Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold

Abstract

WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl.

Fig. 1. Identification of an 84 kDa RII-binding protein that interacts with the SH3 domain of the Abl tyrosine kinase. Rat brain extracts were subjected to pull-down assays using GST fusion proteins expressing the SH3 domains from Abl kinase, the tyrosine kinase Fgr, phospholipase Cγ and the tyrosine kinase vSrc. Control experiments were performed using GST alone and an unrelated GST fusion protein, GST–PP2A/A. RII-binding proteins were detected by a solid-phase overlay assay (Hausken et al., 1998). Detection of immobilized radiolabeled RII was by autoradiogram in the absence (A) and in the presence (B) of RII blocking peptide Ht31 (1 µM). A representative blot from four individual experiments is presented. The source of the GST fusion protein is indicated above each lane. Molecular weight markers are indicated.

Fig. 2. Purification of the 84 kDa ‘Abl-AKAP’ RII-binding protein. (A) The ‘Abl-AKAP’ was isolated from rat brain extracts by a purification strategy that took advantage of its selective interaction with the SH3 domain of Abl. Eluates from the Abl SH3 affinity resin were applied to a MonoQ anion exchange column using FPLC. (B) Proteins were eluted from the MonoQ column with a linear gradient of NaCl and the fractions were analyzed for absorbance (OD₂₈₀). (C) The elution of the 84 kDa RII-binding protein was detected by RII overlay assay in fractions 12–16. (D) The peak fractions were pooled, concentrated, separated by SDS–PAGE and stained with Coomassie Blue dye. Molecular weight markers are indicated and the migration of the ‘Abl-AKAP’ is denoted by an arrow. This protein band was excised from the gel, fragmented by digestion with trypsin and subjected to microsequence analysis using a MALDI-TOF mass spectrometer. (E) BLAST sequence analysis of three peptides obtained from the mass spectrometry (underlined) identified WAVE-1. The mouse WAVE-1 sequence is presented.

Fig. 3. PKA and Abl interact with WAVE inside cells. (A) WAVE-1 protein epitope tagged with the His₆ sequence was expressed in bacteria. Expression of the recombinant protein was detected by the RII overlay assay as described in Materials and methods (Hausken et al., 1998). Molecular weight markers are indicated. (B) Cell extracts prepared from HEK-293 cells expressing recombinant WAVE-1 epitope tagged with the FLAG sequence were subjected to immunoprecipitation using a monoclonal antibody against the FLAG epitope. Control immunoprecipitations were performed with the same antibody using cell extracts prepared from non-transfected HEK-293 cells. Co-purification of Abl kinase (top panel), WAVE (middle panel) and the catalytic subunit of the PKA holoenzyme (bottom panel) were detected by immunoblot using specific antibodies against each protein. Molecular weight markers are indicated. Arrowheads denote the migration position of each protein. (C) Rat brain extracts were immunoblotted with affinity-purified antibodies to WAVE. Molecular weight standards are indicated. (D) Rat brain extracts were subjected to immunoprecipitation using the affinity-purified WAVE antibodies. Co-purification of Abl kinase (top panel), WAVE (middle panel) and the catalytic subunit of the PKA holoenzyme (bottom panel) detected by immunoblotting using specific antibodies against each kinase and RII overlay for WAVE-1. Molecular weight standards are indicated. Arrowheads denote the migration position of each protein. Representative examples of three individual experiments are presented.

Fig. 4. Mapping the Abl-binding domain. (A) A schematic representation of recombinant His-tagged WAVE-1 fusion fragments used in mapping studies is presented. Fragments that interact with the GST–SH3 domain from Abl (filled boxes) and fragments negative for binding (open boxes) are indicated. The first and last residues in each fragment of WAVE-1 are numbered. (B) Recombinant fragments of WAVE-1 were incubated with GST–Abl SH3 fusion protein that was immobilized to glutathione–Sepharose for 1 h at 4°C. Bound proteins were detected by immunoblotting using a monoclonal antibody to the histidine tag present on the WAVE fragments. (C) A control immunoblot indicating the expression levels of each His-tagged WAVE-1 fragment loaded onto the GST–Abl SH3 domain column. WAVE-1 fragments were detected by immunoblotting using a monoclonal antibody to the histidine tag. Molecular weight standards are indicated. Representative examples of three individual experiments are presented.

Fig. 5. Mapping the RII-binding domain. (A) A schematic representation of recombinant WAVE fragments used for the mapping of the RII-binding site. Fragments that interact with RII (filled boxes) and fragments negative for binding (open boxes) are indicated. The first and last residues in each fragment of WAVE-1 are numbered. The amino acid sequence of the putative RII-binding site, which is located between residues 493 and 510 of WAVE-1, is indicated using the one letter code. (B) Fragments of WAVE-1 were separated by electrophoresis on a 6% SDS–polyacrylamide gel, electrotransferred to nitrocellulose and assayed for RII binding by overlay assay (Hausken et al., 1998). Detection of immobilized RII was by autoradiography. Molecular weight standards are indicated. (C) Point mutations were introduced at positions 505 and 509 of the WAVE-1 protein. The RII-binding characteristics of the wild-type and mutant proteins (indicated above each lane) were assessed by the overlay assay (Hausken et al., 1998). (D) Recombinant WAVE-1 was immunoprecipitated from HEK-293 cell lysates using a monoclonal antibody against the FLAG epitope. Control (C) immunoprecipitations were performed with cells not transfected with WAVE-1. Co-purification of the PKA holoenzyme was measured by assaying for the catalytic subunit of PKA using a filter paper assay (Corbin and Reimann, 1974). PKA-specific activity was measured as pmol/min/ml of material. PKA activity was blocked by the specific inhibitor PKI 5–24 peptide. The data represent the results of three independent experiments. (E) The RII-binding region of WAVE-1 is aligned with corresponding regions in the WAVE-2 and WAVE-3 isoforms. Residues are presented using the one letter code. The first and last amino acids in each sequence are indicated. Amino acids not conserved in WAVE-2 and WAVE-3 are boxed and shaded. (F) His epitope-tagged WAVE-1, WAVE-2 and WAVE-3 were expressed and purified from bacteria. Soluble extracts were separated by electrophoresis on a 6% SDS–polyacrylamide gel, electrotransferred to nitrocellulose and assayed for RII binding by overlay assay (top panel) (Hausken et al., 1998). The expression level of each WAVE isoform was detected by western blotting using the anti-His antibody (bottom panel). Molecular weight standards are indicated. Representative examples of three individual experiments are presented. (G) Soluble extracts from HEK-293 cells transfected with FLAG-tagged WAVE-1, WAVE-2 or WAVE-3 and pcDNA3 vector alone (control) were immunoprecipitated using a monoclonal antibody against the FLAG epitope. Co-precipitation of the PKA catalytic subunit (top panel) was detected by immunoblotting using a monoclonal antibody against the kinase. The expression level of each WAVE isoform (bottom panel) was monitored by western blotting using the anti-FLAG monoclonal antibody. Representative examples of three individual experiments are presented.

Fig. 6. RII and actin binding to WAVE is mutually exclusive. (A) Schematic representation of RII- (upper sequence) and actin-binding sites (lower sequence) on WAVE-1. Amino acids are presented using the one letter code. The first and last residues are numbered. (B) Solution-phase binding of GST–WAVE-1 and mutants (indicated above each lane) to actin was detected by immunoblotting (top panel) with a polyclonal antibody against rat actin. The expression level of each GST fusion protein (bottom panel) was confirmed by immunoblotting using monoclonal antibodies against GST. Molecular weight markers are indicated. (C) Solution-phase competition assay was used to examine actin displacement of RII–WAVE-1 binding. The WAVE-1–RII complex was formed on GST–beads and incubated with increasing concentrations of rat brain extract (indicated above each lane). Bound actin (top panel) and RII (middle panel) were detected by immunoblotting with polyclonal antibodies against each protein. Equal levels of the WAVE-1 fragment were detected by immunoblotting using affinity-purified anti-WAVE antibody.

Fig. 7. The subcellular distribution of WAVE. Confocal microscopy of Swiss 3T3 fibroblasts demonstrating the subcellular distribution of endogenous WAVE and cellular markers. Immunocytochemical analysis was carried out using a polyclonal antibody against (A) WAVE (green), (B) a monoclonal antibody against paxillin, a marker for focal adhesions (red), and (C) the nucleus was detected by DNA staining with Hoechst dye (blue). (D) A merged image depicts the distribution of all three stains. Control immunocytochemical analysis showed no antibody signal.

Fig. 8. Dynamic assembly of the WAVE-1–PKA complex in response to PDGF. (A) Cell lysates prepared from Swiss 3T3 fibroblasts were probed for the expression of endogenous WAVE. WAVE was detected by immunoblotting using polyclonal antibodies. Molecular weight markers are indicated. The arrow indicates the migration position of WAVE. (B) Polyclonal antibodies were used to immunoprecipitate endogenous WAVE from Swiss 3T3 fibroblast extracts. Co-precipitation of the PKA holoenzyme was confirmed by immunodetection of the PKA catalytic subunit using monoclonal antibodies. The arrow indicates the migration position of the catalytic subunit. Molecular markers are indicated. Experiments were conducted at least three times. (C–N) Confocal microscopy of Swiss 3T3 fibroblasts demonstrating the subcellular distribution of WAVE and its binding partners. Immunocytochemical analysis was carried out (C, G and K) using a rabbit polyclonal antibody against WAVE (green), (D, H and L) Texas red–phalloidin to detect actin (red) and (E, I and M) mouse monoclonal RII (blue). Merged images (F, J and N) depict the subcellular distribution of all three stains. The cells in (C–F) were serum starved. The cells in (G–J) were treated with PDGF (10 ng/ml) for 10 min prior to fixation. Cells in (K–N) were treated with cytochalasin D (10 µM) for 30 min prior to PDGF (10 ng/ml) for 10 min, and then fixed. Control immunocytochemical analysis showed no antibody signal.

Fig. 9. Dynamic assembly of the WAVE/Abl/PKA signaling scaffold in response to PDGF. Immunocytochemical analysis was carried out (A and E) using a rabbit polyclonal antibody against WAVE (green), (B and F) mouse monoclonal antibody against Abl (red) and (C and G) goat polyclonal antibody to RII (blue). Merged images (D and H) depict the subcellular distribution of all three proteins. The cells in (A–D) were serum starved. The cells in (E–H) were treated with PDGF (10 ng/ml) for 10 min prior to fixation. Control immunocytochemical analysis showed no antibody signal.

Fig. 10. Oligomerization of WAVE isoforms. HEK-293 cells expressing GFP-tagged WAVE-1 or control vector were co-transfected with FLAG-tagged WAVE-1, WAVE-2 or WAVE-3 (indicated above each lane). Soluble HEK-293 cell extracts were subjected to immunoprecipitation using an anti-FLAG monoclonal antibody. Expression of each WAVE isoform (top panel) was confirmed by immunoblotting with monoclonal antibodies against the FLAG epitope. The migration positions of each WAVE isoform are indicated by arrows. Co-precipitation of GFP-tagged WAVE-1 was detected by immunoblotting using antibodies against GFP (bottom panel).