Tyrosine Y189 in the substrate domain of the adhesion docking protein NEDD9 is conserved with p130Cas Y253 and regulates NEDD9-mediated migration and focal adhesion dynamics

Abstract

The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion.

Figure 1. Cas family protein alignment and tyrosine phosphorylation.

A. Cas family protein sequences aligned at the PRALINE website. Shown is the region of alignment encompassing human p130CasY249 (LAPGPQDIyDVPPVRGL) indicated by an asterisk and underlined. Numbers above the alignments refer to amino acid number in the NEDD9 human protein sequence, from human NEDD9 residue G144 to A234. The two highlighted tyrosine residues (Y189, Y214) indicate the two NEDD9 residues of interest. Shading indicates the degree of sequence conservation, with red (10) indicating the greatest conservation as shown in the colour gradient at the bottom of the figure. The full sequence alignment is shown in Figure S1. B. Summary of high throughput tandem mass spectrometry studies from phosphosite plus that report detection of phosphorylated NEDD9 Y189, p130Cas Y249 and NEDD9 Y214. Data accessed and analysed as per the materials and methods. Numbers (n) = the number of non-redundant positive records for each site. Pie charts show the representative distributions of the positive records between the different tumour cell types.

Figure 2. NEDD9 mutations do not affect global tyrosine phosphorylation or targeting to focal adhesions.

A. Schematic representation of NEDD9 protein sequence showing the N-terminal SH3 domain, substrate domain containing 13 consensus tyrosine phosphorylation motifs (Y), the serine rich domain (SRR) and the Focal Adhesion Targeting domain (FAT). The two mutated tyrosine residues are indicated in red in the Y189F and Y214F schematic representations. B. Cells transfected with the indicated expression constructs were held in suspension (–) or suspended and then plated onto fibronectin (+). NEDD9 immunoprecipitates were probed with antibodies to phosphotyrosine (P-Tyr) and with anti-NEDD9 antibodies (GFP fusions). C. Cells transfected with GFP vector (A–C), GFP.NEDD9 (D–F), GFP.NEDD9 Y189F (G–I) and GFP.NEDD9 Y214F (J–L). Left hand panels show GFP fluorescence images, middle panels show paxillin immunostaining and merged images are shown on the right. Arrows point to examples of positive focal adhesions. Each image shows a cropped region of one cell.

Figure 3. Mutation of NEDD9Y189 stimulates faster cell migration.

A. 10 representative migration traces of NEDD9−/− fibroblast cells transfected with the indicated GFP expression plasmids. B. MSD calculated from trajectories of cells expressing exogenous NEDD9 (black squares), NEDD9 Y189F (white triangles) and Y214F (white circles). C. Average speed of NEDD9−/− fibroblasts transfected with the indicated GFP expression plasmids. Graphs show the average (n>50 cells per expression construct) and SEM. **p<0.01, NS = not significant.

Figure 4. Mutation of NEDD9Y189 stimulates faster adhesion dynamics.

A. Time-lapse microscopy of the assembly and disassembly of GFP-positive focal adhesions. Shown are examples of focal adhesions in NEDD9−/− MEFs transfected with GFP.NEDD9, GFP.NEDD9Y189F and GFP.NEDD9 Y214F. Each box shows a 4.7 µm×4.7 µm cropped region. Arrows point to each adhesion at the time of peak fluorescence intensity. B. Focal adhesion assembly rate constants (k) for the indicated fusion proteins. C. As for B, except data showing the disassembly rate constants (k). N>25 individual adhesions were analysed per condition. *p<0.05, N.S. = not significant.

Figure 5. NEDD9Y189D phospho-mimetic inhibits migration and increase focal adhesion lengths.

A. Schematic representation of NEDD9 protein sequence showing the Y189D mutation. B. NEDD9−/− MEFs transfected with GFP.NEDD9 or GFP.NEDD9Y189D, as indicated. Arrows point to examples of positive focal adhesions. Scale bar = 20 µm. C. Average speed of NEDD9−/− fibroblasts transfected with the indicated GFP expression plasmids. Graphs show the average (n>100 cells per expression construct) and SEM. ***p<0.001. D. Fraction of focal adhesions versus adhesion length. Data for GFP.NEDD9 show in black circles (n = 112) and for GFP.Y189D in white circles (n = 96). E. Comparison of the fraction of focal adhesions less than 4 µm long. ***p<0.001.