Phosphorylation of Rab5a protein by protein kinase Cϵ is crucial for T-cell migration

Abstract

Rab GTPases control membrane traffic and receptor-mediated endocytosis. Within this context, Rab5a plays an important role in the spatial regulation of intracellular transport and signal transduction processes. Here, we report a previously uncharacterized role for Rab5a in the regulation of T-cell motility. We show that Rab5a physically associates with protein kinase Cϵ (PKCϵ) in migrating T-cells. After stimulation of T-cells through the integrin LFA-1 or the chemokine receptor CXCR4, Rab5a is phosphorylated on an N-terminal Thr-7 site by PKCϵ. Both Rab5a and PKCϵ dynamically interact at the centrosomal region of migrating cells, and PKCϵ-mediated phosphorylation on Thr-7 regulates Rab5a trafficking to the cell leading edge. Furthermore, we demonstrate that Rab5a Thr-7 phosphorylation is functionally necessary for Rac1 activation, actin rearrangement, and T-cell motility. We present a novel mechanism by which a PKCϵ-Rab5a-Rac1 axis regulates cytoskeleton remodeling and T-cell migration, both of which are central for the adaptive immune response.

Keywords: Cell Migration; Cell Signaling; Immunology; Integrin; Phosphorylation; Rab Proteins; Signal Transduction; T-cell.

FIGURE 1.

Interaction of PKCϵ and Rab5a in migrating T-cells. A, shown is a representative confocal image of HuT-78 T-cells expressing PKCϵ-GFP (green) stimulated to migrate on an anti-LFA-1 coated surface. The right panel shows an intensity plot generated by using LSM software. B, HuT-78 cells were co-transfected with PKCϵ-cherry (red, left panel) and Rab5a-GFP (green, middle panel) expression vectors, stimulated to migrate on an anti-LFA-1-coated surface, and imaged by confocal microscopy. The merged image (right panel) is an overlay of a series of 0.5-μm spacing Z stack images showing the yz (right) and xz (top) planes around the cell centrosomal region. Magnified images are shown in the corresponding insets, and the arrow indicates co-localized PKCϵ-Rab5a. C, co-localization analysis of the above image was performed along the direction of migration, as indicated, using LSM software. D, shown is a representative confocal fluorescent image of resting HuT-78 cells co-transfected with PKCϵ-cherry (red) and Rab5a-GFP (green) expression vectors. E, HuT-78 cells co-expressing PKCϵ-cherry (red) and Rab5a-paGFP (green) were stimulated to migrate on an anti-LFA-1 coated surface. Shown is a representative cell irradiated within the indicated region at the centrosomal region (white circle) and imaged with time-lapse confocal microscopy at 2.44, 20.1, and 28.9 s. F, primary human PBL T-cells were incubated on ICAM-1-coated plates for 30 min and after fixation immunostained for PKCϵ (red), Rab5a (green), and nuclei (blue). Magnified images are shown in the insets. G, PBL T-cells were left unstimulated or stimulated by rICAM-1 for 30 min and lysed. Cell lysates were immunoprecipitated (IP) with mouse monoclonal anti-Rab5a antibody or control IgG and then subjected to Western immunoblot (WB) analysis using rabbit anti-PKCϵ or anti-Rab5a antibody. H, HuT-78 cells were transfected with plasmid constructs encoding wild-type Rab5a-GFP (Rab5a-WT-GFP), constitutively active GTP-bound Rab5aQ79L mutant (Rab5a-Q79L-GFP), or dominant-negative GDP-bound Rab5aS34N mutant (Rab5a-S34N-GFP) and after 24 h stimulated with rICAM-1 for 30 min or left unstimulated. Cells were lysed, and cellular lysates were immunoprecipitated (IP) with mouse monoclonal anti-GFP and then subjected to Western immunoblot (WB) analysis using rabbit anti-PKCϵ or anti-GFP antibody. Relative densitometry values (mean ± S.E.) of protein bands in PKCϵ blots (G and H) are presented. Data represent three independent experiments and for microscopic imaging at least 20 fields per slide were visualized. Scale bar, 5 μm. *, p < 0.01.

FIGURE 2.

PKCϵ-dependent phosphorylations of Rab5a and its involvement in T-cell motility. A, an in vitro kinase assay was performed with recombinant PKCϵ incubated with purified GST alone (control), MBP, or Rab5a-GST. A representative blot from three independent experiments is shown. B, three potential PKC phosphorylation motifs (underlined) were identified in Rab5a amino acid sequence. The predicted serine/threonine residues (indicated in bold) are Thr-7, Ser-84, and Ser-123. C, HuT-78 T-cells were transfected with the control plasmid, wild-type (PKCϵ-WT), or the kinase-dead mutant of PKCϵ (PKCϵ-KD) expression vectors, and the migratory potential of cells was analyzed using trans-well migration assays. D, HuT-78 cells were transfected with wild-type Rab5a (Rab5a-WT) or mutant Rab5a-T7A, Rab5a-S84A, or Rab5a-S123A plasmid constructs and analyzed in a trans-well migration assay. E, HuT-78 cells were transfected with wild-type Rab5a (Rab5a-WT), mutant Rab5a-T7A, or Rab5a-T7E plasmid constructs and analyzed using trans-well migration assay. Data of at least three independent experiments were normalized to respective control and are presented as the mean ± S.E. *, p < 0.01.

FIGURE 3.

Characterization of anti-phospho Rab5a(Thr-7) antibody. A, serial dilutions (1/100 to 1/12,800) of the affinity-purified anti-phospho-Rab5a(Thr-7) antibody was titrated against phospho Thr-7-containing peptide ((p)T7 peptide) and analyzed by ELISA. Preimmune serum and non-phospho (OH)Thr-7-containing peptides ((OH)T7 peptide) were used as controls. B, various dilutions of the purified pRab5a(Thr-7) antibody ranging from 1:1 to 1:1000 were analyzed by immuno dot-blot assay (IB) using Thr(P)-7 peptide. (OH)Thr-7 peptide or pRab5a(Thr-7) antibody preincubated with the Thr(P)-7 peptide was used as the control. C, purified GST, GST-conjugated wild-type Rab5a (Rab5a-WT), or mutant Rab5a-T7A protein was separately incubated in the presence or absence of PKCϵ. The products were analyzed by Western blot using the affinity-purified anti-pRab5a(Thr-7) or anti-Rab5a (loading control). Densitometry analysis of the Western blots was performed and presented (mean ± S.E.). *, p < 0.01. Data represents three independent experiments.

FIGURE 4.

Rab5a undergoes PKCϵ-dependent phosphorylation on Thr-7 in migrating T-cells. A, human primary PBL T-cells were allowed to migrate via LFA-1 stimulation by incubating on ICAM-1-coated plates for 10, 30, 60, or 120 min and lysed. Cell lysates were analyzed by Western immunoblotting using the crude anti-phospho Rab5a(Thr-7) antiserum (pRab5a(T7)). B, PBL T-cells were left unstimulated or stimulated with ICAM-1 for 30 min, SDF-1α for 2 min, or phorbol 12-myristate 13-acetate (PMA) for 30 min and lysed. Cell lysates were analyzed by Western immunoblotting using the crude pRab5a(Thr-7) antiserum. C, PBL T-cells were untreated or pretreated with PKC inhibitor bisindolylmaleimide I for 30 min, stimulated with ICAM-1 for additional 30 min, and lysed. Cell lysates were analyzed by Western immunoblotting using the crude anti-pRab5a(Thr-7) antiserum. D, PBL T-cells were nucleofected with nonspecific siRNA (N/S siRNA) or siRNA against PKCϵ (PKCϵ siRNA) and then lysed after 72 h. Cell lysates were Western-immunoblotted with anti-PKCϵ antibody. E, PBL T-cells nucleofected with nonspecific siRNA or PKCϵ siRNA were unstimulated or stimulated with ICAM-1 for 30 min and Western-immunoblotted using the crude anti-pRab5a(Thr-7) antiserum. All blots were stripped and re-probed with anti-Rab5a or α-tubulin as indicated. Densitometry analysis of the blots were performed and presented. Results are representative of at least three independent experiments. *, p < 0.01.

FIGURE 5.

Subcellular localization of Rab5a and its targeting to T-cell endosomal compartments. A, shown are representative confocal images of HuT-78 T-cells expressing GFP conjugated wild-type Rab5a (Rab5a-WT) or mutant Rab5a-T7A, Rab5a-S84A, or Rab5a-S123A after stimulation by incubating on an anti-LFA-1 coated surface for 1 h. B, HuT-78 cells were transfected with plasmid constructs expressing GFP conjugated wild-type Rab5a (Rab5a-WT), mutant Rab5a-T7A, or Rab5a-T7E and stimulated via LFA-1 as above. Cells were treated with AlexaFluor® 568-labeled transferrin (red) to allow internalization for 10 min and imaged. At least 20 microscopic fields per slide prepared from three independent experiments were visualized, and a representative data is shown. Scale bar, 5 μm.

FIGURE 6.

Involvement of Rab5a T7A phosphorylation in the diffusion of endosomes from the centrosomal region in migrating T-cells. A, HuT-78 T-cells expressing wild-type Rab5a-paGFP (Rab5a-WT) or mutant Rab5a-T7A-paGFP (Rab5a-T7A) were stimulated to migrate on anti-LFA-1 coated coverglass and imaged by confocal microscopy. Photoactivation was achieved with a pulse of 405-nm laser defined within the indicated white circle at the centrosomal region of the cell. B, the integrated fluorescence intensity of the photoactivated region (white circle shown in A) was quantified for each frame of at least 30 movies, calculated relative to the intensity of the frame immediately after photoactivation and corrected for photo-bleaching and plotted against time (s). C, average dispersal rate of Rab5a calculated for the time intervals 0–50, 51–100, and 101–250 s are shown. Values are the mean integrated fluorescence intensity obtained from three independent experiments ± S.E.; n = 30 for Rab5a-WT and n = 40 for Rab5a-T7A. **, p < 0.001; *, p < 0.01.

FIGURE 7.

Photoactivated Rab5a-paGFP- but not Rab5a-T7A-paGFP-associated vesicles redistribute from the centrosomal region to the leading edge of T-cells during migration. A, HuT-78 T-cells expressing wild-type Rab5a-paGFP (Rab5a-WT) or mutant Rab5a-T7A-paGFP (Rab5a-T7A) were stimulated to migrate on anti-LFA-1-coated coverslips and imaged by confocal microscopy. Photoactivation was performed within the indicated white circle around the centrosomal region of the cell. The leading edges of the migrating cell at 200 and 300 s post-photoactivation are shown enlarged in the insets. B, percentage of cells (±S.E.) with Rab5a-WT- or Rab5a-T7A-associated vesicles detected at the leading edge of migrating cells was plotted. n = 19 for Rab5a-WT and 25 for Rab5a-T7A. *, p < 0.01. Results are representative of three independent experiments.

FIGURE 8.

Involvement of Rab5a Thr-7 phosphorylation in the actin cytoskeleton rearrangement at the lamellipodia and Rac1 activation in crawling T-cells. A, HuT-78 T-cells expressing wild-type Rab5a (Rab5a-WT), Rab5a-T7A, or Rab5a-T7E were incubated on anti-LFA-1 coated coverslips for 2 h, stained with phalloidin-TRITC (red) and Hoechst (blue), and imaged. Scale bars, 5 μm. B, percentage of cells showing disrupted lamellipodia (as shown in A) were quantified and presented. Phenotypes were scored blindly by three individuals, and data represent the mean ± S.E., n > 100 cells for each samples. C, cells were seeded on poly-l-lysine (PLL)- or anti-LFA-1 (LFA-1)-coated 96-well plates for 2 h and stained for F-actin and nucleus. Images were automatically acquired and analyzed using an InCell Analyzer^TM by selecting only GFP fusion-expressing cells. Results are representative of at least three independent experiments and are presented as the mean ± S.E. D, serum-starved cells expressing Rab5a-WT, Rab5a-T7A, or Rab5T7E were stimulated with or without ICAM-1 for 30 min. Rac1 activation was measured by PAK-1 PBD pulldown assays. The graph shows densitometric analysis of Rac-GTP levels with respect to input levels of total Rac1 for 3 independent experiments. *, p < 0.01.

FIGURE 9.

A model for signaling via PKCϵ-Rab5a-Rac1 axis in T-cell migration. T-cell stimulation through LFA-1 or CXCR4 triggers PKCϵ-dependent Rab5a Thr-7 phosphorylation in the centrosomal region, which activates Rac1 GTPase. Activated Rac1 regulates actin remodeling and protrusion necessary for T-cell motility.