Fig. 1. Kal-GEF1, but not GEF2, induces lamellipodia. (A) Schematic diagram of Kal12, the longest Kalirin isoform. Kal12 contains putative Sec14p, spectrin, Dbl homology (DH), pleckstrin homology (PH), Src homology 3 (SH3), immunoglobulin (Ig), fibronectin III (FN), and kinase domains as labeled. All Kalirin expression vectors encode an N-terminal His-myc tag (triangle). (B) pEAK Rapid cells transfected with vectors encoding pEAK10.βGal (vector), caRac(Q61L), or myc-Kal-GEF1 were stained simultaneously with a monoclonal antibody specific for Rac or the myc epitope (green), TRITC-phalloidin to visualize filamentous actin (red), and Hoechst to visualize nuclei (blue). Representative images are shown. Arrows indicate processes in control cells with filopodial projections and open arrows indicate filamentous actin spokes in lamellipodial sheets. A pinwheel lamellipodium is shown in the inset (white box). Scale bar = 20 μm. Bar graph shows quantification of morphological changes. Asterisks indicate significant differences in the fraction of cells with flattened lamellipodia compared to control (P < 0.001). The fraction of pinwheel lamellipodia induced by Kal-GEF1 is significantly different compared to control or caRac (^#P < 0.005). (C) Control cells or cells transfected with myc-Kal-GEF2 were visualized as in panel B. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Kal-GEF1 activates Rac and Pak. (A) Kal-GEF1 (2.0 μg) and Kal-GEF2 (3.2 μg) purified from transiently transfected pEAK Rapid cells using His-Bind Resin were fractionated by SDS-PAGE and stained with Coomassie Blue. (B) Fluorescence-based GEF assays were performed by following loss of fluorescence of GTPase bound GDP-MANT. Reactions contained 50 μM GST-Rac1/50 μM GDP-MANT and buffer (X), 0.670 μM Kal-GEF2 (), or 0.670 μM Kal-GEF1(▪). (C) Constitutively active Rac1 (caRac1), Kal-GEF1, and Kal-GEF2 were expressed in pEAK-Rapid cells. Endogenous Pak was immunoprecipitated and Pak kinase activity was assayed by measuring phosphorylation of Histone H4; asterisks indicate significant differences from control (P < 0.001). The p21-Rac/Cdc42 binding domain (PBD) and kinase domains of Pak1 are drawn to scale. (D) pEAK Rapid cells transfected with Kal-GEF1 were immunostained with myc antibody and phospho-Thr⁴²³ Pak antibody (P-Pak). Arrow indicates a non-transfected cell and open arrow indicates co-localization of Kal-GEF1 and P-Pak in the spokes of the lamellipodia. Scale bar = 20 μm.

Fig. 3. Lamellipodia induced by Kal-GEF1 are not blocked by dominant negative Rac1 or RhoG. pEAK Rapid cells were transfected with dn-Rac or dn-RhoG-GFP, or co-transfected with Kal-GEF1 as indicated. Cells were immunostained for transfected dn-Rac or visualized directly (dn-RhoG-GFP) (green) and immunostained for filamentous actin (red) and nuclei (blue). Cells co-transfected with dn-Rac (stained green) and Kal-GEF1 are assumed to express Kal-GEF1. Scale bar = 20 μm. Bar graph shows cells scored as in Fig. 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. GEF activity of Kal-GEF1 is not required for induction of lamellipodia. The catalytic activity of Kal-GEF1(ND/AA) was assessed using in vitro GEF assays (A) and effector binding assays (B). (A) For in vitro GEF assays, 30 μM Rac1/GDP-MANT was incubated with buffer (♦), Kal-GEF1 [170 nM (▪) or 670 nM (□)], or GEF1(ND/AA) [170 nM (•) or 670 nM (○)]. (B) Cell-based GEF assay measuring levels of activated endogenous Rac following transfection of Kal-GEF1 or Kal-GEF1(ND/AA) (0.1 μg DNA). Western blots show equal levels of expression of Kal-GEF1 and GEF1(ND/AA) (myc antibody). GFP was used as the control. (C, D) Cells were transfected with the constructs indicated (0.5 μg dnRac or dnRhoG; 0.1 μg Kal-GEF) and cell morphology was scored as in Fig. 1; *P < 0.001 for induction of pinwheel cells by GEF1(ND/AA) when compared to control cells; ^#P < 0.05 for pinwheel cells in doubly vs. GEF1(ND/AA) transfected cells. (D) Representative images of control cells and cells transfected with GEF1(ND/AA) stained as in Fig. 1 are shown. Scale bar = 20 μm.

Fig. 5. Pak kinase activity is necessary for lamellipodia induced by GEF1(ND/AA). (A) Cells transfected with the indicated vectors were scored for altered cell morphology as in Fig. 1; *P < 0.001 for pinwheel cells in Kal-GEF1(ND/AA) cells with and without Pak-kd. (B) pEAK Rapid cells transfected with a vector encoding myc-Kal-GEF1(ND/AA) were immunostained with myc antibody (green) and P-Pak antibody (P-Pak; red). Arrows indicate actin spokes in the lamellipodia and unfilled arrows indicated non-transfected cells. Scale bar = 20 μm. (C) Kal-GEF1 and ΔKal7 co-immunoprecipitate with Pak. Extracts from cells co-transfected with HA-Pak and myc-Kalirin were immunoprecipitated with an HA antibody and analyzed by Western blotting with a Pak antibody to confirm immunoprecipitation of Pak and with a myc antibody to determine whether Kalirin was co-immunoprecipitated. (D) Kal-GEF1 and GEF1(ND/AA) co-immunoprecipitate with Pak1. Experiment was the same as in panel C except that a Pak antibody was used for the immunoprecipitation. (E) GST-Pak1-PBD assay. Extracts prepared from cells expressing Kal-GEF1 were incubated with GST or GST-Pak1-PBD as indicated; Western blot with myc antibody shows bound epitope-tagged Kal-GEF1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Kal-GEF1 interacts with Filamin A. Filamin A co-immunoprecipitates with Trio-GEF1, Kal-GEF1, and Kal-GEF1(ND/AA). Extracts of pEAK Rapid cells expressing GFP (control) or the indicated myc-tagged GEF domain were immunoprecipitated with monoclonal antibody to myc. Filamin A was detected in immunoprecipitates by Western analysis with a Filamin A antibody. Western blots showing immunoprecipitation of myc-tagged GEF domains and expression of GFP are shown.

Fig. 7. A natural isoform of Kalirin induces lamellipodia and co-localizes with Pak. (A) PEAK Rapid cells transfected with expression vectors for pEAK10.βGal (control) or myc-ΔKal7 were stained for epitope-tagged Kalirin (green) and filamentous actin (red) as in Fig. 1. Images are snapshots from deconvolved Z-stacks. (B) ΔKal7 co-localizes with Pak in ruffles. pEAK Rapid cells immunostained for transfected myc-tagged ΔKal7 with myc (green) and endogenous Pak1 with a Pak antibody (Santa Cruz; red). Arrow indicated membrane ruffles. Scale bar = 10 μm. (C) Flattened lamellipodia induced by expression of ΔKal7 (red) are not inhibited by co-expression of a Pak-AID/GFP chimera (AID = autoinhibitory domain; green). Scale bar = 10 μm. (D) ΔKal7 induces flattened and pinwheel lamellipodia similar to Kal-GEF1. pEAK Rapid cells were transfected with the expression constructs indicated and scored for cell morphology as in Fig. 1. At least 25 cells were counted for each category and error bars are standard deviations for triplicate observations. Asterisks indicate that flattened lamellipodia were significantly different from control cells transfected with a βGal expression vector, and that co-expression of Pak-kd or PakAID did not significantly inhibit formation of flattened lamellipodia in response to ΔKal7 (P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Kal-GEF1 and GEF1(ND/AA) co-localize with Pak, N-Wasp, and Wave2 in lamellipodia. (A) pEAK Rapid cells were transiently transfected with a plasmid encoding myc-Kal-GEF1 or myc-GEF1(ND/AA) and stained with monoclonal antibody to the myc epitope (green) and polyclonal antibodies to endogenous proteins important for lamellipodial formation (red). Arrows indicate membrane ruffles and unfilled arrows indicate spokes of the pinwheel lamellipodia. (B) Model illustrating Kal-GEF1(ND/AA) activity-independent route to lamellipodial formation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)