Fig. 3. PIX forms homodimers. (A) The C terminus of ß1PIX is required for PIX dimerization. GST-ß1PIX-C-ter was transfected with various Flag-PIX constructs into COS-7 cells except for the last lane where full length ß1PIX constructs were used. The GST-tagged protein complex was isolated using glutathione-Sepharose beads (GST pull down), separated by SDS-PAGE and subjected to western blot analysis with anti-Flag antibodies. Only constructs that contained the C terminus could form complexes with GST-ß1PIX-C-ter. (B) The coiled-coil but not the serine-rich C-terminus of PIX is responsible for dimerization. Differently tagged ß1PIX and ß2PIX were transfected into COS-7 cells with: 1, pXJ-GST-ß1PIX and pXJ-Flag-ß1PIX; 2, pXJ-GST-ß1PIX and pXJ-Flag-ß2PIX; 3, pXJ-GST-ß2PIX and pXJ-Flag-ß2PIX. The expressed protein was isolated using glutathione or Flag beads and subjected to western blot analysis with anti-Flag or anti-GST antibodies, respectively. The asterisk marks the GST-ß1PIX fusion protein that was found in the Flag-immunoprecipitated complex. (C) PAK and PIX can form a multimeric complex. COS-7 cells were transiently transfected with (1) GST-PAK and Flag-
N80ß1PIX, (2) GST-PAK, HA-PIX and Flag-N80ß1PIX, (3) GST-PAK, HA-PAK and Flag-ß1PIX1-459, (4) GST-PAK, HA-PAK and Flag-N80ß1PIX, (5) GST-PAK, HA-PAK and Flag-ß1PIX. GST fusion proteins were isolated from the cell lysates using glutathione beads and subjected to western blot analysis. Flag-N80ß1PIX could be found in the isolated GST-PAK complex only when dimerized with full-length HA-ß1PIX (lane 2). HA-PAK was detected in the isolated complex of (5), implying that PAK-PIX-PIX-PAK tetramer could be formed (lane 5). The ß1PIX dimer is required to bring GST-PAK and HA-PAK together into the same complex; a mutant of ß1PIX (ß1PIX1-459) unable to dimerize could not do so (lane 3). The asterisks mark the HA-tagged proteins, the hashes mark the Flag-tagged proteins.
