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ß1PIX, the PAK-interacting exchange factor, requires localization via a coiled-coil region to promote microvillus-like structures and membrane ruffles

¹ Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609
² Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 117609
³ Institute of Neurology, 1 Wakefield Street, London, WC1N 1PJ, UK

View larger version (56K): [in a new window]   Fig. 1. Properties of PIX. (A) Different isoforms of PIX. Abbreviations: CH, calponin homology domain; SH3, Src homology 3; DH, Dbl homology domain; PH, pleckstrin homology domain; inverted triangle, deletion in PIX; I, insert sequence of 30 amino acids in ß1PIX and ß2PIX not present in PIX; II, insert sequence of 60 amino acids present in ß2PIX, ß1PIX-b and ß1PIX-c. The numbers represent the percentage similarity between the domains of PIX and ß1PIX. ß1PIX-b, ß1PIX-c and ß2PIX are all splice variants of ß1PIX. (B) Alignment of the C termini of PIX, ß1PIX and ß2PIX. The C termini of PIX and ß1PIX contain a coiled-coil structure, whereas that of ß2PIX is serine rich. The consensus sequences are boxed. The inverted arrowheads mark the start of the coiled-coil regions of PIX and ß1PIX, and also the serine-rich region of ß2PIX. The major PAK phosphorylation sites are marked with asterisks. The GIT1 binding region is from residues 460 to 555 of ß1PIX. (C) Western analysis of cell lysates of different tissues from rat and also several cell lines. The membranes were probed with anti-PIX-SH3 antibodies (top and middle), which recognized all isoforms of PIX, and with anti-ß2PIX-C-terminus antibodies (bottom), which only recognized the ß2 isoforms. Abbreviations for the rat tissue protein lysates: B, brain; K, kidney; Li, liver; Lu, lung; S, spleen; Te, testis; Th, thymus. Abbreviations for the cell line protein lysates (middle): C, COS-7; H, HeLa; N, NIH 3T3; K, kidney protein lysates; F, pXJ-Flag-ß1PIX transfected COS-7 cell lysate.

View larger version (39K): [in a new window]   Fig. 2. Cellular distribution of PIX and PAK. (A) Distribution of PIX and PAK by cell fractionation. COS-7 cells transfected with pXJ-Flag-ß1PIX and pXJ-Flag-ß2PIX were fractionated, analysed by SDS-PAGE and probed with anti-Flag antibodies. Untransfected COS-7 cell fractions were also analysed by SDS-PAGE and probed with anti-PIX-SH3 or anti-/ß-PAK (PAK1 and Pak3) antibodies. The fractions collected are: T, total protein; S, water-soluble proteins (S100 fraction); Tx, Triton-X-100-soluble proteins (P100); P, SDS-soluble proteins of the pellet from Triton-X-100 extraction, which contain cytoskeletal proteins. (B) Cellular localization of PIX isoforms and mutants. Various PIX constructs were transiently transfected into HeLa cells. The DNA constructs used were pXJ-Flag-PIX, pXJ-Flag-ß1PIX, pXJ-Flag-ß2PIX, pXJ-Flag-ß1PIX1-555, pXJ-Flag-N80ß1PIX and pXJ-GST-ß1PIX-C-ter. The proteins expressed were immunostained using anti-Flag or anti-GST antibodies. Constructs that contained the coiled-coil C terminus were localized at the cell periphery, whereas ß2PIX and the C-terminally truncated mutant ß1PIX1-555 showed cytoplasmic and nuclear staining.

View larger version (31K): [in a new window]   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.

View larger version (37K): [in a new window]   Fig. 4. Phosphorylation by PAK did not affect the exchange activities of PIX. (A) PAK phosphorylates PIX between residues 496 and 555 of ß1PIX. Various Flag-tagged PIX isoforms and mutants were transfected into COS-7 cells and immunoprecipitated. The precipitated proteins were subjected to in vitro phosphorylation by GST-PAK with [-33P]ATP in the reaction buffer. The phosphorylated product was analysed on SDS-PAGE gel and exposed to X-ray film. Top, the Coomassie-Blue-stained gel. Bottom, the autoradiograph. The asterisks mark the bands of interest. The Flag-tagged proteins are: lane 1, ß2PIX; lane 2, ß1PIX; lane 3, ß1PIX1-459; lane 4, ß1PIX1-555; lane 5, N80ß1PIX; lane 6, ß1PIXpro (deletion of amino acids 460-495 of ß1PIX); lane 7, GST-ß1PIX-C-ter; lane 8, GST-PAK alone. (B) Various Flag-tagged isoforms of PIX were transiently transfected into COS-7 cells. The immunoprecipitated PIX was assayed for exchange activity using GST-Rac1 as substrate with and without prior in vitro phosphorylation by GST-PAK (the kinase prepared as a GST fusion protein from E. coli is constitutively active). Each exchange assay was repeated with four determinants for each time point and the average is shown. (C) GST-PAK was transfected with wild-type Cdc42 and various PIX DNA constructs into COS-7 cells. GST-PAK fusion protein from the different transfection cell lysates was isolated by glutathione-Sepharose beads. Kinase activity was then measured by in vitro kinase assay using [-33P]ATP and myelin basic protein as substrates. The figure shows results from three different sets of transfection experiments and kinase assays. Top, western blot probed with anti-GST antibody to show the amount of GST-PAK from the different transfection cell lysates isolated by glutathione beads. Middle, autoradiograph showing the corresponding MBP phosphorylation by the GST-PAK pulled down in top panel. Bottom, normalized kinase activity of the GST-PAK from the corresponding transfection cell lysates. There was mobility shift of the GST-PAK band when PAK was transfected with wild-type Cdc42 and active Cdc42.

View larger version (108K): [in a new window]   Fig. 5. PIX and ß1PIX induced cell ruffling via Rac1. (A) PIX and ß1PIX promote the formation of phase-dark ruffle-like structures. HeLa cells were microinjected with DNA constructs of the various PIX and PIX mutants in pXJ-40 vector. The cells were then viewed by phase-contrast microscopy. The injected cells are marked by arrowheads. The DNA construct used were pXJ-Flag-PIX, pXJ-Flagß1PIX, pXJ-Flag-ß2PIX, pXJ-Flag-N80ß1PIX, pXJ-Flag-ß1PIX1-555, pXJ-GST-ß1PIX-C-ter, pXJ-Flag-ß1PIX-DHm and pXJ-GST vector. (B) The formation of these ruffles could be blocked by Rac1N17 but not Cdc42N17. HeLa cells were microinjected with pXJ-40 DNA construct of ß1PIX together with Rac1N17 or Cdc42N17. The injected cells are marked by arrow heads. (C) ß1PIX and ß2PIX could induce filopodia and cell rounding when wild-type Cdc42 was also overexpressed. HeLa cells were microinjected with the plasmid DNA constructs in pXJ-40 vector and the cells were observed 2-2.5 hours after microinjection. The plasmid DNA injected are: 1, hPem2; 2, Cdc42V12; 3, ß1PIX and Cdc42 wild type (w/t); 4, ß2PIX and Cdc42 w/t; 5, Cdc42 w/t alone.

View larger version (77K): [in a new window]   Fig. 6. ß1PIX induced the formation of microvillus-like structures by activating Cdc42. (A) The formation of microvillus-like structures was blocked by Cdc42N17 but not Rac1N17. Scanning electron micrographs (SEMs) of HeLa cells microinjected with various plasmid DNA. The DNAs were pXJ-GST vector, pXJ-Flag-ß1PIX, pXJ-Flag-ß2PIX, pXJ-HA-CDC42V12, pXJ-Flag-ß1PIX plus pXJ-Flag-RacN17 and pXJ-Flag-ß1PIX plus pXJ-Flag-Cdc42N17. The apical surface structures induced by ß1PIX are unlike the filopodia induced by Cdc42V12. (B) PIX mutants did not induce microvillus-like structures. SEMs of HeLa cells microinjected with plasmid constructs of the PIX mutant constructs pXJ-Flag-N80ß1PIX, pXJ-Flag-ß1PIX-DHm and pXJ-Flag-ß1PIX1-555. (C) The morphological effect of localized activation of Rac1 and Cdc42. Apical protrusions induced by ß1PIX are a combination of microvillus-like structures and lamellipodia (marked by arrowheads), seen here as higher-magnification SEM. Scale bar, 10 µm.

View larger version (26K): [in a new window]   Fig. 7. A proposed scheme for the morphological changes elicited by the PAK-PIX-GIT1 complex. PIX is localized at the cell periphery, possibly by the localization signal found in the PIX coiled-coil C terminus or via formation of PIX dimers that interact with other proteins or phospholipids found at the plasma membrane-cytoskeleton interface. PAK is recruited to the focal complexes (FCs) through its binding to membrane-bound PIX, which also binds to GIT1, which in turn binds paxillin (Turner et al., 1999; Zhao et al., 2000a). Active PIX could then induce membrane ruffles by activating Rac1 as well as the formation of filopodium- and microvillus-like structures by activating Cdc42. Upon autophosphorylation, PAK dissociates from the PIX-GIT1 complex, whereas PIX-GIT1 remains at the cell periphery.