RYQ/AAA (+RYQ) compared with control cells expressing endogenous TBC1D9B (–). Left, the precipitate from the pull down was analyzed by Western blot, using anti-GST and anti-Rab11a antibodies. Right, 2% of each lysate was analyzed by Western blot, using anti-GFP and anti-Rab11a antibodies. (G) GST-Sec15A pull down of activated Rab11a in MDCK cells expressing CFP-TBC1D9B-WT (+WT) or -RYQ/AAA (+RYQ). CFP-TBC1D9B-WT and -RYQ/AAA were detected using anti-GFP antibody, endogenous Rab11a was detected using anti-Rab11a antibody, and GST-Sec15A was detected using GST antibody. (H) Data from G are quantified. In B, C, E, and H, data were obtained from at least three independent experiments, and mean ± SEM is shown. Values different from the control, assessed by ANOVA, are indicated (*p < 0.05).
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Figure 9: TBC1D9B has GAP activity in cellula. (A) Localization of endogenous Rab11a and endogenous Sec15A in cells transduced with adenovirus encoding CFP-TBC1D9B-WT or the inactive mutant (-RYQ/AAA). Top, from the subapical region of the cell; bottom, xz-sections. (B, C) Coefficient of colocalization for the fraction of Rab11a that colocalizes with Sec15A (or vice versa) in MDCK cells transduced with adenovirus encoding CFP-TBC1D9B-WT or -RYQ/AAA (B) or in cells expressing shRNA-1 or shRNA-2 against canine TBC1D9B (C). In B, GFP expression was used as control, and in C, scrambled shRNA was used as control. (D) GST-Sec15A–mediated pull down of activated Rab11a in MDCK cells expressing scrambled shRNA (Control) or shRNA-2 specific for canine TBC1D9B. Left, immunoprecipitate from the pull down was analyzed by Western blot, using anti-GST and anti-Rab11a antibodies. Right, 2% of each lysate was analyzed by Western blotting, using anti-TBC1D9B and anti-Rab11a antibodies. (E) Data from D and F are quantified and normalized to control incubations. (F) GST-Sec15A pull down of activated Rab11a in MDCK cells expressing CFP-TBC1D9B-RYQ/AAA (+RYQ) compared with control cells expressing endogenous TBC1D9B (–). Left, the precipitate from the pull down was analyzed by Western blot, using anti-GST and anti-Rab11a antibodies. Right, 2% of each lysate was analyzed by Western blot, using anti-GFP and anti-Rab11a antibodies. (G) GST-Sec15A pull down of activated Rab11a in MDCK cells expressing CFP-TBC1D9B-WT (+WT) or -RYQ/AAA (+RYQ). CFP-TBC1D9B-WT and -RYQ/AAA were detected using anti-GFP antibody, endogenous Rab11a was detected using anti-Rab11a antibody, and GST-Sec15A was detected using GST antibody. (H) Data from G are quantified. In B, C, E, and H, data were obtained from at least three independent experiments, and mean ± SEM is shown. Values different from the control, assessed by ANOVA, are indicated (*p < 0.05).
Mentions: We also determined whether TBC1D9B colocalized to a greater extent with the GFP-tagged Rab11aQ70L mutant. Indeed, we observed higher degree of colocalization between endogenous TBC1D9B and GFP-tagged Rab11a-QL than with GFP-tagged Rab11a-WT (coefficient of colocalization of 0.73 ± 0.03 and 0.61 ± 0.02, respectively; Figure 7, E–G). We also determined whether the localization of Rab11a in MDCK cells was altered by expressing CFP-TBC1D9B-WT or CFP-TBC1D9B-RYQ/AAA. However, we found no difference in the Rab11a distribution (Supplemental Figure S5, A and B; see Figure 9A later in this article), which is consistent with previous reports that the Rab11a-SN mutant remains membrane bound in MDCK cells (Wang et al., 2000b). There was also no change in Rab11a colocalization with these TBC1D9B variants (coefficient of colocalization of 0.72 ± 0.06 and 0.70 ± 0.06 for WT and RYQ/AAA, respectively; Supplemental Figure S5C). In contrast, in HEK cells, there was a significant redistribution of Rab11a from recycling endosomes to the cytosol when TBC1D9B-WT, but not when the inactive mutant TBC1D9B-RYQ/AAA, was expressed (Supplemental Figure S2, A and B). This is the expected result for HEK cells if the GDP-bound form of Rab11a increased as a result of expressing TBC1D9B-WT. Finally, when Rab11a expression was depleted using a specific shRNA, endogenous TBC1D9B's normal accumulation in the apical half of the cell was altered, and its distribution became more dispersed throughout the cell (Supplemental Figure S5D).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.

Show MeSH
Related in: MedlinePlus