TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells

<div><p>Recycling endosomes are generally thought to play a central role in endocytic recycling, but recent evidence has indicated that they also participate in other cellular events, including cytokinesis, autophagy, and neurite outgrowth. Rab small GTPases are key regulators in membrane trafficking, and although several Rab isoforms, e.g., Rab11, have been shown to regulate recycling endosomal trafficking, the precise mechanism by which these Rabs regulate recycling endosomes is not fully understood. In this study, we focused on a Rab-GTPase-activating protein (Rab-GAP), one of the key regulators of Rabs, and comprehensively screened 43 mammalian Tre-2/Bub2/Cdc16 (TBC)/Rab-GAP-domain-containing proteins (TBC proteins) for proteins that specifically localize on recycling endosomes in mouse embryonic fibroblasts (MEFs). Four of the 43 mammalian TBC proteins screened, i.e., TBC1D11, TBC1D12, TBC1D14, and EVI5, were found to colocalize well with transferrin receptor, a well-known recycling endosome marker. We further investigated the biochemical properties of TBC1D12, a previously uncharacterized TBC protein. The results showed that TBC1D12 interacted with active Rab11 through its middle region and that it did not display Rab11-GAP activity <i>in vitro</i>. The recycling endosomal localization of TBC1D12 was found to depend on the expression of Rab11. We also found that TBC1D12 expression had no effect on common Rab11-dependent cellular events, e.g., transferrin recycling, in MEFs and that it promoted neurite outgrowth, a specialized Rab11-dependent cellular event, of PC12 cells independently of its GAP activity. These findings indicated that TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells.</p></div

Identification of TBC proteins that specifically localize on recycling endosomes in MEFs.

<p>(A) Typical images of four TBC proteins that clearly colocalized with TfR (a recycling endosome marker). MEFs transiently expressing EGFP-TBC proteins were immunostained with anti-TfR antibody (1/250 dilution), and the stained cells were examined with a confocal fluorescence microscope. The insets show magnified views of the boxed areas. The line scan profiles (broken arrows in the far right column) were acquired by using ImageJ software. (B) A typical image of TBC protein showing no colocalization with TfR. Scale bars, 40 μm. The images of all other TBC proteins are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174883#pone.0174883.s001" target="_blank">S1 Fig</a>.</p

TBC1D12 interacts with Rab11 through its middle region.

<p>(A) TBC1D12 interacts with a constitutively active form that mimics the GTP-bound form of Rab11. Total lysates of COS-7 cells expressing T7-TBC1D12 were incubated with glutathione-Sepharose beads coupled with GST alone, GST-Rab11A-Q70L (indicated by QL), or GST-Rab11A-S25N (indicated by SN). A 2% volume of the reaction mixture (input) and proteins bound to the glutathione-Sepharose beads were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-T7 tag antibody (top and middle panels), and the GST-fusion proteins were stained with amido black (bottom panel). The positions of the molecular mass markers (in kDa) are shown on the left. (B) Schematic representation of three deletion mutants of TBC1D12 (TBC1D12-N, -M, and -TBC). TBC domains and coiled-coil (CC) domains are shown as blue boxes and green boxes, respectively. (C) Total lysates of COS-7 cells expressing T7-TBC1D12 (indicated by FL, full-length), T7-TBC1D12-N, T7-TBC1D12-M, or T7-TBC1D12-TBC were incubated with glutathione-Sepharose beads coupled with GST alone (indicated by G) or GST-Rab11A-Q70L (indicated by 11). A 2% volume of the reaction mixture (input) and proteins bound to the beads were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-T7 tag antibody (top three panels). The third panel corresponds to the longer exposure of the second panel. The GST-fusion proteins were stained with amido black (bottom panel). The asterisk indicates non-specific bands, and the positions of the molecular mass markers (in kDa) are shown on the left.</p

Overexpression of TBC1D12 in PC12 cells promotes neurite outgrowth independently of its GAP activity.

<p>(A) Overexpression of either TBC1D12 wild-type (WT) or its GAP-activity-deficient mutant (RK) promoted neurite outgrowth of PC12 cells. Typical images of PC12 cells expressing EGFP alone (control), EGFP-TBC1D12-WT, or EGFP-TBC1D12-RK. After NGF stimulation for 36 h the cells were fixed, immunostained with anti-GFP antibody (1/500 dilution), and examined with a fluorescence microscope. Scale bars, 30 μm. (B) The total neurite length of each cell in (A) after NGF stimulation for 36 h was measured with MetaMorph software (n >100). The total neurite length of each sample was normalized to that of the control cells. Error bars indicate the SEMs of the data from three independent experiments. No significant difference was observed between the WT and RK mutant. (C) TBC1D12 promotes neurite outgrowth in a Rab11-dependent manner. Typical images of PC12 cells expressing EGFP alone (control) or EGFP-TBC1D12-WT together with siControl or siRab11A/B. After NGF stimulation for 36 h the cells were fixed, immunostained with anti-GFP antibody (1/500 dilution), and examined with a fluorescence microscope. Scale bars, 30 μm. (D) The total neurite length of each cell in (C) after NGF stimulation for 36 h was measured with MetaMorph software (n >100). The total neurite length of each sample was normalized to that of the control cells. Error bars indicate the SEMs of the data from three independent experiments. *, <i>p</i> <0.05; **, <i>p</i> <0.01; ***, <i>p</i> <0.001; NS, not significant; and a.u., arbitrary unit. (E) Rab11-S25N (SN), but not Rab11-Q70L (QL), inhibits neurite outgrowth of both control cells and TBC1D12-expressing cells. Typical images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174883#pone.0174883.s007" target="_blank">S7 Fig</a>. The total neurite length of each cell after NGF stimulation for 36 h was measured as described in (D). Error bars indicate the SEMs of the data from ≥79 cells. ***, <i>p</i> <0.001; **, <i>p</i> <0.01; NS, not significant. Although both positive and negative effects of Rab11-Q70L (or a positive effect of S25N) on neurite/axon outgrowth have been reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174883#pone.0174883.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174883#pone.0174883.ref039" target="_blank">39</a>], under our experimental conditions expression of Rab11-Q70L and Rab11-S25N in PC12 cells promoted and inhibited, respectively, NGF-stimulated neurite outgrowth.</p

Rab11 recruits TBC1D12 to Rab11-positive recycling endosomes.

<p>(A) Overexpression of Rab11 altered TBC1D12 localization from around the nucleus to the cell protrusions (arrowheads). MEFs transiently expressing both EGFP-TBC1D12 and mStr-Rab11A (or mStr alone as a control) were examined with a confocal fluorescence microscope. Scale bars, 10 μm. (B) Knockdown of Rab11A/B caused dispersion of TBC1D12 into the cytosol. MEFs stably expressing T7-TBC1D12 were transfected with control siRNA (siControl) or Rab11A/B siRNAs (siRab11A/B) and immunostained with anti-TBC1D12 antibody (1/300 dilution) and anti-Rab11 antibody (1/200 dilution), and the stained cells were examined with a confocal fluorescence microscope. Scale bars, 20 μm. (C) Knockdown of Rab11A/B in MEFs as revealed by immunoblotting. The band intensity of Rab11 in siRab11A/B-treated cells was 6.7% of its band intensity in the control cells. Total cell lysates of siControl-treated cells (lane 1) and siRab11A/B-treated cells (lane 2) were analyzed by 8% SDS-PAGE and immunoblotting with anti-GFP antibody (top panel; 1/500 dilution), anti-Rab11 antibody (middle panel; 1/500 dilution), and anti-β-actin antibody (bottom panel; 1/15,000 dilution). The positions of the molecular mass markers (in kDa) are shown on the left.</p

Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

<p>MAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.</p

Cdk5-mediated <i>in vivo</i> Ser34 phosphorylation.

<p>(A) Phosphorylation of AATYK1 in mouse brain. AATYK1 was immunoprecipitated from mouse brain extracts and incubated in the presence (+) or absence (–) of bacterial alkaline phosphatase (BAP). The anti-pS34 reaction is shown in the upper panel and the anti-AATYK1 reaction is shown in the lower panel. (B) Phosphorylation of AATYK1 in mouse brain during early postnatal development. AATYK1 was immunoprecipitated from mouse brain extracts at P2, P5, P10, and six weeks of age (Ad) using the anti-AATYK1 antibody. The immunoprecipitates were immunoblotted using the anti-pS34 (top panel) and anti-AATYK1 (second panel) antibodies. Immunoblots of brain extracts using anti-AATYK1 and anti-actin antibodies are also shown in the lower panels. (C) Phosphorylation of AATYK1 at Ser34 in Cdk5^–/– mouse brain. AATYK1 was immunoprecipitated from brain extracts of Cdk5^–/– mice at embryonic day 18.5 (E18.5) and immunoblotted using the anti-pS34 antibody (top panel). Immunoblots of the brain extracts using anti-AATYK1 (third panel), anti-Cdk5 (fourth panel), and anti-actin (bottom panel) antibodies. Quantification of AATYK1 and pS34 is shown in (D) and (E), respectively. Bars indicate the means ± S.E. of three independent experiments (n = 3; * <i>P</i><0.05; <i>t</i> test).</p

Binding of AATYK1A to Cdk5/p35.

<p>(A) Coimmunoprecipitation of Cdk5/p35 with AATYK1A in HEK293 cells. AATYK1A-Flag was coexpressed with p35 and/or Cdk5 in HEK293 cells and immunoprecipitated from cell lysates using the anti-Flag antibody. p35 and Cdk5 were detected in the anti-Flag immunoprecipitates by immunoblotting using anti-p35 and anti-Cdk5 antibodies. (B) Coimmunoprecipitation of Cdk5/p35 from mouse brain extracts using the anti-AATYK1 antibody. AATYK1 was immunoprecipitated from a mouse brain extract (10 weeks). p35 and Cdk5 were detected in the AATYK1 immunoprecipitates using the anti-p35 and anti-Cdk5 antibodies.</p

Colocalization of p35 with AATYK1A in early and recycling endosomes.

<p>(A) Colocalization of AATYK1A and p35 in COS-7 cells. COS-7 cells were transfected with AATYK1A-Myc together with p35 and Cdk5. AATYK1A and p35 were detected by immunostaining with the anti-Myc antibody and anti-p35 antibody, followed by incubation with Alexa 488-conjugated anti-mouse IgG and Alexa 548-conjugated anti-rabbit antibody, respectively. A merged image is shown in the right panel. Insets represent higher magnifications and arrows indicate the colocalization. Scale bar, 20 µm. (B) Localization of AATYK1A and p35 in early and recycling endosomes. COS-7 cells were transfected with AATYK1A-Myc, p35, Cdk5, and either EGFP-Rab5A (as an early-endosome marker) or EGFP–Rab11A (as a recycling-endosome marker). After 24 h of transfection, cells were fixed and stained with anti-Myc and anti-p35 antibodies, as described above, and were observed using a confocal microscope. Scale bar, 10 µm. (C) Localization of AATYK1 and p35 in endosomes in cultured cortical neurons. Rat brain cortical neurons at DIV5 were transfected with EGFP-Rab11A (middle panels). The cells were immunostained with anti-AATYK1 and p35 (C19) 24 h after transfection, followed by Alexa 546-conjugated anti-rabbit secondary antibody (left panels). Merge is shown in right panels. Bar, 10 µm.</p

Generation of the anti-pSer34-specific antibody and Ser34 phosphorylation of AATYK1 in PC12D cells.

<p>(A) Amino-acid sequences of mouse, rat, and human AATYK1A around Ser34. A synthetic peptide corresponding to the mouse AATYK1A amino-acid residues 29–39 (the Ser34 phosphorylation site is underlined) was used for rabbit immunization. (B) Specificity of the anti-pS34 antibody. COS-7 cells were transfected with AATYK1A or its S34A mutant in the presence (+) or absence (–) of Cdk5/p35. Cell extracts were immunoblotted with the anti-pS34 antibody or anti-Myc antibody for AATYK1A. (C) Phosphorylation of AATYK1 at Ser34 in PC12D cells. PC12D cells were treated with 50 ng/ml NGF for 24 h in the presence or absence of 20 µM roscovitine (Ros). AATYK1 was immunoprecipitated in PC12D cells using the anti-AATYK1 antibody and was subjected to immunoblotting with the anti-pS34 or anti-AATYK1 antibodies. (D) Immunofluorescent staining of PC12D cells using the anti-pS34 antibody. PC12D cells expressing AATYK1A-Myc were treated with NGF for 24 h and double labeled with the anti-pS34 (top panel) and anti-Myc (AATYK1A, middle panel) antibodies. A merged image is shown in the lower panel. The growth cone is indicated by an arrow. Scale bar, 20 µm.</p