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Fig. 8. PAK-Pbd interferes with the recruitment of the PIX/GIT1 complex at large cytoplasmic structures. (A) Scheme of PAK1 and of the PAK-Pbd construct. 1-4 indicate the four proline-rich regions in the amino-terminal portion of PAK1; region 4 (asterisk) is the one involved in binding to the SH3 domain of PIX. (B) Immunoprecipitation from lysates of COS7 cells transfected with ßPIX (IP1) or ßPIX and PAK-Pbd (IP2). Unbound fractions (Ub) and lysates (Lys) from the two samples were also loaded. Portions of the same filters were immunoblotted for PIX (PIX_tr), endogenous PAK (PAKe), or PAK-Pbd, as indicated. (C) Immunoprecipitation with anti-Myc antibodies from lysates of COS7 cells transfected with FLAG-GIT1, HA-ßPIX and Myc-PAK (Lys1) or Myc-PAK-Pbd (Lys2). 50 µg of each lysate were also loaded. Portions of the same filters were immunoblotted for GIT1, PIX or PAK, as indicated. (D-G) Effects of PAK expression on the distribution of PIX and GIT1. CEFs were triple transfected with PAK, ßPIX and GIT1 (D,E), or with PAK-Pbd, PIX and GIT1 (F,G). Bar, 20 µm. (H) Quantification of the effects of PAK-Pbd expression on the localization of GIT1 and PIX at large cytoplasmic structures in CEFs cotransfected with GIT1 and ßPIX (n=385), triple transfected with PAK, PIX and GIT1 (n=200), or with PAK-Pbd, PIX and GIT1 (n=200). Error bars represent the s.d. from at least two independent experiments. Statistical significance was assessed by the Student's t-test (P<0.05 considered significant). The values from cells triple transfected with GIT1, ßPIX and PAK-Pbd were compared to those from cells triple transfected with GIT1, ßPIX and wild-type PAK. *P<0.005. (I) A431 cells were cotransfected with PIX and PAK. Endogenous GIT proteins (GITe, green) colocalized with transfected PIX (red) at cytoplasmic structures. Bar, 25 µm.

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