Journal:

Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells

doi: 10.1128/MCB.25.17.7743-7757.2005

Figure Lengend Snippet: Caveolin-1 complexes with Tollip and IRAK-1 in monocytes. A. After being pulsed with TT, freshly isolated monocytes were lysed, and IP assays were conducted with anti-Tollip, anti-IRAK-1 antibodies, or control Ig. IP complexes were then separated using 5 to 20% SDS-PAGE followed by Western blotting and were immunoblotted with the indicated antibodies. Shown are the representative results obtained in five independent experiments. IgH and IgL denote immunoglobulin heavy chain and immunoglobulin light chain, respectively. B. TT-loaded monocytes were attached to poly-l-lysine-coated coverslips, fixed with paraformaldehyde, and permeabilized using 0.05% Triton X-PBS. Cells were then stained with anti-caveolin PAb (a and d), anti-Tollip MAb (b and g), or anti-IRAK-1 PAb (e and h), followed by staining with fluorescein isothiocyanate- or Texas red-conjugated secondary antibodies. Cells were visualized by confocal laser microscopy. Observations were made on 50 cells in each of five independent experiments. The micrographs are representative of more than 75% of the cells observed. Bars indicate a 10-μm scale. C. TT-loaded monocytes were stimulated with rsCD26-wt-coated beads for the indicated time periods. Membrane proteins were extracted and immunoprecipitated with anti-caveolin-1 antibody, and immune complexes were resolved by 5 to 20% SDS-PAGE and immunoblotted with anti-phospho-caveolin-1 (a), anti-IRAK-1 (b), or anti-Tollip (c) antibodies, followed by stripping and reprobing with anti-caveolin-1 antibody (d). Total cell lysates from monocytes stimulated as described above were also resolved by 5 to 20% SDS-PAGE and immunoblotted with anti-IRAK (e), anti-Tollip (f), or anti-IκBα (g) antibodies. Position of IRAK-1 bands in e was indicated by an open triangle, and supershifted bands of IRAK-1 were indicated by a solid triangle. The reciprocal intensities of phospho-caveolin-1, caveolin-1, Tollip, and IRAK-1 in membrane proteins that were immunoprecipitated by anti-caveolin-1 were demonstrated (h). The reciprocal intensity of IκBα in total cell lysates was also demonstrated (h). Similar results were obtained in five independent experiments. WB, Western blot.

Article Snippet: Anti-caveolin-1 rabbit polyclonal antibody (PAb), anti-IRAK rabbit PAb, anti-IκBα MAb, anti-glutathione- S -transferase (GST) MAb, antihemagglutinin (anti-HA) MAb, and anti-HA rabbit PAb agarose-conjugated antibodies were purchased from Santa Cruz Biotechnology Inc.; anti-phospho-caveolin-1 MAb was from BD Transduction; anti-Tollip rat MAb was from ALEXIS Biochemicals; anti-vesicular stomatitis virus (VSV) rabbit PAb was from Medical & Biological Laboratory Co. Ltd.; and anti-FLAG (M2) MAb, 3× FLAG peptide, and poly- l -lysine were from Sigma-Aldrich.

Techniques: Isolation, SDS Page, Western Blot, Staining, Microscopy, Immunoprecipitation, Stripping Membranes

Journal:

Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells

doi: 10.1128/MCB.25.17.7743-7757.2005

Figure Lengend Snippet: Determination of the binding domains involved in the interaction among caveolin-1, Tollip, and IRAK-1. A. Schematic representation of HA-tagged or GST-fused caveolin-1, FLAG-tagged or GST-fused Tollip, VSV-tagged IRAK-1, and their mutants. In caveolin-1, residues 1 to 81 comprised the N-terminal region (NT) (open square), residues 82 to 101 comprised the scaffolding domain (SCD) (black square), residues 102 to 134 comprised the transmembrane region (memb; striped square), and residues 135 to 178 comprised the C-terminal region (dotted square). In Tollip, residues 47 to 178 comprised the protein kinase C conserved region 2 (C2 domain) (black square), and residues 179 to 274 comprised coupling of ubiquitin conjugation to endoplasmic reticulum degradation domain (CUE) (dotted square). In IRAK-1, residues 523 to 712 comprised the C-terminal domain (CT) (dotted square). H, G, and F denote HA, GST, and FLAG, respectively. B. GST-fused caveolin-1 and deletion mutants on glutathione- Sepharose (GSH) beads were incubated with THP-1 cell lysate after preclearing with GST on GSH beads. Bound proteins and a 1% amount of input lysate were resolved by 5 to 20% SDS-PAGE and immunoblotted with anti-Tollip MAb, followed by stripping and reprobing with anti-GST MAb. Similar results were obtained in three independent experiments. C. GST-fused Tollip and deletion mutants on GSH beads were incubated with THP-1 cell lysate after preclearing with GST on GSH beads. Bound proteins and a 1% amount of input lysate were resolved by 5 to 20% SDS-PAGE and immunoblotted with anti-caveolin-1 PAb, followed by stripping and reprobing with anti-GST MAb. Similar results were obtained in three independent experiments. D. COS cells were transfected with FLAG-tagged Tollip mutants, HA-tagged caveolin-1-wt, and VSV-tagged IRAK-1-wt, lysed, and immunoprecipitated with anti-FLAG (M2) MAb. Elutions of the FLAG-fusion protein complex were conducted by adding 150 ng/ml of 3× FLAG peptide. The eluted samples were separated using 5 to 20% SDS-PAGE and immunoblotted with anti-HA (caveolin-1-wt) or anti-VSV (IRAK-1-wt) PAbs, followed by stripping and reprobing with anti-FLAG (M2) MAb. Similar results were obtained in three independent experiments. E. COS cells were transfected with HA-tagged caveolin-1, FLAG-tagged Tollip, VSV-tagged IRAK-1, and mutants, lysed, and immunoprecipitated with agarose-conjugated anti-HA MAb. IPs were separated using 5 to 20% SDS-PAGE and immunoblotted with anti-FLAG (M2) (Tollip) MAb or anti-VSV (IRAK-1) PAb, followed by stripping and reprobing with anti-HA (caveolin-1) MAb (top three panels). Whole lysates of COS cells transfected as described above were separated using 5 to 20% SDS-PAGE to resolve expression of transfected FLAG-tagged Tollip and VSV-tagged IRAK-1 and mutants (bottom two panels). Similar results were obtained in three independent experiments. WB, Western blot.

Article Snippet: Anti-caveolin-1 rabbit polyclonal antibody (PAb), anti-IRAK rabbit PAb, anti-IκBα MAb, anti-glutathione- S -transferase (GST) MAb, antihemagglutinin (anti-HA) MAb, and anti-HA rabbit PAb agarose-conjugated antibodies were purchased from Santa Cruz Biotechnology Inc.; anti-phospho-caveolin-1 MAb was from BD Transduction; anti-Tollip rat MAb was from ALEXIS Biochemicals; anti-vesicular stomatitis virus (VSV) rabbit PAb was from Medical & Biological Laboratory Co. Ltd.; and anti-FLAG (M2) MAb, 3× FLAG peptide, and poly- l -lysine were from Sigma-Aldrich.

Techniques: Binding Assay, Scaffolding, Conjugation Assay, Incubation, SDS Page, Stripping Membranes, Transfection, Immunoprecipitation, Expressing, Western Blot

Journal:

Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells

doi: 10.1128/MCB.25.17.7743-7757.2005

Figure Lengend Snippet: Subcellular colocalization of caveolin-1, Tollip, and IRAK-1 in living cells. HEK293FT cells were transfected with GFP-fused (a, b, d, e, g, and h) or HA-tagged (c, f, and i) caveolin-1-wt, FLAG-tagged Tollip (wt or ΔC2), and VSV-tagged IRAK-1 (wt or ΔCT). Cells were then fixed and permeabilized with acetone-methanol and stained with anti-FLAG (M2) MAb or anti-VSV PAb, followed by staining with anti-mouse Ig (fluorescein isothiocyanate- or Texas red-conjugated) or anti-rabbit Ig (Texas red-conjugated) antibodies. Stained cells were mounted using a Prolong Antifade kit. Observations were made with 10 to 15 cells in each of five different experiments. The micrographs are representative of more than 75% of the cells observed. Bars indicate a 10-μm scale.

Article Snippet: Anti-caveolin-1 rabbit polyclonal antibody (PAb), anti-IRAK rabbit PAb, anti-IκBα MAb, anti-glutathione- S -transferase (GST) MAb, antihemagglutinin (anti-HA) MAb, and anti-HA rabbit PAb agarose-conjugated antibodies were purchased from Santa Cruz Biotechnology Inc.; anti-phospho-caveolin-1 MAb was from BD Transduction; anti-Tollip rat MAb was from ALEXIS Biochemicals; anti-vesicular stomatitis virus (VSV) rabbit PAb was from Medical & Biological Laboratory Co. Ltd.; and anti-FLAG (M2) MAb, 3× FLAG peptide, and poly- l -lysine were from Sigma-Aldrich.

Techniques: Transfection, Staining

Journal:

Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells

doi: 10.1128/MCB.25.17.7743-7757.2005

Figure Lengend Snippet: Enhanced CD86 promoter activity by increasing doses of Tollip or IRAK-1 in response to exogenous CD26 stimulation. A. Twelve hours after HEK293 cells were cotransfected with human CD86-promoter luciferase constructs, caveolin-1 (wt or deleting tyrosine at residue 14 mutant [Y14−]) and Tollip (wt, ΔC2, or ΔCUE) vectors, wild-type-soluble CD26 (rsCD26-wt), or rsCD26 lacking the caveolin-binding domain (rsCD26-Δ201) was added to the culture medium and incubated for an additional 20 h. Cells were harvested for measurement of luciferase activity and protein concentration. Lucif-erase activity is shown as being relative to 1 μg of applied protein. Data represent means ± standard errors (SE) from triplicate experiments. B. HEK293 cells were cotransfected with human CD86-promoter luciferase constructs, caveolin-1 (wt or Y14−), and IRAK-1 (wt or ΔCT) vectors, treated, and prepared for luciferase assay as described above. RLU, relative light units.

Article Snippet: Anti-caveolin-1 rabbit polyclonal antibody (PAb), anti-IRAK rabbit PAb, anti-IκBα MAb, anti-glutathione- S -transferase (GST) MAb, antihemagglutinin (anti-HA) MAb, and anti-HA rabbit PAb agarose-conjugated antibodies were purchased from Santa Cruz Biotechnology Inc.; anti-phospho-caveolin-1 MAb was from BD Transduction; anti-Tollip rat MAb was from ALEXIS Biochemicals; anti-vesicular stomatitis virus (VSV) rabbit PAb was from Medical & Biological Laboratory Co. Ltd.; and anti-FLAG (M2) MAb, 3× FLAG peptide, and poly- l -lysine were from Sigma-Aldrich.

Techniques: Activity Assay, Luciferase, Construct, Mutagenesis, Binding Assay, Incubation, Protein Concentration

Journal:

Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells

doi: 10.1128/MCB.25.17.7743-7757.2005

Figure Lengend Snippet: Model for CD26-caveolin-1 interaction leading to upregulation of CD86. A. Caveolin-1 in monocytes (APC) resides at the inner membrane in the presence or absence of Tollip and IRAK-1 (1). Afteruptake of tetanus toxoid into monocytes via caveolae, some population of caveolin-1 is exposed on the outer cell surface of TT-loaded monocytes (2). Migration of CD26+ antigen-specific memory T cells to areas of antigen-loaded APCs results in contact with TT APC, leading to the association of CD26 and caveolin-1 (3). Aggregation of caveolin-1 in the contact area occurs, presumably by homo-oligomerization (via its residues 61 to 101), followed by its phosphorylation. Phosphorylated caveolin-1 (phospho-caveolin-1) dissociates complexed Tollip and IRAK-1, presumably due to conformational changes, and IRAK-1 is then phosphorylated in the cytosol (4). After IRAK is phosphorylated, NF-κB is activated to lead to upregulation of CD86 (5). B. Antigens such as tetanus toxoid are loaded into monocytes and are then processed and presented with MHC class II (MHC II) on the cell surface along with exposure of caveolin-1 N terminus (1). Memory T cells expressing CD26 have contact with these antigen-presenting cells, and maturation of the immunological synapse occurs via T-cell receptor (TCR)-MHC class II, CD28-CD86/CD80, and CD26-caveolin-1 interactions (2). T cells and APC are then activated and cytokines are secreted (3). CD86 upregulation therefore leads to greater T-cell-APC interaction and the development of activated T cells locally and activated immune response, resulting in potential autoimmune diseases (4).

Article Snippet: Anti-caveolin-1 rabbit polyclonal antibody (PAb), anti-IRAK rabbit PAb, anti-IκBα MAb, anti-glutathione- S -transferase (GST) MAb, antihemagglutinin (anti-HA) MAb, and anti-HA rabbit PAb agarose-conjugated antibodies were purchased from Santa Cruz Biotechnology Inc.; anti-phospho-caveolin-1 MAb was from BD Transduction; anti-Tollip rat MAb was from ALEXIS Biochemicals; anti-vesicular stomatitis virus (VSV) rabbit PAb was from Medical & Biological Laboratory Co. Ltd.; and anti-FLAG (M2) MAb, 3× FLAG peptide, and poly- l -lysine were from Sigma-Aldrich.

Techniques: Migration, Expressing

Journal:

Article Title: Phospho-Caveolin-1 Mediates Integrin-Regulated Membrane Domain Internalisation

doi: 10.1038/ncb1293

Figure Lengend Snippet: Caveolin-1 internalisation. (a) Adherent 3T3 cells or cells suspended for the indicated times were fixed, permeabilized and stained for caveolin-1. Cell surface (arrows) vs. intracellular (arrowheads) staining is indicated. (b) Internalisation of caveolin in images from (a) was quantified as described in Methods. Caveolin fluorescence within 3.5 microns of the cell surface is considered exterior; fluorescence internal to this zone, further from the plasma membrane, is considered interior. Values are means ± S.E.M. from 10 cells in 3 independent experiments. Differences between 30s and 1h are statistically significant (p<2.5×10-4 for the interior region and p<7×10-3 for the exterior). (c) Attached cells or cells suspended for the indicated times were fixed and processed for electron microscopy to detect caveolae in the plasma membrane. Caveolae are indicated by arrows. Images are representative of areas within cells where caveolae are concentrated. (d) Cells suspended for 2 min. were fixed and processed for either (L) immunogold labelling of caveolin-1 or (R) electron microscopy. (L) A region of caveolae internalisation showing a vacuole surrounded by multiple α-Cav-1 IgG gold particles. To the right of this vacuole (bracket) is a cluster of caveolae emanating from the vacuole. (R) A similar region of a cell processed for regular TEM microscopy. This image shows several caveolae-rich vacuoles (asterisks) and associated clusters of caveolae and caveolae-derived vesicles (arrows). Bar=0.2 μm. (n=3).

Article Snippet: Double caveolin-1/pY14cav-1 staining was performed using a rabbit pAb anti-caveolin-1 (BD Transduction Labs) followed by an Alexa 488-conjugated goat anti-rabbit pAb (Molecular Probes) and a mouse anti-pY14cav-1 (BD Transduction Labs) followed by a Rhodamine Red X-conjugated goat anti-mouse pAb (Molecular Probes).

Techniques: Staining, Fluorescence, Electron Microscopy, Microscopy, Derivative Assay

Journal:

Article Title: Phospho-Caveolin-1 Mediates Integrin-Regulated Membrane Domain Internalisation

doi: 10.1038/ncb1293

Figure Lengend Snippet: Pathway of integrin-regulated GM1 internalisation. (a) Serum deprived NIH-3T3 fibroblasts were surface labelled on ice with CTxB-Alexa 568, detached and held in suspension (Sus) for the indicated times, or plated on FN for 2hours (Adherent). Cells were then fixed and stained for caveolin-1 in green. Cells shown are observed at the equatorial plane and representative of up to 50 cells at each time point. (b) To quantify co-localization between GM-1 and caveolin, pixels that were positive for GM1 stain were identified and the fraction of the caveolin-1 stain within these zones calculated. Values are means ± S.E.M. from 3 independent experiments. (c) wt MEFs expressing HA-tagged dominant negative (K44A) or wt dynamin-2 were held in suspension for 90 min, surface labelled with Alexa 488-CTxB, and then stained with anti-HA antibody. (d) wt MEFs expressing GFP alone, GFP-tagged Eps15DIII, or the Cdc42-binding domain (CBD) of WASP were surface labelled with Alexa 568-CTB, then detached and held in suspension for the indicated times. (e) Control and GFP-tagged Eps15DIII expressing wt MEFs adherent on FN were stained for the transferrin receptor. Cells shown are representative of three independent experiments.

Article Snippet: Double caveolin-1/pY14cav-1 staining was performed using a rabbit pAb anti-caveolin-1 (BD Transduction Labs) followed by an Alexa 488-conjugated goat anti-rabbit pAb (Molecular Probes) and a mouse anti-pY14cav-1 (BD Transduction Labs) followed by a Rhodamine Red X-conjugated goat anti-mouse pAb (Molecular Probes).

Techniques: Staining, Expressing, Dominant Negative Mutation, Binding Assay

Journal:

Article Title: Phospho-Caveolin-1 Mediates Integrin-Regulated Membrane Domain Internalisation

doi: 10.1038/ncb1293

Figure Lengend Snippet: Requirement for Caveolin-1. (a) Lysates from M21L melanoma or M21L-cav cells were analysed by Western blotting with anti-caveolin polyclonal Ab. (b) Cells detached for 2h (top panels) were stained live with FITC-CTxB to label surface GM1. Middle panels: cells expressing GFP-V12Rac. Bottom panels: Cells stained with rhodamine-phalloidin. Arrows indicate protrusions and ruffle-like structures (c) GFPV12Rac localization to the membrane in (b) was assessed by measuring pixel intensity starting at the cell edge. Values are means ± SEM. (d) Confocal z-section series of actin-stained cells. (e) Serum-starved caveolin-1-/- MEFs (KO) or caveolin-1+/+ MEFs (WT), adherent or suspended for 2h, were stimulated with 10% serum for 10 min, and PAK kinase activity assayed. (f) Caveolin-1-/- MEFs (KO) or caveolin-1+/+ MEFs (WT) were kept adherent or suspended for 8h in the presence of 10% serum. Cell lysates were analysed by Western blotting with phospho-specific antibodies to Erk1/2, Akt, and FAK, or with phospho-independent antibodies to the same proteins for normalization. Values in (e) and (f) are means ± S.D. of the specific enzymatic activity (kinase activity or amount of phospho-protein normalized to total protein) in non-adherent cells normalized to that of adherent cells (n=4).

Article Snippet: Double caveolin-1/pY14cav-1 staining was performed using a rabbit pAb anti-caveolin-1 (BD Transduction Labs) followed by an Alexa 488-conjugated goat anti-rabbit pAb (Molecular Probes) and a mouse anti-pY14cav-1 (BD Transduction Labs) followed by a Rhodamine Red X-conjugated goat anti-mouse pAb (Molecular Probes).

Techniques: Western Blot, Staining, Expressing, Activity Assay

Journal:

Article Title: Phospho-Caveolin-1 Mediates Integrin-Regulated Membrane Domain Internalisation

doi: 10.1038/ncb1293

Figure Lengend Snippet: Phospho-caveolin-1 mediates integrin-dependent membrane domain internalisation. (a) Adherent NIH-3T3 cells were doubly stained for phospho-caveolin-1 plus either vinculin or total caveolin-1. wt MEFs (b) or caveolin-1-/- MEFs (c) in suspension were stained for total caveolin-1 plus pY14cav-1. (d) NIH-3T3 cells were kept adherent or placed in suspension for the indicated times. Cell lysates were analysed by Western blotting with anti-caveolin-1 or anti-phosphocaveolin-1 (n=10). (e) Western blotting for caveolin-1 and flag in caveolin-1 +/+ MEFs (WT), caveolin-1 -/- MEFs (KO), and KO MEFs transiently transfected with flag-tagged WT or Y14F caveolin-1. (f). Caveolin-null MEFs transiently transfected with WT or Y14F caveolin-1 were detached for 2h and stained live with FITC-CTxB to label surface GM1, then fixed and stained with anti-flag mAb to detect caveolin expression. Arrows denote transfected cells. Images are single confocal sections (n=4). (g) Quantitation of surface GM1. The level of surface GM1 was quantified by measuring total staining intensity of suspended, surface CTxB-labelled untransfected caveolin-null cells and cells expressing WT or Y14F caveolin from (f). Values are means ± S.E.M. from 10 cells in 4 independent experiments. (h) NIH-3T3 cells, untreated COS-7 cells, and COS-7 cells incubated with 1μM sodium pervanadate for 1 h were lysed, and analysed by western blotting with antibodies against caveolin-1, pY14cav-1, and β1 integrin. (i) Adherent and suspended cells treated as in (h) were surface labelled with CTxB-Alexa 568. Asterisks denote cells not readily visible due to low surface labelling. Results are representative of three independent experiments.

Article Snippet: Double caveolin-1/pY14cav-1 staining was performed using a rabbit pAb anti-caveolin-1 (BD Transduction Labs) followed by an Alexa 488-conjugated goat anti-rabbit pAb (Molecular Probes) and a mouse anti-pY14cav-1 (BD Transduction Labs) followed by a Rhodamine Red X-conjugated goat anti-mouse pAb (Molecular Probes).

Techniques: Staining, Western Blot, Transfection, Expressing, Quantitation Assay, Incubation