spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 12 May 2009
doi: 10.1242/jcs.039057

Journal of Cell Science 122, 1882-1894 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Neel, N. F.
Right arrow Articles by Richmond, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Neel, N. F.
Right arrow Articles by Richmond, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis

Nicole F. Neel1,2, Melanie Barzik5, Dayanidhi Raman1,2, Tammy Sobolik-Delmaire1,2, Jiqing Sai1,2, Amy J. Ham3,4, Raymond L. Mernaugh3, Frank B. Gertler5 and Ann Richmond1,2,*

1 Department of Veterans Affairs, Nashville, TN 37212, USA
2 Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
3 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
4 Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
5 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA


Figure 1
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 1. CXCR2 and VASP interact in dHL-60-CXCR2 cells. (A) Lysates from cells stimulated with vehicle (Mock IgG, Untreated) or cells stimulated with 100 ng/ml CXCL8 for 1 or 5 minutes were incubated with normal rabbit IgG-(Mock IgG) or rabbit anti-CXCR2 antibody-coupled Sepharose. Immunoprecipitated proteins were eluted and analyzed by SDS-PAGE and western blot (IB) for CXCR2 and VASP. (B) Quantification of unphosphorylated and Ser157-phosphorylated VASP coimmunoprecipitating with CXCR2. Values are normalized to total VASP levels and represented as mean ± s.e.m. Significant differences between the unphosphorylated and phosphorylated VASP in the IP are indicated (*P≤0.05, Mann Whitney U-test). (C) dHL-60-CXCR2 cells not treated (lane 2) or treated (lane 3) with 100 ng/ml CXCL8 for 5 minutes were lysed, subjected to immunoprecipitation as above, and analyzed by SDS-PAGE and western blot (IB) for Ser157-P, Ser239-P or P-Thr278-P VASP using ECL, or for total VASP using the Odyssey system or ECL. One set of immunoprecipitated lysates stimulated with CXCL8 was incubated with lambda phosphatase (lane 4) as described in Materials and Methods. Data shown are representative of three separate experiments.

 

Figure 2
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 2. CXCR2 and VASP localize to plasma membrane ruffles upon global and directional CXCL8 stimulation. (A) Immunofluorescence confocal images of CXCR2 and VASP staining in dHL-60-CXCR2 cells stimulated globally with vehicle (0 minutes) or 100 ng/ml of CXCL8 for 1 or 5 minutes. Colocalization after both 1 and 5 minutes of stimulation is indicated by arrows. Images represent single z-sections of 0.38 µm. Overlay images are pseudocolored where green is CXCR2 and red is VASP. Images after 0 minutes (a), 1 minute (b), and 5 minutes (c) stimulation are enlarged x5 from the boxed areas on the left. (B) Immunofluorescence confocal images of CXCR2, VASP and F-actin staining in dHL-60-CXCR2 cells stimulated directionally with 50 ng/ml CXCL8 in a Zigmond chamber for 15 minutes. The arrow indicates the direction of CXCL8 gradient (0-50 ng/ml CXCL8). Image represents a single z-section of 0.35 µm pseudocolored, where green is VASP, red is CXCR2, and blue is F-actin. (C) Immunofluorescence confocal images of CXCR2 and VASP phosphorylated on Ser157 (pPSer157 VASP) or Ser239 (pSer239) following 1 minute of global CXCL8 (100 ng/ml) stimulation. Overlay images are pseudocolored, where green is CXCR2 and red is pVASP. Image represents a single z-section of 0.28 µm. Insets are enlarged x2 from boxed areas in originals. Images were processed using AdobePhotoshop. Scale bars: 5 µm. Data shown are representative of three separate experiments.

 

Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 3. Purified His6-VASP interacts with amino acids 331-355 from the CXCR2 C-terminus. (A) 96-well plates were coated with 3 µg/ml GST, GST-CXCR2 311-330, or GST-CXCR2 331-355 and incubated with various concentrations of His6-VASP (0.06-60 ng/ml). Bound His6-VASP was detected using nickel-activated HRP (Abs 405 nm). Triplicate experiments show His6-VASP binding (mean ± s.e.m.) to GST-CXCR2 331-355 was statistically higher than to GST alone or GST-CXCR2 311-330 (*P≤0.05, Mann Whitney U-test). Data shown are representative of at least three separate experiments. (B) Representative Coomassie-blue-stained gel of 5 µg purified GST, GST-CXCR2 311-330 and GST-CXCR2 331-355. (C) Representative Coomassie-blue-stained gel of 5 µg purified His6-VASP.

 

Figure 4
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 4. VASP is phosphorylated on Ser157, Ser239 and Thr278 specifically in response to CXCL8 stimulation. HL-60-CXCR2 cells were stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 1 or 5 minutes. Lysates were subjected to SDS-PAGE and western blot analysis (IB) using antibodies specific for Ser157-P (A), Ser239-P (B) or Thr278-P VASP (C). HL-60-CXCR2 cells were stimulated with vehicle (Untreated) or 100 ng/ml CCL3 for 1 minute. Lysates were subjected to SDS-PAGE and western blot analysis (IB) for Ser157-P (D), Ser239-P (E) or Thr278-P VASP (F). Data shown are representative of three separate experiments.

 

Figure 5
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 5. Phosphorylation of VASP upon CXCL8 stimulation is mediated through PKA and PKC. Lysates from HL-60-CXCR2 cells pretreated with various inhibitors, stimulated with vehicle (0 minutes) or 100 ng /ml CXCL8 for 1 or 5 minutes and analyzed by SDS-PAGE and western blot. Statistical difference of mean ± s.e.m. in DMSO versus treatment is indicated (*P≤0.05, Mann Whitney U-test). (A) Cells pretreated with DMSO or 20 µM H89 for 60 minutes and analyzed by western blot using antibodies specific for VASP and VASP Ser157-P. (B) Quantification of normalized density of VASP Ser157-P in DMSO-versus H89-treated samples. (C) Cells pretreated with DMSO or 5 µM rottlerin for 15 minutes and analyzed by western blot analysis for VASP and VASP Ser157-P. (D) Quantification of normalized density of VASP Ser157-P in DMSO-versus rottlerin-treated samples. (E) Cells pretreated with DMSO or 5 µM rottlerin for 15 minutes and analyzed for VASP and VASP Ser239-P (F). Quantification of normalized density of VASP Ser239-P in DMSO-versus rottlerin-treated samples. (G) Cells pretreated with DMSO or 20 µM H89 for 60 minutes and analyzed using antibodies for VASP and VASP Ser239-P. (H) Quantification of normalized density of VASP Ser239-P in DMSO-versus H89-treated samples. Data shown represent average quantification from at least three separate experiments.

 

Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 6. Phosphorylation of VASP regulates its interaction with CXCR2. (A) Western blot analysis for GFP (IB) of GST pull-downs with GST or GST-CXCR2 C-terminus using lysates from MVD7 cells stably expressing GFP-VASP WT, S153A-S235A, S153-T274A and S235A-T274A. (B) Western blot analysis of purified His6-VASP EVH2 domain incubated with catalytic subunit of PKA with and without ATP using a Ser235-P-specific antibody. (C) Binding (mean ± s.e.m.) of His6-VASP EVH2 domain incubated with and without ATP to either GST or GST-CXCR2 331-355. Binding was detected by measuring absorbance at 405 nm from three separate experiments. Statistical significance of binding to GST-CXCR2 with or without ATP is indicated by *P≤0.05 (Mann Whitney U-test). (D) Representative Coomassie-blue-stained gel of 5 µg purified His6-VASP EVH2. (E) Representative Coomassie-blue-stained gel of 5 µg of purified GST or GST-CXCR2 331-355.

 

Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 7. CXCR2 preferentially interacts with VASP as opposed to Mena or EVL. Western blot analysis for GFP (IB) of GST pull-downs with GST or GST-CXCR2 C-terminus using lysates from MVD7 cells stably expressing GFP-VASP, GFP-Mena or GFP-EVL. Data shown are representative of three separate experiments.

 

Figure 8
View larger version (48K):
[in this window]
[in a new window]

  		Fig. 8. CXCR2 interaction with VASP occurs through the VASP-EVH2 domain and requires the coiled-coil region. (A) Schematic of murine VASP domain structure and fragments used in pull-down experiments. Numbers indicate the amino acids phosphorylated in murine VASP. Western blot analysis for GFP (IB) to detect GFP-VASP of GST pull-downs with GST or GST-CXCR2 C-terminus using: (B) lysates from MVD7 cells stably expressing GFP-VASP-EVH1, GFP-VASP-EVH2 or GFP-VASP-{Delta}Pro (deletion of proline-rich region) or (C) lysates from MVD7 cells stably expressing GFP-VASP-EVH2 WT, GFP-VASP-{Delta}COCO (coiled-coil deletion), or GFP-VASP-{Delta}FAB (F-actin binding motif deletion). Data shown are representative of three separate experiments.

 

Figure 9
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 9. F-actin is necessary for localization of VASP to the membrane and interaction with CXCR2. dHL-60 cells were pretreated for 30 minutes with 25 nM cytochalasin D (CytoD) or DMSO (vehicle), then stimulated with vehicle (Mock, Unt) or 100 ng/ml CXCL8 for 1 minute. (A) Cell lysates were incubated with either normal rabbit IgG (Mock IgG) or rabbit anti-CXCR2 antibody-coupled Sepharose. Eluted immunoprecipitated proteins were analyzed by SDS-PAGE and western blot (IB) for CXCR2 and VASP. (B) Western blot analysis (IB) using antibodies specific for VASP P-Ser157 or VASP Ser239-P of lysates. (C) Immunofluorescence confocal images of CXCR2, VASP and F-actin staining in dHL-60-CXCR2 cells and stimulated directionally with 50 ng/ml CXCL8 in Zigmond chamber for 15 minutes. The arrow indicates direction of CXCL8 gradient (0-50 ng/ml CXCL8). Image represents a single z-section of 0.49 µm. Enlarged panel images are magnified x4 from original images. Overlay image is pseudocolored where green is VASP, red is CXCR2, and blue is F-Actin. Images were processed using Adobe Photoshop. Scale bars: 5 µm. (D) Quantification of mean ± s.e.m. percentage of cells per x63 field exhibiting VASP immunofluorescence staining at the plasma membrane versus cytoplasm. Ten microscopic fields were examined for DMSO- and CytoD-treated cells with 2-8 cells per field Statistical significance of DMSO-versus CytoD-treated cells is indicated (*P<0.0005, Student's t-test). Data shown are representative of three separate experiments.

 

Figure 10
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 10. Expression of VASP shRNA specifically impairs CXCR2-mediated chemotaxis. (A) Western blot analysis of VASP in lysates from HL-60-CXCR2 cells stably expressing non-silencing shRNA and VASP shRNA. Average percentage knockdown ± s.e.m. of VASP in western blots from three separate experiments. (B) Boyden chamber assay assessing chemotaxis of HL-60-CXCR2 cells stably expressing non-silencing or VASP shRNA in response to CXCL8 (B) or (C) in response to CCL3. Graphs represent the mean ± s.e.m. chemotactic index from three separate experiments. Statistical difference in dose response curve for CXCL8-mediated chemotaxis in non-silencing versus VASP shRNA expressing cells (*P<0.01, one-way ANOVA). There was no significant difference (ns) in CCL3-mediated chemotaxis between non-silencing versus VASP shRNA expressing cells (one-way ANOVA).

 

Figure 11
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 11. Cells expressing VASP shRNA exhibit impaired CXCL8-induced cell polarization. Confocal images of cells expressing non-silencing or VASP shRNA stimulated directionally with 50 ng/ml of CXCL8 (A) or 50 ng/ml of CCL3 (B) in a Zigmond chamber for 15 minutes and stained using anti-VASP antibody (green) and phalloidin (red). Arrows indicate the direction of the gradients (0-50 ng/ml CXCL8 or CCL3). Enlarged panels are magnified x2.5 original images. Scale bars: 5 µm (C). Quantification of mean ± s.e.m. number of cells per field polarized in the direction of the CXCL8 or CCL3 gradients. Statistical significance of non-silencing versus VASP shRNA is indicated (*P<0.001, Mann Whitney U-test). (D) Quantification of the mean ± s.e.m. number of polarized cells from images of 20 x63 fields, each containing 25-50 cells. Data shown are representative of two separate experiments.

 

Figure 12
View larger version (44K):
[in this window]
[in a new window]

  		Fig. 12. Model illustrating the roles of VASP phosphorylation in mediating the VASP-CXCR2 interaction. (A) CXCR2 and VASP in unstimulated cells. VASP is predominantly located in the cytoplasm but might have some loose basal association with CXCR2. (B) Upon ligand binding, uncoupling of the G proteins occurs, activating PKA- and PKC-signaling pathways. VASP localizes to the plasma membrane. It is likely that there is a change in G{alpha} coupling to CXCR2 to activate PKA, which phosphorylates VASP on Ser157 in the EVH1 domain. (C) Uncoupling of the Gβ{gamma} subunit activates PKC, which phosphorylates Ser239 and possibly Thr278 in the EVH2 domain of VASP. These phosphorylations allow strong interaction between VASP and CXCR2. CXCR2 binding to VASP is likely mutually exclusive with the F-Actin–VASP interaction but it is possible that when phosphorylated VASP is bound to CXCR2, sustained interaction with F-actin occurs.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2009