WIP, a protein associated with wiskott-aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells

Abstract

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.

Figure 1

WIP cDNAs, deduced amino acid sequence of WIP and its alignments. (A) Schematic representation of full-length WIP, WIP2, WIP4, and Prpl2 cDNAs. The open box in Prpl2 represents the seven amino acids that replace the C-terminal 17 amino acids in WIP. (B) Deduced amino acid sequence of WIP. The two APPPPP motifs implicated in profilin binding are denoted by ∗. A line is drawn over the KLKK motif implicated in acting binding. (C) Sequence alignment of the N-terminal regions of WIP and verprolin. The two verprolin homology regions are boxed. (D) Sequence alignment of GRSGPXPPXP motifs in WIP and WASP. Numbers refer to amino acid positions.

Figure 2

In vitro binding and coimmunoprecipitation of WIP and WASP. (A) In vitro binding of WIP and WASP fusion proteins. WASP_1–270, WIP4, and the control protein M were expressed in E. coli as fusion proteins with GST or MBP. Soluble extracts from induced bacteria were mixed, and the fusion proteins were affinity-precipitated by using glutathione beads, or amylose resin, run on 4–15% gradient SDS/PAGE, and examined for the presence of MBP fusion proteins by Western blotting with rabbit anti-MBP antiserum. The blots were developed by using protein A conjugated to horseradish peroxidase followed by ECL. The position of MBP-WIP is indicated by a dot. Note that the molecular masses of MBP-WIP and the control MBP-M proteins are similar. The lower molecular bands represent degradation products of the fusion proteins. The presence of GST-WASP_1–270 in lanes 3 and 5 and GST in lane 7 was confirmed by Western blotting with anti-GST mAb (data not shown). (B) Affinity precipitation of endogenous WASP by GST-WIP. Lysates of BJAB cells were incubated with GSH-Sepharose beads coupled to GST-WIP2 or GST. Bound proteins were eluted, run on 10% SDS/PAGE, and examined for the presence of WASP by immunoblotting with rabbit anti-WASP peptide antibody. The blots were developed as described in A. (C) In vivo association of WIP and WASP. FLAG-tagged full-length WIP cDNA was transfected into BJAB cells, and the cell lysates were immunoprecipitated with M2 anti-FLAG mAb, blotted, and probed with anti-WASP peptide antibody.

Figure 3

Mapping of the binding domains of WIP and WASP. (A) Mapping of the WIP binding site of WASP. The domains of WASP are indicated. PH, Pleckstrin homology domain (amino acids 6–105); WH1, WH1 domain (amino acids 47–137); pppppp, proline-rich region; GBD, GTPase binding domain (amino acids 238–257); WH2, WH2 domain (amino acids 423–449); AAD, actin association domain (amino acids 443–502). The numbers under the bar at the bottom of the diagram represent the amino acids of WASP. Truncation mutants of WASP, generated either by PCR or cleavage with appropriate restriction enzymes, were cloned into the pGBT9 vector and examined for WIP binding in the yeast two-hybrid system. Blue color development by β-galactosidase activity was used to score the interaction of WIP with WASP truncations. +++ represents color change in 30 min or less, + represents color change in 3 hr, and − indicates no color change and lack of growth in His⁻ medium. For each mutant at least three independent colonies were tested in the β-galactosidase assay. (B) Mapping of the WASP binding site of WIP. Truncation mutants of WIP4 (amino acids 321–503), generated by cleavage with appropriate restriction enzymes, were cloned into the pGAD424 vector and examined for WASP binding in the yeast two-hybrid system. Interactions were scored as in A.

Figure 4

Expression of WIP transcripts in human tissues. Multiple tissue Northern blots containing 2 μg of human mRNA per lane were hybridized with ³²P-labeled full-length WIP cDNA probe (Upper). The filter was exposed for 12 hr. As a control for loading, the blot was reprobed for glyceraldehyde-3-phosphate dehydrogenase probe (Lower). PBMC, peripheral blood mononuclear cells.

Figure 5

Effect of WIP overexpression on F-actin distribution in BJAB cells. (A) Expression of WIP4 and WIP in BJAB clones. (Left) Expression of FLAG-WIP4 protein in clone I is demonstrated by the presence of a specific band corresponding to FLAG-WIP4 in immunoblots of anti-FLAG immunoprecipitates developed with anti-FLAG mAb followed by peroxidase labeled goat anti-mouse Ig and ECL. This band is absent in the control clone A.11 transfected with pcDNA3 alone. The heavy (H) and light (L) chains of the immunoprecipitating mAb are visualized. (Center) Full-length FLAG-WIP did not transfer in immunoblotting. (Right) Expression of FLAG-WIP protein in clone 3 transfected with FLAG-WIP is inferred by the presence of a specific band corresponding to WASP in immunoblots of anti-FLAG immunoprecipitate developed with rabbit anti-WASP followed by protein A and ECL. This band is absent in the control clone A.11. (B) F-actin distribution in BJAB cells. Untransfected cells (control), a pcDNA3 transfected clone (A.11), and representative clones expressing WIP (clone 3) and WIP4 (clone I) were permeabilized, stained with rhodamine-conjugated phalloidin, and examined by immunofluorescence microscopy. (Bar, 5 μm.)

Figure 6

Affinity precipitation of endogenous profilin by GST-WIP2. Lysates of BJAB cells were incubated with GSH-Sepharose beads coupled to GST-WIP2 or GST. Bound proteins were eluted, run on 4–15% gradient SDS/PAGE, and examined for the presence of profilin by immunoblotting with rabbit anti-profilin antiserum. The blots were developed as described in Fig. 2A.