FIGURE 4.

Pak1 recruitment to the Golgi membrane is enhanced by Golgi-localized PIP₂ and Cdc42. A, HEK293 cells were cotransfected with plasmids encoding Tgn38-FRB-CFP (Golgi targeting protein), PIP5K-FKBP-mRFP (PIP5 kinase), and a GFP-tagged PIP₂-specific binding protein (GFP-PH(PLCδ)). Transfected cells were stimulated by iRap for 20 min to induce FRB-FKBP dimerization (bottom panels). The white bars indicate 10 μm. B, Pearson correlation coefficients for the cellular colocalization of FRB and PIP5K were calculated for 5 randomly selected cells before (−) or after (+) 20 min of iRap treatment; colocalization of FRB and GFP-PH before (−) and after (+) iRap treatment was analyzed similarly. The iRap-stimulated colocalization of these proteins is highly significant for both FRB/PIP5K (***, p = 0.0007) and FRB/GFP-PH (***, p = 0.0001). The distribution of Pearson coefficients in each case are shown as a box and whisker plot, where whiskers represent the maximum and minimum values, the box represents the 25th to 75th percentiles, and the central line depicts the median. C, HEK293 cells were transfected with Tgn38-FRB-CFP, PIP5K-FKBP-mRFP, and HA-tagged Pak1, 8T-Pak1 (cannot bind PIP₂), or LL-Pak1 (cannot bind Cdc42). Cells were treated for 20 min with iRap and then immunostained for Pak1 using a total Pak1 antibody (Invitrogen). The white bars indicate 10 μm. D, for each condition, 18 cells were randomly chosen and Pearson correlation coefficients were determined for colocalization of Pak1 and FRB. The distribution of Pearson correlation coefficients is shown as box and whisker plots as in B. *, p = 0.0148 and ***, p = 0.0009.