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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 interacts with Rab4a and Rab11b. (A) Left, coimmunoprecipitation of flag-tagged TBC1D9B with GFP-Rab11b-WT (11b-WT), GFP-Rab11b-Q70L (11b-QL), or GDP-locked GFP-Rab11bS25N (11b-SN) coexpressed in HEK cells. Anti-flag antibody was used to immunoprecipitate flag-TBC1D9B, and anti-GFP antibody was used to detect the coimmunoprecipitated GFP-tagged Rab protein. IgG was used as a nonspecific control. Right, 2% of each lysate was resolved by SDS–PAGE and overexpressed proteins detected by Western blot. Values were normalized to the total expression of each protein and then normalized to the amount of Rab11b-WT lysate. (B) Same as in A, but HEK cells coexpressed flag-TBC1D9B and GFP-Rab4a-WT (4a-WT), GFP-Rab4aQ72L (4a-QL), or GFP-Rab4aS27N (4a-SN). (C) Same as A, but the cells expressed Rab8aQ67L (8a-QL) or Rab11aQ70L (11a-QL), and after lysis, the incubations were performed in the absence of MgCl2. (D) Same as in A, but HEK cells coexpressed flag-TBC1D9B with GFP-Rab8a-WT (8a-WT), GFP-Rab8a-Q67L (8a-QL), or GFP-Rab8a-T22N (8a-TN). (E–G) Top left, GST alone or GST-TBC1D9B-(301-810) (#2 fragment) was used to affinity capture GFP-Rab11a-QL and GFP-Rab11b-QL (E), GFP-Rab4a-QL (F). or GFP-Rab8a-QL (G). Right, 2% of each lysate was resolved by SDS–PAGE and GFP-Rab-QL detected by Western blot using anti-GFP antibody. Bottom left, GST constructs were resolved by SDS–PAGE and proteins detected on Western blots using an anti-GST antibody. (H) Quantification of data from E–G. Values are normalized to the total expression of the protein first and then to the values obtained for the GFP-Rab11a-WT pull down. Data were obtained 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).
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Figure 5: TBC1D9B interacts with Rab4a and Rab11b. (A) Left, coimmunoprecipitation of flag-tagged TBC1D9B with GFP-Rab11b-WT (11b-WT), GFP-Rab11b-Q70L (11b-QL), or GDP-locked GFP-Rab11bS25N (11b-SN) coexpressed in HEK cells. Anti-flag antibody was used to immunoprecipitate flag-TBC1D9B, and anti-GFP antibody was used to detect the coimmunoprecipitated GFP-tagged Rab protein. IgG was used as a nonspecific control. Right, 2% of each lysate was resolved by SDS–PAGE and overexpressed proteins detected by Western blot. Values were normalized to the total expression of each protein and then normalized to the amount of Rab11b-WT lysate. (B) Same as in A, but HEK cells coexpressed flag-TBC1D9B and GFP-Rab4a-WT (4a-WT), GFP-Rab4aQ72L (4a-QL), or GFP-Rab4aS27N (4a-SN). (C) Same as A, but the cells expressed Rab8aQ67L (8a-QL) or Rab11aQ70L (11a-QL), and after lysis, the incubations were performed in the absence of MgCl2. (D) Same as in A, but HEK cells coexpressed flag-TBC1D9B with GFP-Rab8a-WT (8a-WT), GFP-Rab8a-Q67L (8a-QL), or GFP-Rab8a-T22N (8a-TN). (E–G) Top left, GST alone or GST-TBC1D9B-(301-810) (#2 fragment) was used to affinity capture GFP-Rab11a-QL and GFP-Rab11b-QL (E), GFP-Rab4a-QL (F). or GFP-Rab8a-QL (G). Right, 2% of each lysate was resolved by SDS–PAGE and GFP-Rab-QL detected by Western blot using anti-GFP antibody. Bottom left, GST constructs were resolved by SDS–PAGE and proteins detected on Western blots using an anti-GST antibody. (H) Quantification of data from E–G. Values are normalized to the total expression of the protein first and then to the values obtained for the GFP-Rab11a-WT pull down. Data were obtained 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).

Mentions: We were initially surprised that Rab8a-QL served as a substrate for TBC1D9B in the GAP assays (Figure 3B) but did not coimmunoprecipitate with flag-TBC1D9B (Figure 2). Additional experimentation revealed that these differences were likely related to the amount of Mg2+ used in these assays. Whereas the in vitro GAP assays were initially performed in minimal concentrations of Mg2+ (GTP bound with Mg2+ was used in the assays at a final concentration of 0.5 mM), similar to previous reports (Krugmann et al., 2004; Haas et al., 2005; Fuchs et al., 2007; Yoshimura et al., 2008), the coimmunoprecipitation studies in Figure 2 were performed in the presence of an additional 2.5 mM Mg2+ (Oztan et al., 2007; Tsun et al., 2013). Of interest, when we removed Mg2+ from the coimmunoprecipitation studies, we observed interactions between GFP-Rab8a-QL and flag-TBC1D9B (see later discussion of Figure 5C). In contrast, supplementing the concentration of Mg2+ in the GAP assays by 5 mM MgCl2 (Du et al., 1998; Albert et al., 1999; Casanova et al., 1999) evoked a significant increase in Rab11a GTPase activity (Figure 3E) but not for Rab8a (Figure 3E) or any of the other Rab GTPases tested (Supplemental Figure S2C). Finally, we expressed full-length flag-TBC1D9B in HeLa cells and then recovered the protein by immunoprecipitation. Like TBC1D9B-(301-810), the full-length protein showed GTPase activity only against Rab11a and not Rab8a (Figure 3F).


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 interacts with Rab4a and Rab11b. (A) Left, coimmunoprecipitation of flag-tagged TBC1D9B with GFP-Rab11b-WT (11b-WT), GFP-Rab11b-Q70L (11b-QL), or GDP-locked GFP-Rab11bS25N (11b-SN) coexpressed in HEK cells. Anti-flag antibody was used to immunoprecipitate flag-TBC1D9B, and anti-GFP antibody was used to detect the coimmunoprecipitated GFP-tagged Rab protein. IgG was used as a nonspecific control. Right, 2% of each lysate was resolved by SDS–PAGE and overexpressed proteins detected by Western blot. Values were normalized to the total expression of each protein and then normalized to the amount of Rab11b-WT lysate. (B) Same as in A, but HEK cells coexpressed flag-TBC1D9B and GFP-Rab4a-WT (4a-WT), GFP-Rab4aQ72L (4a-QL), or GFP-Rab4aS27N (4a-SN). (C) Same as A, but the cells expressed Rab8aQ67L (8a-QL) or Rab11aQ70L (11a-QL), and after lysis, the incubations were performed in the absence of MgCl2. (D) Same as in A, but HEK cells coexpressed flag-TBC1D9B with GFP-Rab8a-WT (8a-WT), GFP-Rab8a-Q67L (8a-QL), or GFP-Rab8a-T22N (8a-TN). (E–G) Top left, GST alone or GST-TBC1D9B-(301-810) (#2 fragment) was used to affinity capture GFP-Rab11a-QL and GFP-Rab11b-QL (E), GFP-Rab4a-QL (F). or GFP-Rab8a-QL (G). Right, 2% of each lysate was resolved by SDS–PAGE and GFP-Rab-QL detected by Western blot using anti-GFP antibody. Bottom left, GST constructs were resolved by SDS–PAGE and proteins detected on Western blots using an anti-GST antibody. (H) Quantification of data from E–G. Values are normalized to the total expression of the protein first and then to the values obtained for the GFP-Rab11a-WT pull down. Data were obtained 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).
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Figure 5: TBC1D9B interacts with Rab4a and Rab11b. (A) Left, coimmunoprecipitation of flag-tagged TBC1D9B with GFP-Rab11b-WT (11b-WT), GFP-Rab11b-Q70L (11b-QL), or GDP-locked GFP-Rab11bS25N (11b-SN) coexpressed in HEK cells. Anti-flag antibody was used to immunoprecipitate flag-TBC1D9B, and anti-GFP antibody was used to detect the coimmunoprecipitated GFP-tagged Rab protein. IgG was used as a nonspecific control. Right, 2% of each lysate was resolved by SDS–PAGE and overexpressed proteins detected by Western blot. Values were normalized to the total expression of each protein and then normalized to the amount of Rab11b-WT lysate. (B) Same as in A, but HEK cells coexpressed flag-TBC1D9B and GFP-Rab4a-WT (4a-WT), GFP-Rab4aQ72L (4a-QL), or GFP-Rab4aS27N (4a-SN). (C) Same as A, but the cells expressed Rab8aQ67L (8a-QL) or Rab11aQ70L (11a-QL), and after lysis, the incubations were performed in the absence of MgCl2. (D) Same as in A, but HEK cells coexpressed flag-TBC1D9B with GFP-Rab8a-WT (8a-WT), GFP-Rab8a-Q67L (8a-QL), or GFP-Rab8a-T22N (8a-TN). (E–G) Top left, GST alone or GST-TBC1D9B-(301-810) (#2 fragment) was used to affinity capture GFP-Rab11a-QL and GFP-Rab11b-QL (E), GFP-Rab4a-QL (F). or GFP-Rab8a-QL (G). Right, 2% of each lysate was resolved by SDS–PAGE and GFP-Rab-QL detected by Western blot using anti-GFP antibody. Bottom left, GST constructs were resolved by SDS–PAGE and proteins detected on Western blots using an anti-GST antibody. (H) Quantification of data from E–G. Values are normalized to the total expression of the protein first and then to the values obtained for the GFP-Rab11a-WT pull down. Data were obtained 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).
Mentions: We were initially surprised that Rab8a-QL served as a substrate for TBC1D9B in the GAP assays (Figure 3B) but did not coimmunoprecipitate with flag-TBC1D9B (Figure 2). Additional experimentation revealed that these differences were likely related to the amount of Mg2+ used in these assays. Whereas the in vitro GAP assays were initially performed in minimal concentrations of Mg2+ (GTP bound with Mg2+ was used in the assays at a final concentration of 0.5 mM), similar to previous reports (Krugmann et al., 2004; Haas et al., 2005; Fuchs et al., 2007; Yoshimura et al., 2008), the coimmunoprecipitation studies in Figure 2 were performed in the presence of an additional 2.5 mM Mg2+ (Oztan et al., 2007; Tsun et al., 2013). Of interest, when we removed Mg2+ from the coimmunoprecipitation studies, we observed interactions between GFP-Rab8a-QL and flag-TBC1D9B (see later discussion of Figure 5C). In contrast, supplementing the concentration of Mg2+ in the GAP assays by 5 mM MgCl2 (Du et al., 1998; Albert et al., 1999; Casanova et al., 1999) evoked a significant increase in Rab11a GTPase activity (Figure 3E) but not for Rab8a (Figure 3E) or any of the other Rab GTPases tested (Supplemental Figure S2C). Finally, we expressed full-length flag-TBC1D9B in HeLa cells and then recovered the protein by immunoprecipitation. Like TBC1D9B-(301-810), the full-length protein showed GTPase activity only against Rab11a and not Rab8a (Figure 3F).

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