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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 in vitro GAP activity. (A) TBC1D4 (PDB: 3QYB) was used as a template (gray) to model the 3D structure of the TBC1D9B TBC domain (green). (B) GTP hydrolysis by the indicated wild-type, GST-tagged Rab loaded with [γ-32P]GTP and incubated with 2 μM of either wild-type or mutant TBC1D9B-(301-810) for 60 min at 30°C. Data are corrected for reactions lacking the TBC1D9B fragment. Those values significantly different from the group means, assessed by ANOVA, are indicated (*p < 0.05). (C) Kinetics of Rab11a GTP hydrolysis loaded with [γ-32P]GTP and incubated with 0, 0.5, 1, 2, or 4 μM recombinant TBC1D9B-(301-810). (D) Initial rates of Rab11a GTP hydrolysis plotted against the concentration of wild-type or mutant TBC1D9B-(301-810). (E) In vitro GAP assays performed in the presence of Rab11a or Rab8a. In these reactions, Mg2+ mixed at a 1:1 M ratio with GTP, was added at a final concentration of 0.5 mM. Alternatively, the reaction was supplemented with 5 mM MgCl2. Reactions were incubated for 30 min at 30°C. Data were normalized to control reactions in which no TBC1D9B-(301-810) was added. Values for TBC1D9B-(301-810) that were significantly different (p < 0.05) from matched incubations performed in the presence of TBC1D9B-RYQ/AAA (301-810) or between the indicated reactions are identified with an asterisk. (F) Top, full-length flag-TBC1D9B-WT or flag-TBC1D9B-RYQ/AAA was immunoprecipitated from HeLa cell lysates and a GAP assay performed using Rab11a or Rab8a loaded with [γ-32P]GTP and incubated for 60 min at 30°C. Lysates from nontransfected cells were used as controls. Bottom, Western blot of a 5-μl aliquot of the immunoprecipitates used in the in vitro assay was detected using an anti-flag antibody. (B–F) Data were obtained from three or more independent experiments performed in duplicate, and the mean ± SEM is shown. In E and F, values significantly different between TBC1D9B-WT and TBC1D9B-RYQ/AAA are indicated with an asterisk (p < 0.05).
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Figure 3: TBC1D9B in vitro GAP activity. (A) TBC1D4 (PDB: 3QYB) was used as a template (gray) to model the 3D structure of the TBC1D9B TBC domain (green). (B) GTP hydrolysis by the indicated wild-type, GST-tagged Rab loaded with [γ-32P]GTP and incubated with 2 μM of either wild-type or mutant TBC1D9B-(301-810) for 60 min at 30°C. Data are corrected for reactions lacking the TBC1D9B fragment. Those values significantly different from the group means, assessed by ANOVA, are indicated (*p < 0.05). (C) Kinetics of Rab11a GTP hydrolysis loaded with [γ-32P]GTP and incubated with 0, 0.5, 1, 2, or 4 μM recombinant TBC1D9B-(301-810). (D) Initial rates of Rab11a GTP hydrolysis plotted against the concentration of wild-type or mutant TBC1D9B-(301-810). (E) In vitro GAP assays performed in the presence of Rab11a or Rab8a. In these reactions, Mg2+ mixed at a 1:1 M ratio with GTP, was added at a final concentration of 0.5 mM. Alternatively, the reaction was supplemented with 5 mM MgCl2. Reactions were incubated for 30 min at 30°C. Data were normalized to control reactions in which no TBC1D9B-(301-810) was added. Values for TBC1D9B-(301-810) that were significantly different (p < 0.05) from matched incubations performed in the presence of TBC1D9B-RYQ/AAA (301-810) or between the indicated reactions are identified with an asterisk. (F) Top, full-length flag-TBC1D9B-WT or flag-TBC1D9B-RYQ/AAA was immunoprecipitated from HeLa cell lysates and a GAP assay performed using Rab11a or Rab8a loaded with [γ-32P]GTP and incubated for 60 min at 30°C. Lysates from nontransfected cells were used as controls. Bottom, Western blot of a 5-μl aliquot of the immunoprecipitates used in the in vitro assay was detected using an anti-flag antibody. (B–F) Data were obtained from three or more independent experiments performed in duplicate, and the mean ± SEM is shown. In E and F, values significantly different between TBC1D9B-WT and TBC1D9B-RYQ/AAA are indicated with an asterisk (p < 0.05).

Mentions: We next sought to determine whether TBC1D9B had GAP activity against any of its binding partners. We first performed comparative protein structure modeling using the known three-dimensional (3D) structure of the TBC1D4 TBC domain as a template (Protein Data Bank [PDB] file 3QYB; Park et al., 2011). Figure 3A shows the predicted structure of the TBC domain of TBC1D9B superimposed on the known structure of the TBC1D4 TBC domain. The structural overlap parameter obtained was 64%, confirming that the two structures were structurally related (Geourjon et al., 2001). In addition, the putative catalytic residues (R559, Y592, and Q594) of TBC1D9B were exposed similarly to those of TBC1D4 and other TBC proteins such as Gyp1p (unpublished data). Thus, the sequence homologies and predicted structure are consistent with the possibility that TBC1D9B was a functional GAP.


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 in vitro GAP activity. (A) TBC1D4 (PDB: 3QYB) was used as a template (gray) to model the 3D structure of the TBC1D9B TBC domain (green). (B) GTP hydrolysis by the indicated wild-type, GST-tagged Rab loaded with [γ-32P]GTP and incubated with 2 μM of either wild-type or mutant TBC1D9B-(301-810) for 60 min at 30°C. Data are corrected for reactions lacking the TBC1D9B fragment. Those values significantly different from the group means, assessed by ANOVA, are indicated (*p < 0.05). (C) Kinetics of Rab11a GTP hydrolysis loaded with [γ-32P]GTP and incubated with 0, 0.5, 1, 2, or 4 μM recombinant TBC1D9B-(301-810). (D) Initial rates of Rab11a GTP hydrolysis plotted against the concentration of wild-type or mutant TBC1D9B-(301-810). (E) In vitro GAP assays performed in the presence of Rab11a or Rab8a. In these reactions, Mg2+ mixed at a 1:1 M ratio with GTP, was added at a final concentration of 0.5 mM. Alternatively, the reaction was supplemented with 5 mM MgCl2. Reactions were incubated for 30 min at 30°C. Data were normalized to control reactions in which no TBC1D9B-(301-810) was added. Values for TBC1D9B-(301-810) that were significantly different (p < 0.05) from matched incubations performed in the presence of TBC1D9B-RYQ/AAA (301-810) or between the indicated reactions are identified with an asterisk. (F) Top, full-length flag-TBC1D9B-WT or flag-TBC1D9B-RYQ/AAA was immunoprecipitated from HeLa cell lysates and a GAP assay performed using Rab11a or Rab8a loaded with [γ-32P]GTP and incubated for 60 min at 30°C. Lysates from nontransfected cells were used as controls. Bottom, Western blot of a 5-μl aliquot of the immunoprecipitates used in the in vitro assay was detected using an anti-flag antibody. (B–F) Data were obtained from three or more independent experiments performed in duplicate, and the mean ± SEM is shown. In E and F, values significantly different between TBC1D9B-WT and TBC1D9B-RYQ/AAA are indicated with an asterisk (p < 0.05).
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Figure 3: TBC1D9B in vitro GAP activity. (A) TBC1D4 (PDB: 3QYB) was used as a template (gray) to model the 3D structure of the TBC1D9B TBC domain (green). (B) GTP hydrolysis by the indicated wild-type, GST-tagged Rab loaded with [γ-32P]GTP and incubated with 2 μM of either wild-type or mutant TBC1D9B-(301-810) for 60 min at 30°C. Data are corrected for reactions lacking the TBC1D9B fragment. Those values significantly different from the group means, assessed by ANOVA, are indicated (*p < 0.05). (C) Kinetics of Rab11a GTP hydrolysis loaded with [γ-32P]GTP and incubated with 0, 0.5, 1, 2, or 4 μM recombinant TBC1D9B-(301-810). (D) Initial rates of Rab11a GTP hydrolysis plotted against the concentration of wild-type or mutant TBC1D9B-(301-810). (E) In vitro GAP assays performed in the presence of Rab11a or Rab8a. In these reactions, Mg2+ mixed at a 1:1 M ratio with GTP, was added at a final concentration of 0.5 mM. Alternatively, the reaction was supplemented with 5 mM MgCl2. Reactions were incubated for 30 min at 30°C. Data were normalized to control reactions in which no TBC1D9B-(301-810) was added. Values for TBC1D9B-(301-810) that were significantly different (p < 0.05) from matched incubations performed in the presence of TBC1D9B-RYQ/AAA (301-810) or between the indicated reactions are identified with an asterisk. (F) Top, full-length flag-TBC1D9B-WT or flag-TBC1D9B-RYQ/AAA was immunoprecipitated from HeLa cell lysates and a GAP assay performed using Rab11a or Rab8a loaded with [γ-32P]GTP and incubated for 60 min at 30°C. Lysates from nontransfected cells were used as controls. Bottom, Western blot of a 5-μl aliquot of the immunoprecipitates used in the in vitro assay was detected using an anti-flag antibody. (B–F) Data were obtained from three or more independent experiments performed in duplicate, and the mean ± SEM is shown. In E and F, values significantly different between TBC1D9B-WT and TBC1D9B-RYQ/AAA are indicated with an asterisk (p < 0.05).
Mentions: We next sought to determine whether TBC1D9B had GAP activity against any of its binding partners. We first performed comparative protein structure modeling using the known three-dimensional (3D) structure of the TBC1D4 TBC domain as a template (Protein Data Bank [PDB] file 3QYB; Park et al., 2011). Figure 3A shows the predicted structure of the TBC domain of TBC1D9B superimposed on the known structure of the TBC1D4 TBC domain. The structural overlap parameter obtained was 64%, confirming that the two structures were structurally related (Geourjon et al., 2001). In addition, the putative catalytic residues (R559, Y592, and Q594) of TBC1D9B were exposed similarly to those of TBC1D4 and other TBC proteins such as Gyp1p (unpublished data). Thus, the sequence homologies and predicted structure are consistent with the possibility that TBC1D9B was a functional GAP.

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