Rab5C depletion significantly inhibits cell migration.

<p>A) DIC images of stable Rab5 isoform knock-down (KD) HeLa cells taken with light microscope at 40X magnification (left panel). Arrows indicate membrane ruffles. KD of Rab5 isoforms (right panel) in these stable cell lines is shown in the immunoblots following SDS-PAGE as described in Experimental Procedures. B) 0.5–1 mm width wounds were made on a monolayer of HeLa stable control or Rab5 isoform KD cells. 5–7 wounded spots in each dish were imaged with time-lapse microscope every 5 minutes for 20 hours. C) The percentage of wound closure (left panel) was calculated from images acquired at time 0 and 16 hours with ImageJ. For each sample, at least 5 images were used to calculate the percentage of wound closure in each experiment. The graph represents the Mean± S.E. from four independent experiments. U937 cells (right panel) transiently transfected with siRNA against Rab5 isoforms were seeded in the upper chamber of the Transwell plates and allowed to migrate towards 10% FBS in the bottom chamber for 24 hours. Migrated cells were measured as indicated in Material and Methods. Data are normalized to initial seeding cell numbers. The graph represents the Mean± S.E. from four independent experiments. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test.(*P<0.05, ***P<0.001)</p

PI3K signaling in response to Rab5 isoform depletion.

<p>A) HeLa cells were transfected with GFP (as negative control) or Rab5 isoform-specific siRNA. 48 hours post-transfection, cells were starved and then stimulated with EGF for indicated times. Cell lysates were subjected to SDS-PAGE and probed with antibodies as indicated. Band intensity was quantified with AlphaEaseFc 4.0 software. Bars represent the mean value ± S.E. from four independent experiments. Analysis was carried out with a two-way ANOVA, Bonferroni’s post-test. P<0.05. B) HeLa cells were transfected with indicated siRNA. 48 hours post-transfection, cells were seeded onto micropatterned coverslips coated with fibronectin, and then allowed to spread out for 2 hours in starvation medium. Starved cells were stimulated with 10 % FCS for 3 minutes and then fixed for PIP₃-FITC antibody immuno-staining. Images shown here are average projections of PIP₃ staining from 30–35 cells.</p

Depletion of Rab5C reduces cell adhesion.

<p>A) HeLa cells were seeded on coverslips O/N and then transfected with GFP or Rab5 isoform siRNAs. The focal adhesion complex was visualized by immunostaining with vinculin antibody. The numbers of focal adhesion complexes were determined with ImageJ. The graph represents Mean± S.E. from 30 cells. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test. P<0.0001. B) HeLa cells transfected with GFP or Rab5 isoform siRNAs were re-suspended and re-plated on fibronectin-coated plates for indicated times. At the end of each time point, cell lysates were extracted and prepared for SDS-PAGE and Western bloting. The activation of focal adhesion kinase was determined with phospho-FAK antibody. Total levels of FAK were not determined. The data represents Mean± S.E. from three independent experiments. Analysis was carried out with a two-way ANOVA, Bonferroni’s post-test. P<0.05.</p

Rab5 Isoforms Orchestrate a “Division of Labor” in the Endocytic Network; Rab5C Modulates Rac-Mediated Cell Motility

<div><p>Rab5, the prototypical Rab GTPase and master regulator of the endocytic pathway, is encoded as three differentially expressed isoforms, Rab5A, Rab5B and Rab5C. Here, we examined the differential effects of Rab5 isoform silencing on cell motility and report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. Initial observations revealed that silencing of Rab5C expression, but neither Rab5A nor Rab5C, led to spindle-shaped cells that displayed reduced formation of membrane ruffles. When subjected to a scratch wound assay, cells depleted of Rab5C, but not Rab5A or Rab5B, demonstrated reduced cell migration. U937 cells depleted of Rab5C also displayed reduced cell motility in a Transwell plate migration assay. To examine activation of Rac, HeLa cells stably expressing GFP-Rac1 were independently depleted of Rab5A, Rab5B or Rab5C and seeded onto coverslips imprinted with a crossbow pattern. 3-D GFP-Rac1 images of micro-patterned cells show that GFP-Rac1 was less localized to the cell periphery in the absence of Rab5C. To confirm the connection between Rab5C and Rac activation, HeLa cells depleted of Rab5 isoforms were starved and then stimulated with EGF. Rac1 pull-down assays revealed that EGF-stimulated Rac1 activity was significantly suppressed in Rab5C-suppressed cells. To determine whether events upstream of Rac activation were affected by Rab5C, we observed that EGF-stimulated Akt phosphorylation was suppressed in cells depleted of Rab5C. Finally, since spatio-temporal assembly/disassembly of adhesion complexes are essential components of cell migration, we examined the effect of Rab5 isoform depletion on the formation of focal adhesion complexes. Rab5C-depleted HeLa cells have significantly fewer focal adhesion foci, in accordance with the lack of persistent lamellipodial protrusions and reduced directional migration. We conclude that Rab5 isoforms selectively oversee the multiple signaling and trafficking events associated with the endocytic network.</p></div

Loss of Rab5C reduced translocation of Rac1 to cell periphery and Rac1 activation in response to EGF stimulation.

<p>A) HeLa cells stably expressing GFP-Rac1 were transfected with scrambled or Rab5 isoform siRNAs. 48 hours post-transfection, cells were seeded onto coverslips imprinted with crossbow micro-patterns. 3-D GFP-Rac1 images of at least 40 micro-patterned cells were acquired for each sample. Each GFP-Rac1 3-D image stack was subjected to Maximum intensity projection and then grey scale normalization. Next, the max projections of 88 GFP-Rac1 images (from two independent experiments) were aligned and averaged (upper panel, in pseudo-color). Image subtraction was carried out between averaged image of scramble control and that of individual Rab5 isoform siRNA-treated sample. The resulting image after subtraction (siScr minus siRab5) is shown in pseudo-color (bottom panel). B) HeLa cells stably expressing GFP-Rac1 were transfected with scrambled or Rab5 isoform siRNAs. 48 hours post-transfection, cells were separated into membrane (Mem) and cytosolic (Cyt) fractions as described in Material and Method. Relative amounts of GFP-Rac1 in each fraction were analyzed by SDS-PAGE and Western blot. Densitometry of the bands was quantified using AlphaEaseFC 4.0 software. The numbers represent the ratio of GFP-Rac1 in cytosol or membrane/total. C) HeLa cells were transfected with scrambled or Rab5 isoform-specific siRNA. 48 hours post-transfection, cells were starved and then stimulated with EGF for two minutes. Cell lysates were prepared and subjected to Rac1 pull-down assays. Proteins were eluted, separated by SDS-PAGE and blotted for Rac. Total lysates were also probed for Rac1 to determine the total Rac1 level is equal in all samples. The intensity of the bands from western blots was quantified with AlphaEaseFc 4.0 software. The relative amount of Rac-GTP from pull-downs was normalized to that of total Rac1 from total cell lysates. The adjacent graph represents the mean value ± S.E. from four independent experiments. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test. (*P<0.05, **P<0.01)</p

Model for the regulation of IRS-1 degradation by TBC1D3 expression.

<p>We propose that TBC1D3 suppresses the degradation of IRS-1 by regulating the phosphorylation of S6K at T389. In this model, mTOR phosphorylates S6K in response to insulin signaling. TBC1D3 interacts, directly or indirecly, with PP2A B56γ to enhance the dephosphorylation of S6K:T389 thereby reducing the S6K-dependent phosphorylation of IRS-1 at key sites which are required for IRS-1 ubiquitination and degradation.</p

PP2A B56γ mediates S6K phosphorylation in response to TBC1D3 expression.

<p>(A) DU145 cells with GFP (control) or PP2A B56γ knockdown were transfected with myc-TBC1D3 or empty vector, serum-starved, and stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of S6K were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of S6K:T389 phosphorylation normalized to S6K protein levels (* <i>p</i><0.05, ** <i>p</i><0.01). (B) Reduction of IRS-1:S636/639 phosphorylation induced by TBC1D3 expression is rescued by a rapamycin-resistant mutant of S6K. pCIS2-IRS-1, myc-TBC1D3 or empty vector were co-transfected with HA-S6K-ED₃E or vector in DU145 cells. Cells were stimulated with insulin (10 nM) for 0, 5 or 30 min after serum-starvation. Phosphorylation and protein levels of IRS-1 were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of IRS-1:S636/639 phosphorylation normalized to IRS-1 protein levels (* <i>p</i><0.05, ** <i>p</i><0.01). The data are presented as means ± SD of three independent experiments. (C) TBC1D3 co-immunoprecipitates with PP2A B56γ. Hek293 cells were transfected with myc-TBC1D3 or empty vector. Cell lysates were immunoprecipitated with anti-myc or anti-B56γ antibodies, respectively. Co-IP samples were separated by SDS-PAGE and analyzed by Western blotting with specific antibodies.</p

TBC1D3 reduces S6K activation, but does not affect mTOR-C1 pathway.

<p>(A) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of S6K were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of S6K:T389 phosphorylation normalized to S6K protein levels (** <i>p</i><0.01). (B) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, pre-treated with Rapamycin (50 nM) for 2 h, and stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of 4EBP1 and S6K were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of 4EBP1 phosphorylation normalized to GAPDH protein levels. The data are presented as means ± SD of three independent experiments.</p

TBC1D3 expression blocks IRS-1 degradation.

<p>IRS-1 degradation is delayed in cells expressing TBC1D3. DU145 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for the indicated times. Protein levels of IRS-1 were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of IRS-1 normalized to GAPDH protein levels. The value of IRS-1 at time 0 was set at 1.0. The data are presented as means ± SD of three independent experiments.</p

TBC1D3 selectively decreases IRS-1 Serine phosphorylation.

<p>(A) Empty vector or myc-TBC1D3 was co-transfected with pCIS2-IRS-1 in Hek293 cells. Cells were stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of IRS-1 were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of IRS-1 phosphorylation normalized to IRS-1 protein levels (* <i>p</i><0.05, ** <i>p</i><0.01). (B) Hek293 cells were transfected with myc-TBC1D3 or empty vector, serum-starved and stimulated with insulin (10 nM) for 5 or 30 min. Phosphorylation of endogenous IRS-1:S636/639 was analyzed by Western blotting. (<i>Right panel</i>) Quantification data of endogenous IRS-1 phosphorylation normalized to IRS-1 protein levels (* <i>p</i><0.05). The data are presented as means ± SD of three independent experiments.</p