Figure 4

MMP-2 directly interacts with PAK4. (a) Western blotting was performed using whole-cell lysates of pSV- and PAK4si-treated cells. (b) Modulation of EGF receptor phosphorylation levels after PAK4si treatment in 4910 and 5310 cells. Whole-cell lysates of pSV- and PAK4si-treated cells were subjected to EGFR phosphorylation array and representative array images from three independent experiments were presented. (c) Densitometric analysis of EGFR array showing the mean±S.E. values obtained from three repetitions and significance is denoted by ** at P<0.01. (d) The formation of a PAK4/MMP-2 complex in 4910 cells was analyzed by co-immunoprecipitation (IP) with whole-cell lysates (500 μg) using specific antibodies against MMP-2 and non-specific IgG. Immunoprecipitates were subjected to western blotting and probed with anti-PAK4 antibody (upper panel). Immunoprecipitation with PAK4 and subsequent immunoblotting with MMP-2 also confirmed the PAK4/MMP-2 complex formation (lower panel). Input samples indicate 10% of pre-immunoprecipitated samples (50 μg). (e) The formation of a PAK4/MMP-2 complex in MMP-2-FL-overexpressing cells was analyzed by IP with anti-Myc antibody, followed by immunoblotting using anti-PAK4 and anti-MMP-2 antibodies. (f) Identification of MMP-2 interaction domain within PAK4. Bacterially expressed GST and GST-MMP-2 fusion protein were purified using MagneGST Pull-Down System following the manufacturer’s instructions, separated on SDS-PAGE and verified by Ponceau staining (left, represented by “*”). Biotin-labeled full-length and truncated mutants of PAK4 were synthesized using a TNT Quick Coupled Transcription/Translation Systems and incubated with GST-MMP-2 (middle-top). The mixtures were washed and elutes were separated by SDS-PAGE, transferred to nitrocellulose membrane and interacting partners were then detected as mentioned in Materials and Methods. Input samples (10% input) were analyzed by SDS-PAGE (middle bottom). Right panel: schematic representations of PAK4 (full length, 1–591 aa) and its truncation mutants (1–288 aa, 290–591 aa and 330–591 aa) used in GST pull-downs to localize the MMP-2 interaction domain within PAK4. Various domains of PAK4 protein are represented by KD (kinase domain), CRIB (Cdc42Hs/Rac-interactive binding domain), GID (GEF–H1 interaction domain) and IBD (integrin-binding domain). (g) PAK4 is involved in complex formation with MMP-2 and αvβ3-integrin in glioma xenograft cells. Co-immunoprecipitation experiments were performed with anti-MMP-2 and anti-PAK4 antibodies. Non-specific IgG or no antibody (beads only) precipitates were loaded as negative controls, and blots were probed with specific antibodies against MMP-2, αv, β3 and PAK4. Representative blots from three independent experiments were presented. (h) Representative confocal microscopic images from three independent experiments indicate the expression and colocalization of PAK4 and MMP-2 in pSV- and PAK4si-treated 4910 and 5310 cells. (i). Western blotting was performed with whole-cell lysates and representative blots from three experimental replicates were shown