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First published online 12 April 2005
doi: 10.1242/jcs.02313

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PAK4 mediates morphological changes through the regulation of GEF-H1

Marinella G. Callow^1,*, Sergey Zozulya^1,, Mikhail L. Gishizky^1,, Bahija Jallal^1,¶ and Tod Smeal^1,**,

¹ SUGEN Incorporated, 230 East Grand Avenue, South San Francisco, CA 94080, USA

View larger version (48K): [in a new window]   Fig. 1. Lysates were prepared from 293T cells overexpressing (A) Myc-tagged PAK4 alleles or sub domains and incubated with GST, GST-GEF-H1 (787-921) and GST-Maguin-like bound to glutathione beads (1-4 bead designation) or (B) GFP-GEF-H1 and incubated with PAK4 biotinylated peptides (291-355) and (276-324) linked to streptavidin beads. An equivalent amount of beads and excess lysates (500 µg) were used. Eluates of beads and total lysates were analyzed by SDS-PAGE and immunoblotted with Myc or GEF-H1 antiserum. Panel C represents a PAK4 gene schematic outlining the regions of PAK4 used to identify the GEF-H1 interaction domain (GID). (D) Alignment of the GID defined by the minimal binding region of PAK4 with PAK5 and PAK6.

View larger version (75K): [in a new window]   Fig. 2. (A) Human tumor cell lysate from H1299 cells were immunoprecipitated with protein G beads linked with anti-PAK4, anti-GEF-H1 and anti-PAK4 preimmune sera. Bead eluates were analysed by western blotting using anti-GEF-H1 antiserum (upper panel). The same beads were subject to a kinase reaction and separated by SDS-PAGE and autoradiographed to detect incorporation of [-32P]ATP (lower panel). (B) H1299 cells were stained for PAK4 (FITC) and the Golgi marker ß-COP (Texas Red) and F-actin (Coumarin-Phalloidin). The merge shows co-localization of PAK4 with Golgi-like structures. (C) PAK4 (FITC) localizes to the Golgi (arrow a), and MT-like structures (arrow b) (I and V). GEF-H1 (Texas Red) localizes to the cytoplasm (arrow c) and co-stains with PAK4 on Golgi and MT structures (I and II). Staining for phosphorylated GEF-H1 (Marina Blue) shows localization to MTs surrounding the nucleus, cytoplasm and Golgi-like structures (compare III and IV). ß-tubulin (FITC) staining was used to show MTs of the cytoskeleton throughout the cell (IV). In the presence of DSP crosslinking PAK4 is more concentrated in the Golgi and some MT-like structures (compare V and VIII). GEF-H1 staining retains its localization to all compartments consistent with conventional crosslinking (compare VI with II). Phosphorylated GEF-H1 is clearly cytoplasmic bearing no spatial relationship to MTs, although MTs are still present (compare VII with VIII). Briefly, for DSP treatment, H1299 cells on coverslips were removed from growth medium and incubated in D-PBS (Dulbecco's PBS) containing 1 mM DSP at 37°C. After 20 minutes the coverslips were incubated in MTSB (containing 50 mM glycine) to quench the DSP and stabilize the MTs. Washes and further fixation is described in Materials and Methods.

View larger version (36K): [in a new window]   Fig. 3. (A) An in vitro kinase assay using purified GST, GST-GEF-H1 (763-921) and GST-Maguin-like protein as substrates for PAK4. The negative control autophosphorylation reaction is the reaction mix without substrate. A concentration gradient of reaction substrate is indicated by the triangles. (B) In vivo phosphorylation of GEF-H1 by PAK4 is demonstrated in western blots of co-transfected lysates probed with phosphospecific antisera (lower panel). Anti-HA serum detects the level of GEF-H1 in these lysates (upper panel). Increased phosphorylation is seen in the presence of PAK4 and PAK4 S474E (lanes 2-3 compared with lane 1). Basal level activity is further reduced by kinase-inactive PAK4 K350,351A. (C) PAK4 alleles, including wild-type, kinase domain and H19,22L (CRIB mutant), co-expressed with GEF-H1 induce phosphorylation of GEF-H1 (lanes 3-5) whereas Cdc42 and vector retain basal activity (lanes 1-2), middle panel. Upper and lower panels are probed with tag-specific serum to detect the levels of GEF-H1 and PAK4 alleles present in these lysates.

View larger version (81K): [in a new window]   Fig. 4. (A-F) NIH-3T3 cells were transfected with vector EGFP, and EGFP-tagged GEF-H1 alleles: GEF-H1S, GEF-H1S-S810A, GEF-H1-Q312M,R313G, GEF-H1S-S67A and GEF-H1S-S67,810A. GFP fluorescence was used to detect expression of GEF-H1 (left panels) and fluorescent-phallicidin was used to monitor cytoskeletal rearrangements (right panels). Localization of GEF-H1S proteins in NIH-3T3 fibroblasts was detected by expressing as EGFP fusion proteins and analyzing by fluorescence microscopy. (G-L) Overlapping F-actin localization. (M) Regulation of Rho activity by GEF-H1 proteins was measured by precipitation of Rho-GTP with GST-RBD from lysates co-expressing GEF-H1 and Rho. The amount of RhoA bound to RBD and the level of RhoA expression in whole cells lysates were analyzed by western blotting and detected with anti-Myc antibody. GEF-H1 was detected with anti-HA antibody. (N) Histogram displaying fold activation of Rho. Measurements were by densitometry using the western blots of two individual experiments.

View larger version (99K): [in a new window]   Fig. 5. NIH-3T3 cells were transfected with PAK4 and GEF-H1S alleles and examined by fluorescence imaging of EGFP for GEF-H1 alleles, PAK4 constructs were identified by immunostaining using PAK4 antiserum followed by anti-rabbit fluorescent rhodamine conjugate for PAK4 and fluorescent coumarin-phallicidin to label actin. (A-C) GEF-H1S co-transfected with PAK4 alleles WT,S474E and K350,351A. (D-F) GEF-H1S-S810A, GEF-H1S-S67A and GEF-H1S-Q312M,R313G together with PAK4-S474E. Arrow a highlights filopodia; arrow b highlights lamellipodia induction for cells co-expressing PAK4-S474E and GEF-H1S or GEF-H1S-S67A; arrow c marks cells with stress fibers induction. Bar, 10 µm.

View larger version (87K): [in a new window]   Fig. 6. NIH-3T3 cells were co-transfected with GEF-H1 and PAK4. Expression of GEF-H1 and PAK4 was detected by fluorescence imaging of EGFP for GEF-H1 and PAK4 antiserum (#933) followed with anti-rabbit fluorescent rhodamine for PAK4. Fluorescent coumarin-phallicidin was used to examine the actin cytoskeleton. Anti-ß-tubulin followed by anti-mouse Marina Blue conjugate was used to stain for the MT cytoskeleton. (A) GEF-H1M together with PAK4 and the mutant PAK4-S474E. Arrow a highlights an actin halo seen with overexpressed GEF-H1M; arrow b indicates stress fibers organized with actin cables; arrow c highlights actin-rich lamellipodia in cells co-expressing GEF-H1M and the mutant PAK4-S474E. (B) GEF-H1M together with vector and the mutants PAK4-S474E and PAK4 K350,351A. Note the non-polarized partial dissolution of the MT cytoskeleton in the presence of the mutant PAK4-S474E. Arrow d indicates lack of MTOC. Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 7. (A) GEF-H1 in a non-phosphorylated state retains basal constitutive Rho activity. (B) PAK4 can regulate GEF-H1 by phosphorylation leading to simultaneous Rac activation and inhibition of constitutive Rho signaling.

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