Figure 7.
PKL, but not GIT1, requires paxillin, Src and FAK for efficient localization to focal adhesion in MEFs. (A) MEF null cells were plated on fibronectin followed by transfection with GFP-PKL and constitutively active G12VRac1. (A) The capacity of GFP-PKL to localize to focal adhesions was examined by fluorescence microscopy. The actin cytoskeleton was visualized by labeling with rhodamine phalloidin. A significant attenuation in the localization of PKL to focal adhesions in Src, FAK, PTP-PEST, and paxillin but not Nck null cells was observed. (B) The null MEF cells were plated on fibronectin followed by transfection with GFP-GIT1 and G12VRac1 revealing the efficient localization of GIT1 to focal adhesions in all null cells examined. (C) Quantitation of cells, processed as described above, showing focal adhesion localization of GFP-PKL or GFP-GIT1 (expressed as percentage of transfected cells). Unlike PKL, GIT1 retains the capacity to localize efficiently to G12VRac1 focal adhesions irrespective of the loss of Paxillin, Src, or FAK. Bars, 20 μm. (D) Reintroduction of WT Src into the SYF null cells rescues GFP-PKL targeting to focal adhesions.