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First published online 24 June 2008
doi: 10.1242/jcs.026187

Journal of Cell Science 121, 2394-2405 (2008)
Published by The Company of Biologists 2008
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Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells

Andrea Dorfleutner1,2,*, YoungJin Cho1,2, Deanne Vincent1,2, Jess Cunnick1,3, Hong Lin1,2, Scott A. Weed1,4, Christian Stehlik1,2,* and Daniel C. Flynn1,2,{ddagger}

1 The Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA
2 The Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV, USA
3 The Department of Pathology, West Virginia University, Morgantown, WV, USA
4 The Department of Neurobiology and Anatomy, West Virginia University, Morgantown, WV, USA


Figure 1
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  		Fig. 1. (A) AFAP-110 localizes to PE-induced podosomes in A7r5 smooth muscle cells. A7r5 cells plated on fibronectin coated glass coverslips were either left untreated (a,b) or were treated with 1 µM PDBu for 1 hour (c,d). Cells were stained with Rhodamine-labeled phalloidin to label F-actin (a,c) and with a polyclonal AFAP-110 antibody (b,d). Confocal microscopy was used to scan 0.7-µm-thick images along the ventral membrane of the cells to image the podosomes. (B) GFP-AFAP-110 was transiently transfected into A7r5 cells and immunofluorescence images were obtained using antibodies to detect endogenous vinculin (b) and cortactin (c), to contrast with GFP fluorescence (a) in the merged image (d). (C) Monomeric dsRed-AFAP-110 was transiently expressed in A7r5 cells that had been plated on an FITC-gelatin matrix containing 50 µg/ml fibronectin.

 

Figure 2
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  		Fig. 2. PE treatment causes a gel shift of the GFP-AFAP-110 protein band in western blot analysis that is dependent upon Ser277. (A) Western blot analysis of GFP-AFAP-110 transfected COS-7 cell lysates. 48 hours post transfection cells were either left untreated (–) or treated for 1 hour with 100 nM PMA (+), directly lysed in SDS sample buffer and subjected to SDS-PAGE and subsequent western blotting with GFP antibody. PMA treatment causes a gel shift of the GFP-AFAP-110 protein band. The horizontal line is shown to contrast the gel shift. (B) Protein sequence alignment of the Ser/Thr rich region between different species. Conserved Ser residues that represent putative PKC target sites – [R/K]XX[S/T] and [R/K]X[S/T] (Pearson and Kemp, 1991Go) – and are underlined and have subsequently been mutated to Ala. (C,D) Western blot analysis of GFP-AFAP-110 phosphorylation mutants. (C) Ser277, Ser278, Ser282 and Ser283 (as indicated in 2B) were mutated to Ala to create the 4A mutant (lanes 1 and 2). For double mutants Ser277 and Ser278 or Ser282 and Ser283 were mutated to Ala to create S277A-S278A or S282A-S283A mutants (277A278A and 282A283A lanes 5,6 and 7,8, respectively). (D) For single mutants, Ser277 or Ser278 were mutated to Ala to create S277A or S278A (277A or 278A, respectively). All constructs were transfected into COS-7 cells and 48 hours post transfection treated as indicated with PMA for 1 hour, while inhibition of PKC was accomplished using the PKC inhibitor GF109203X (GF) for 30 minutes. All blots were probed with anti-GFP antibody and the gelshifted protein bands are indicated by an asterisk. Molecular mass markers are indicated on the left. The horizontal line is shown to contrast the gel shift.

 

Figure 3
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  		Fig. 3. Characterization of the anti-pSer277 antibody as a potential phospho-specific AFAP-110 antibody. (A) Specificity of the newly generated phospho-specific AFAP-110 antibody was determined by dot-blot assay as described in Materials and Methods. NP, non-phosphorylated peptides; P, phosphorylated peptides. (B) Western blot analysis with the newly generated phospho-specific AFAP-110 antibody anti-pSer277. Samples were prepared as described in Fig. 2A,C, where a gel shift of the AFAP-110 protein band was observed in response to PMA treatment in western blot analysis. Here the western blot was probed with anti-pSer277 phospho-specific AFAP-110 antibody ({alpha}(P) AFAP, upper panel) or with an antibody against total AFAP-110 ({alpha}AFAP, lower panel) to control for equal expression of AFAP-110. (C) Time course of PMA-induced immunoreactivity with anti-pSer277 antibodies. GFP-AFAP-110 transfected COS-7 cells were treated with 100 nM PMA for the indicated time, prior to lysis in sample buffer, separation by SDS-PAGE and subsequent western blot analysis with anti-pSer277 ({alpha}(P) AFAP, upper panel). After stripping the membrane it was re-probed with a GFP antibody (lower panel), to control for equal loading. (D) Anti-pSer277 antibodies are immunoreactive with AFAP-110 in cells treated with PMA/PDBu. Anti-pSer277 antibody ({alpha}(P) AFAP, upper panel) recognizes PMA/PDBu-induced phosphorylation of overexpressed avian and human AFAP-110 as well as of endogenous human and rat AFAP-110 in western blot analysis. Overexpression of AFAP-110 was performed in COS-7 cells that had been lysed directly after PMA treatment. Endogenous AFAP-110 was immunoprecipitated after PMA treatment, as indicated, and SDS-PAGE and western blot analysis was performed. Membranes were probed with anti-pSer277 antibodies ({alpha}(P) AFAP, upper panel) and with a anti-AFAP antibody (lower panel), to control for equal loading. All western blots are labeled with molecular mass markers on the left.

 

Figure 4
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  		Fig. 4. The PH domain is required for phosphorylation of Ser277. (A) Schematic of mutants used for the mapping of domains that are indispensable for AFAP-110 phosphorylation on Ser277. P71, Pro71 of the SH3-interaction domain; 71A, Pro71 to Ala mutation; PH, pleckstrin homology; LZ, leucine zipper; ABD, actin-binding domain. (B) Western blot analysis of AFAP-110 mutants described in Fig. 4A. 48 hours post transfection cells were treated for 1 hour with 100 nM PMA as indicated, directly lysed in sample buffer and SDS-PAGE and western blot analyses were performed. Western blot results using anti-pSer277 antibody ({alpha}(P) AFAP; upper panel), and anti-GFP antibody (lower panel) to control for equal loading. Molecular mass markers on the left of the blots.

 

Figure 5
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  		Fig. 5. PKC{alpha} is involved in phosphorylation of AFAP-110 on Ser277. (A,B) Kinase inhibitors were added to GFP-AFAP-110 transfected COS-7 cells before PMA treatment for 1 hour. Staurosporine (STS) to inhibit Ser/Thr kinases and PP2 for Src kinase (A), and Safingol (Saf) and rottlerin (Rot) to selectively inhibit certain PKC isoforms as well as other Ser/Thr kinases (B). Cell lysates were separated by SDS-PAGE, and western blotted with antibodies against anti-pSer277 ({alpha}(P) AFAP, upper panel), or anti-GFP (lower panel) to control for equal loading. (C) PKC{alpha} or the dominant-active myristoylated (myr-) form of PKC{alpha} were co-expressed with GFP-AFAP-110 in COS-7 cells and cells were stimulated with 100 nM PMA for 1 hour as indicated. Control cells were left untransfected and also subjected to PMA treatment as indicated. Cells were directly lysed in sample buffer and subjected to SDS-PAGE and western blot analysis using anti-pSer277 ({alpha}(P) AFAP; top panel), anti-GFP ({alpha} GFP; middle panel) or anti-PKC ({alpha} PKC; bottom panel). All blots are labeled with molecular mass markers on the left. (D) Similarly, cells expressing GFP-AFAP-110 were pre-treated with GF109203X (GF), followed by stimulation with 100 nM PMA for 1 hour as indicated. Cells were directly lysed in sample buffer and subjected to SDS-PAGE and western blot analysis using anti-pSer277 ({alpha}(P) AFAP, top panel) or anti-GFP ({alpha} GFP; bottom panel).

 

Figure 6
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  		Fig. 6. Phosphorylated AFAP-110 localizes to podosomes. A7r5 cells were transfected with GFP-AFAP-110 constructs and either left untreated (panels a-d) or treated with 1 µM PDBu for 1 hour (panels e-h). Immunofluorescence staining was performed as described in Materials and Methods, with actin pseudocolored in red, GFP-AFAP-110 in green and the phosphorylation-specific AFAP-110 antibody anti-pSer277 (P-AFAP) in blue. Merged pictures show colocalization of phosphorylated AFAP-110 with AFAP-110 and actin in podosomes.

 

Figure 7
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  		Fig. 7. GFP-AFAP-110S277A expression is associated with an increased number of cells that contain podosomes. (A) Immunofluorescence staining of A7r5 cells, transfected with wild-type GFP-AFAP-110 and treated with PDBu for 1 hour (A, A', A") to show cellular morphologies demonstrating (A) stress fibers, (A') a mix of stress filaments and podosomes or (A") podosomes only. (B) Quantification of GFP-AFAP-110-expressing or GFP-AFAP-110S277A-expressing A7r5 cells (uninduced and PDBu-treated) that display stress fibers (black), podosomes (white) or a mixed population of stress fibers and podosomes (checkerboard) correlating to A, A" or A', respectively. (C) GFP-AFAP-110S277A expression (pseudocolored green) in A7r5 cells contrasted with TRITC-phalloidin (pseudocolored red) and cortactin detected with monoclonal antibody 4F11 (pseudocolored white). Cells were either untreated or treated with PDBu for 60 minutes. Cells that expressed GFP-AFAP-110S277A expressed either predominantly podosomes or a mix of stress filaments and podosomes.

 

Figure 8
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  		Fig. 8. GFP-AFAP-110S277A expression increases the average lifespan of a podosome. (A) Percentages of podosomes were plotted against their lifespan in incremental 10-minute time brackets. The mean percentages of podosomes observed in cells from three different experiments is given ± s.d. (B) The average lifespan of podosomes was 17.9±17 minutes in cells that express GFP-AFAP 110 wild type (gray bar) and 28.6±21 minutes in cells that overexpress GFP-AFAP-110S277A (black bar); the difference is statistically significant (P<0.0001).

 

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© The Company of Biologists Ltd 2008