Title: Disabled-2 is transcriptionally regulated by ICSBP and augments macrophage spreading and adhesion
Abstract: Article1 February 2002free access Disabled-2 is transcriptionally regulated by ICSBP and augments macrophage spreading and adhesion Frank Rosenbauer Frank Rosenbauer Present address: Hematology/Oncology Division, Harvard Institutes of Medicine, Boston, MA, 02155 USA Search for more papers by this author Axel Kallies Axel Kallies Department of Molecular Genetics, Institute of Molecular Pharmacology, and Benjamin Franklin Medical Center, Free University of Berlin, Krahmerstrasse 6, D-12207 Berlin, Germany Search for more papers by this author Marina Scheller Marina Scheller Department of Molecular Genetics, Institute of Molecular Pharmacology, and Benjamin Franklin Medical Center, Free University of Berlin, Krahmerstrasse 6, D-12207 Berlin, Germany Search for more papers by this author Klaus-Peter Knobeloch Klaus-Peter Knobeloch Present address: GenPat77 Pharmacogenetics, D-10115 Berlin, Germany Search for more papers by this author Charles O. Rock Charles O. Rock Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Search for more papers by this author Maike Schwieger Maike Schwieger Heinrich-Pette-Institute for Experimental Virology and Immunology, D-20251 Hamburg, Germany Search for more papers by this author Carol Stocking Carol Stocking Heinrich-Pette-Institute for Experimental Virology and Immunology, D-20251 Hamburg, Germany Search for more papers by this author Ivan Horak Corresponding Author Ivan Horak Department of Molecular Genetics, Institute of Molecular Pharmacology, and Benjamin Franklin Medical Center, Free University of Berlin, Krahmerstrasse 6, D-12207 Berlin, Germany Search for more papers by this author Frank Rosenbauer Frank Rosenbauer Present address: Hematology/Oncology Division, Harvard Institutes of Medicine, Boston, MA, 02155 USA Search for more papers by this author Axel Kallies Axel Kallies Department of Molecular Genetics, Institute of Molecular Pharmacology, and Benjamin Franklin Medical Center, Free University of Berlin, Krahmerstrasse 6, D-12207 Berlin, Germany Search for more papers by this author Marina Scheller Marina Scheller Department of Molecular Genetics, Institute of Molecular Pharmacology, and Benjamin Franklin Medical Center, Free University of Berlin, Krahmerstrasse 6, D-12207 Berlin, Germany Search for more papers by this author Klaus-Peter Knobeloch Klaus-Peter Knobeloch Present address: GenPat77 Pharmacogenetics, D-10115 Berlin, Germany Search for more papers by this author Charles O. Rock Charles O. Rock Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Search for more papers by this author Maike Schwieger Maike Schwieger Heinrich-Pette-Institute for Experimental Virology and Immunology, D-20251 Hamburg, Germany Search for more papers by this author Carol Stocking Carol Stocking Heinrich-Pette-Institute for Experimental Virology and Immunology, D-20251 Hamburg, Germany Search for more papers by this author Ivan Horak Corresponding Author Ivan Horak Department of Molecular Genetics, Institute of Molecular Pharmacology, and Benjamin Franklin Medical Center, Free University of Berlin, Krahmerstrasse 6, D-12207 Berlin, Germany Search for more papers by this author Author Information Frank Rosenbauer2, Axel Kallies1, Marina Scheller1, Klaus-Peter Knobeloch3, Charles O. Rock4, Maike Schwieger5, Carol Stocking5 and Ivan Horak 1 1Department of Molecular Genetics, Institute of Molecular Pharmacology, and Benjamin Franklin Medical Center, Free University of Berlin, Krahmerstrasse 6, D-12207 Berlin, Germany 2Present address: Hematology/Oncology Division, Harvard Institutes of Medicine, Boston, MA, 02155 USA 3Present address: GenPat77 Pharmacogenetics, D-10115 Berlin, Germany 4Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN, 38105 USA 5Heinrich-Pette-Institute for Experimental Virology and Immunology, D-20251 Hamburg, Germany ‡F.Rosenbauer and A.Kallies contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:211-220https://doi.org/10.1093/emboj/21.3.211 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mice lacking transcription factor interferon consensus sequence binding protein (ICSBP) develop a syndrome similar to human chronic myeloid leukemia and are immunodeficient. In order to define the molecular mechanisms responsible for the cellular defects of ICSBP−/− mice, we used bone marrow-derived macrophages (BMM) to identify genes deregulated in the absence of ICSBP. Here, we report that disabled-2 (Dab2), a signal phosphoprotein, is transcriptionally up-regulated and accumulates in the cytoskeleton/membrane fraction of ICSBP−/− BMM. Moreover, our results revealed Dab2 as a novel IFN-γ-response gene. Both ICSBP and the Ets-transcription factor PU.1 bind to the Dab2 promoter, whereby ICSBP represses PU.1-induced Dab2 promoter transactivation in vitro. Notably, repression of Dab2 expression by ICSBP is also found in myeloid progenitors. Overexpression of Dab2 leads to accelerated cell adhesion and spreading, accompanied by enhanced actin fiber formation. Furthermore, cell adhesion induces transient Dab2 phosphorylation and its translocation to the cytoskeletal/membrane fraction. Our results identify a novel role of Dab2 as an inducer of cell adhesion and spreading, and strongly suggest that the up-regulation of Dab2 contributes to the hematopoietic defect seen in ICSBP−/− mice. Introduction Progression of pluripotent hematopoietic stem cells to mature, terminally differentiated effector cells of the hemato-lymphoid system proceeds through a process of sequential changes in the gene expression pattern, driven by multiple extrinsic and intrinsic signals. Transcription factors are shared mediators of both extrinsic and intrinsic signals and control gene activity directly. Both general transcription factors, indispensable for development of all hematopoietic cells, as well as lineage specific factors have been described (Tenen et al., 1997; Orkin, 2000). Analysis of mice deficient for interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor (IRF) family of transcription factors, has provided evidence for a critical role of ICSBP in hematopoiesis, in particular as a regulatory switch in myeloid differentiation. ICSBP deficiency in mice results in a complex phenotype, dominated by immunodeficiency and a myelo-lymphoproliferative syndrome that resembles chronic myeloid leukemia (CML) (Holtschke et al., 1996; Fehr et al., 1997). Several lines of evidence support the notion of ICSBP involvement in human CML, a disease caused by the Bcr–Abl fusion protein (reviewed in Deininger et al., 2000). Patients with CML have strongly reduced levels of ICSBP, which can be restored after interferon (IFN)-α therapy (Schmidt et al., 1998). Furthermore, ICSBP is significantly reduced in mice with a Bcr–Abl-induced CML-like disease, and forced overexpression of ICSBP inhibits the myeloproliferative syndrome in that system (Hao and Ren, 2000). The hematopoietic precursor cells from ICSBP−/− mice, similar to those from CML patients, reveal gross alterations in their response to growth factors and altered adhesion. The most remarkable characteristic of the ICSBP−/− bone marrow progenitors is their altered response to CSF-1, suggesting a critical role of ICSBP in the proliferation and differentiation of the myeloid cell lineage (Scheller et al., 1999). This notion was confirmed and further extended by Tamura et al. (2000), who showed that ICSBP directed macrophage differentiation of a myeloid progenitor cell line established from ICSBP−/− mice. Recently, we have found that in the absence of ICSBP, CSF-1R signaling is attenuated (Kallies et al., 2002), as seen from a rapid termination of MAP kinase phosphorylation and reduced cell growth. This coincides with enhanced accumulation of c-Cbl, which is known to down-regulate CSF-1R signaling by its ubiquitin-ligase activity. Our results indicate that c-Cbl is proteolytically degraded and that this proteolytic activity is reduced in ICSBP−/− bone marrow-derived macrophages (BMM). However, the primary target genes directly deregulated by the lack of the transcription factor ICSBP remain to be found. In view of the profound effects of the ICSBP deficiency on myeloid differentiation and proliferation, the identification of ICSBP target genes should contribute to our understanding of normal myelopoiesis and could reveal insights into novel mechanisms underlying myeloproliferative diseases. Here we have identified disabled-2 (dab2) as a gene differentially transcribed in myeloid cells from ICSBP−/− and ICSBP+/+ mice. Mouse Dab2 is a mitogen-responsive phosphoprotein with sequence homology to Dab proteins of humans as well as Caenorhabditis elegans and Drosophila melanogaster (Xu et al., 1995; Albertsen et al., 1996). Dab2 contains a phosphotyrosine-interacting domain, a C-terminal proline-rich domain and a potential actin-binding motif, KKEK (Xu et al., 1995). It has been reported that Dab2 interaction with Grb2 reduces the binding between Grb2 and Sos and thus could modulate growth factor/Ras pathways (Xu et al., 1998). Recently, Dab2 was characterized as a critical link between transforming growth factor (TGF)-β receptors and the Smad proteins (Hocevar et al., 2001). Results presented here identify Dab2 as a novel ICSBP down-regulated target of the IFN-γ pathway and show for the first time that Dab2 induces macrophage adhesion and spreading. Results Dab2 expression is enhanced in BMM from ICSBP−/− mice In order to identify the molecular mechanisms responsible for altered myeloid differentiation, we used BMM from ICSBP+/+ and ICSBP−/− mice to search for genes differentially expressed in the absence of ICSBP. cDNA expression was compared by expression arrays, which allow the comparison of expression levels of almost 1200 different genes in parallel. After normalizing the expressed signals to a set of housekeeping genes, only six genes (1.3% of the positive signals) were differentially expressed by >2-fold in ICSBP−/− BMM (data not shown). The low numbers of differentially expressed genes confirmed the high comparability of both BMM populations. The most pronounced difference in expression was observed for dab2, with a 3.2-fold increase in mRNA levels in ICSBP−/− BMM (Figure 1A). This result was confirmed also by semiquantitative RT–PCR (Figure 1B). Dab2 is a mitogen-responsive phosphoprotein with signal transduction capability (Xu et al., 1995), and therefore an interesting candidate gene, potentially involved in molecular pathways leading to altered cellular properties of ICSBP−/− BMM. Figure 1.Dab2 is overexpressed in ICSBP−/− BMM. (A) The hybridization patterns from ICSBP+/+ and ICSBP−/− BMM from a section of the cDNA expression arrays are shown. The arrows indicate the position of the Dab2 cDNA and of two control genes (β-actin and a 40S ribosomal protein). The arrays show a strong hybridization signal of Dab2 in ICSBP−/− but a weak signal in ICSBP+/+ BMM. (B) Semiquantitative RT–PCR analysis demonstrating the mRNA expression levels of the Dab2 gene in BMM from ICSBP+/+ and ICSBP−/− mice. (C) Western blot analysis of total protein extracts showing the different protein expression levels of Dab2 in ICSBP+/+ and ICSBP−/− BMM. Bac1.2F5 cells, expressing p96 and p67 (Xu et al., 1995), served as a control to identify the Dab2 isoforms expressed in BMM. The Dab2 proteins were detected by M2 antiserum. Equal protein loading was confirmed by the protein p80 detected by ICSBP antiserum. Download figure Download PowerPoint Dab2 is expressed as two isoforms, p96 and p67, in vivo (Xu et al., 1995); both of them were also present in ICSBP+/+ and ICSBP−/− BMM (Figure 1C). However, a striking accumulation of both isoforms was found in ICSBP−/− cells. Thus, our results demonstrate a significant up-regulation of Dab2 at both RNA and protein levels in ICSBP−/− BMM. ICSBP represses PU.1-induced transactivation of the Dab2 promoter To analyze whether ICSBP regulates Dab2 transcription directly, a luciferase reporter plasmid under the transcriptional control of the Dab2 promoter (pGL3-Dab2p) was transfected into two cell lines, K562 and CV-1, both lacking endogenous ICSBP expression (Rosenbauer et al., 1999; data not shown). As demonstrated in Figure 2A, transcription of the reporter gene driven by Dab2 regulatory sequences was repressed by the co-transfection of ICSBP expression vector (pcDNA-ICSBP) in K562 cells. Consequently, ICSBP acts as a negative transcriptional regulator of the Dab2 promoter. Figure 2.ICSBP down-regulates PU.1 induced transactivation of the Dab2 promoter. (A) K562 cells were transfected with pGL3-Basic containing a 2 kb fragment of the mouse Dab2 promoter together with pcDNA or pcDNA-ICSBP and the reference vector pRL-TK. Luciferase reporter gene activities were measured as described in Materials and methods. (B) CV-1 cells were transiently transfected with a Dab2 promoter driven luciferase reporter gene (300 ng) along with the following expression vectors: PU.1 200 ng; ICSBP 200 ng or 600 ng. Empty vector DNA was added so that each reaction contained a total of 800 ng of expression plasmid. (C) Endogenously expressed PU.1 binds to the Dab2 regulatory sequence. K562 nuclear extracts were incubated with the immobilized Dab2 promoter DNA, and the bound fraction was analyzed by western blotting using antibodies as indicated. (D) ICSBP and PU.1 bind to the Dab2 promoter. Magnetic beads containing the 2 kb mouse Dab2 promoter were incubated with nuclear extracts of CV-1 cells, which were supplemented with 10 μl of 35S-labeled ICSBP, PU.1, or both ICSBP and PU.1. The beads were washed and the eluates were subjected to SDS–PAGE and autoradiography. 35S-labeled protein (1 μl) was used as an input control. Probing of the membrane with an anti-Stat3 antibody confirmed the specificity of the employed assay. Download figure Download PowerPoint In CV-1 cells only very low levels of Dab2-directed luciferase expression could be detected, which was not affected by the co-transfection of the ICSBP expression vector. These data suggest that other co-factors, which are required for Dab2 promoter activation, might be absent in CV-1 cells. In contrast to K562 cells, CV-1 cells do not express the hematopoietic-specific Ets-transcription factor PU.1 (Behre et al., 1999). Since PU.1 is a well-defined binding partner of ICSBP (Brass et al., 1996), we analyzed the effect of PU.1 on the Dab2 promoter by co-transfecting pGL3-Dab2p along with a PU.1 expression vector into CV-1 cells. PU.1 transactivated the Dab2 promoter >5-fold, indicating that PU.1 is a potent positive-regulator of Dab2. Interestingly, when increasing amounts of ICSBP were co-transfected together with constant amounts of pGL3-Dab2p and PU.1, a repression of PU.1-induced Dab2 promoter transactivation was observed (Figure 2B). Together, these results indicate that ICSBP down-regulates the PU.1-dependent transcription of the Dab2 promoter. To test whether ICSBP and PU.1 can physically interact with the Dab2 promoter, we employed a DNA affinity binding assay that has previously been used as a sensitive method for the analysis of protein recruitment to regulatory DNA elements (Wang et al., 2000). The Dab2 promoter DNA was conjugated to magnetic beads and incubated with nuclear extracts from CV-1 cells, which were supplemented with [35S]methionine-labeled ICSBP, PU.1, or both ICSBP and PU.1. The proteins bound to the immobilized DNA were separated from the unbound fraction, resolved by SDS–PAGE and visualized by autoradiography. Although both ICSBP and PU.1 were recruited to the Dab2 promoter individually, ICSBP binding increased >6-fold in the presence of PU.1 (Figure 2D). The specificity of the assay was tested by probing the membrane with an antibody against Stat3 as a negative control. Although Stat3 was highly expressed in CV-1 cells, it was not recruited to the conjugated beads. Furthermore, endogenous PU.1 expressed by K562 cells was also found to bind strongly to the Dab2 promoter (Figure 2C). Thus, the employed DNA affinity binding assay revealed specific binding of both PU.1 and ICSBP to the Dab2 regulatory DNA, and indicated that ICSBP binding is supported by PU.1. ICSBP suppresses Dab2 expression in myeloid progenitors The role of ICSBP as a transcriptional repressor of Dab2 was directly confirmed by its enforced expression in two mouse myeloid progenitor cell lines. IC34L cells, a line derived from ICSBP−/− mice, infected with either a control retroviral vector or a vector expressing a hydroxytamoxifen (TAM)-inducible ICSBP, express Dab2 but no endogenous ICSBP. After ICSBP induction by TAM, Dab2 expression was no longer detectable in these cells, whereas TAM induction of control infected IC34L cells had no effect on Dab2 levels (Figure 3A). Similar results were obtained with FDC-P1Mac11 cells, a line that expresses Dab2 but only very limited amounts of ICSBP (Figure 3B). After transfection with an ICSBP expression plasmid, Dab2 expression was significantly reduced. Further evidence supporting a direct regulation of Dab2 gene expression by ICSBP in myeloid progenitor cells was revealed by DNA affinity binding. As shown in Figure 3C, both ICSBP and PU.1 were specifically recruited to the Dab2 promoter when incubated with nuclear extracts of IC34L cells. Figure 3.ICSBP regulates Dab2 expression in myeloid progenitor cells. (A) TAM-inducible ICSBP represses Dab2 expression in IC34L cells. The IC34L precursor cell line, isolated from ICSBP−/− mouse, was transfected with a TAM-inducible estrogen (ER)–ICSBP fusion construct or with control vector by retroviral infection as described in Materials and methods. Cells were treated with TAM for 16 h and total cell extracts were analyzed by western blotting using an anti-Dab2 antibody and ICSBP antiserum. (B) Overexpression of ICSBP in the bipotential myeloid precursor cell line FDC-P1Mac11 down-regulates endogenous Dab2 expression. FDC-P1Mac11 cells were either mock-transfected or transiently transfected with pcDNA-ICSBP. After 24 h, total protein was extracted and analyzed by western blotting using a Dab2 antiserum (M2) and ICSBP antiserum. (C) ICSBP and PU.1 bind to the Dab2 promoter in myeloid progenitor cells. The Dab2 promoter DNA was immobilized by magnetic beads and incubated with nuclear extracts of IC34L cells transfected with the TAM-inducible ER–ICSBP fusion protein or a control vector. Proteins recruited to the Dab2 promoter were analyzed by western blotting using antibodies as indicated. Download figure Download PowerPoint The fact that ICSBP represses Dab2 expression in myeloid progenitors indicates that Dab2 gene regulation by ICSBP is not limited to more mature monocytic cells, e.g. BMM, but also plays a role in myeloid progenitors. dab2 is a novel IFN-γ-responsive gene The expression of ICSBP is induced by IFN-γ and also, to a lesser extent, by LPS (Wang et al., 2000). Whether dab2 expression is controlled by IFN-γ was tested in the experiment shown in Figure 4. As expected, Dab2 transcription, as well as protein expression, was repressed in ICSBP+/+ BMM stimulated with IFN-γ. In contrast, no reduction in dab2 mRNA and protein levels was observed in ICSBP−/− cells in response to IFN-γ. Furthermore, ICSBP recruitment to the dab2 promoter was strongly increased after IFN-γ induction, as revealed by DNA affinity binding (Figure 4C). In contrast, no effect of IFN-γ on PU.1 binding to the dab2 promoter was observed. Taken together, our results show that dab2 is a novel downstream target of the IFN-γ pathway, transcriptionally regulated by ICSBP. Figure 4.Dab2 is a novel IFN-γ-responsive protein. (A and B) BMM from ICSBP+/+ and ICSBP−/− mice were cultivated for 19 h in the absence (−) or presence (+) of 200 U/ml IFN-γ. (A) Total RNA was isolated and subjected to semiquantitative RT–PCR analysis using primers for Dab2, β-actin and ICSBP. (B) Total cell extracts were analyzed by western blotting with an anti-Dab2 antibody and an anti-ICSBP antiserum. (C) IFN-γ induces ICSBP binding to the Dab2 promoter. Nuclear extracts of the macrophage-like cell line Bac1.2F5, either untreated (−) or treated with IFN-γ (200 U/ml) (+) for 16 h, were incubated with immobilized Dab2 promoter DNA. The bound fraction was analyzed by western blotting using antibodies as indicated. Download figure Download PowerPoint Dab2 is phosphorylated, and accumulates in the cytoskeleton/membrane fraction of ICSBP−/− BMM Mitogen stimulation, such as treatment with CSF-1 or tetradecanoylphorbol-13-acetate (TPA), leads to serine-phosphorylation of Dab2 by PKC (Xu et al., 1995). This modification seems to be important for at least some Dab2 functions, since only the phosphorylated protein can inhibit AP-1 activity (Tseng et al., 1999). As shown by others, phosphorylated Dab2 can easily be detected by its retarded electrophoretic mobility in gels compared with the unphosphorylated protein (Xu et al., 1995). To investigate whether the increase of Dab2 protein expression in ICSBP−/− BMM is also accompanied by its phosphorylation, we analyzed the phosphorylation of p96 after CSF-1 stimulation. We found that the slower migrating phospho-p96 (pp96) appears 5 min after stimulation of both ICSBP+/+ and ICSBP−/− BMM (Figure 5A). Figure 5.Phosphorylation and subcellular distribution of Dab2 in BMM. (A) Phosphorylation of p96 Dab2 in ICSBP+/+ and ICSBP−/− BMM following CSF-1 stimulation. Cells were deprived of CSF-1 for 16 h, left untreated or were stimulated with 150 ng/ml CSF-1 at 37°C for 5 min. Total cell extracts were analyzed by western blotting with a Dab2 antiserum (M15). To obtain a better separation of p96 and its phosphorylated form (P-p96), only 30% of the ICSBP−/− protein extract, compared with the ICSBP+/+ extract, was loaded on the gel. (B) Subcellular distribution of p96 Dab2 in BMM from ICSBP+/+ and ICSBP−/− mice. Membrane/cytoskeleton (ME/CS) and cytosolic (CY) fractions (25 μg each) were loaded on SDS–PAGE and subjected to western blotting using M15 Dab2 and ICSBP antisera. Download figure Download PowerPoint It has been reported previously that Dab2 is found mainly in the cytosol but appears in the particulate fraction after stimulation with TPA (Tseng et al., 1999). We therefore analyzed the subcellular distribution of Dab2 in asynchronously growing BMM by separating the cytosolic from the cytoskeleton/membrane-associated proteins. Comparable amounts of Dab2 were present in the cytosol of both ICSBP+/+ and ICSBP−/− cells (Figure 5B). However, a strongly enhanced accumulation of Dab2 was seen in the cytoskeleton/membrane fraction of ICSBP−/− BMM. Together, these results indicate that loss of ICSBP in BMM leads to enhanced expression of Dab2, which becomes phosphorylated after CSF-1 stimulation and accumulates in the membrane/cytoskeletal fraction. Enhanced expression of Dab2 augments spreading and adhesion of macrophages We reported previously that myeloid progenitors from ICSBP−/− mice have altered adhesion properties to extracellular matrix (ECM) components (Scheller et al., 1999). Sheng et al. (2000) suggested a role of Dab2 in cell positioning control and in mediating the exigency for basement membrane attachment of epithelial cells. Therefore, we have compared the adhesion of BMM from ICSBP−/− and ICSBP+/+ mice to laminin and collagen IV using an adhesion assay. These two compounds are the main components of the basement membrane and other ECM. As seen in Figure 6A, an increased adhesion of ICSBP−/− BMM to both substrates was observed. Figure 6.Overexpression of Dab2 leads to enhanced adhesion of macrophages to laminin and collagen type IV. (A) BMM from ICSBP+/+ and ICSBP−/− mice, and (B) RAW cells stably transfected with a pcDNA vector containing the complete Dab2-cDNA or the empty pcDNA were used in an adhesion assay. Indicated numbers of enzymatically labeled cells were plated on 96-well plates coated with laminin or collagen type IV. After 15 min incubation, non-adherent cells were removed by washing, and the adherent cells were stained and processed according to the manufacturer's protocol. The graphs show the results of a representative experiment; three experiments were performed in triplicate. (C) Total cell extracts of RAW cells stably transfected with pcDNA-Dab2, untransfected or transfected with the empty vector were subjected to western blotting with a Dab2 antibody. Extracts of 5 × 105 cells per lane were loaded on the gel. Download figure Download PowerPoint To investigate whether the enhanced adhesion of ICSBP−/− BMM to laminin or collagen IV could be linked directly to Dab2 overexpression, the murine macrophage cell line RAW 264.1 was transfected with a Dab2 expression vector, and four stably transfected lines overexpressing Dab2 were established. Their independent origin was confirmed by Southern blots (data not shown). Only a slight expression of the endogenous Dab2 gene was detected in non-transfected RAW cells, as well as in cells stably transfected with the empty plasmid (Figure 6C). The adhesion of Dab2-overexpressing RAW cells to laminin and collagen IV was markedly enhanced, similar to what is seen in ICSBP−/− BMM (Figure 6B). Thus Dab2 is a potent inducer of cell adhesion and it is very likely that its deregulated expression is, at least in part, directly responsible for the altered adhesion properties of ICSBP−/− myeloid cells. In addition to the enhanced adhesion, we noted an accelerated spreading of ICSBP−/− BMM. This phenomenon was even more prominent in Dab2-overexpressing RAW cells (Figure 7A). We therefore compared the adhesion-induced morphological changes of Dab2 and control transfected RAW cells. Cell spreading and the adhesion-induced reorganization of the actin cytoskeleton was monitored by plating the cells on laminin-coated glass coverslips, followed by rhodamin-labeled phalloidin staining at different time points (Figure 7B). When maintained in suspension, both types of cells had a rounded, contracted shape and displayed a cortical ring of actin filaments. Notably, intensification of adhesion-induced lamellae and filopodia formation was more prominent in cells overexpressing Dab2. Furthermore, cell spreading and formation of lamellae and filopodia started earlier and was clearly visible as early as 30 min after plating the cells. Figure 7.Overexpression of Dab2 leads to accelerated spreading and formation of pseudopodia in macrophages. (A) Phase-contrast microscopy of RAW cells transfected with Dab2-pcDNA or the empty vector, and plated on the plastic surface of tissue culture plates. (B) Confocal immunofluorescence microscopy of actin cytoskeletal organization of RAW cells transfected with Dab2-pcDNA or the empty vector. Cells were trypsinized and plated on laminin-coated coverslips, cultured for 30 and 60 min, fixed and processed for F-actin staining using rhodamine-conjugated phalloidin. Scale bar = 5 μm. Download figure Download PowerPoint Taken together, we found that Dab2 overexpression augments macrophage adhesion and spreading and is associated with reorganization of the actin cytoskeleton. Dab2 is phoshorylated and translocates to cytoskeleton/membrane upon macrophage adhesion Adhesion is known to induce phosphorylation and translocation of several cytosolic signal proteins to components of the cytoskeleton and cell membrane (Machesky and Hall, 1997). To further examine the role of Dab2 in adhesion-induced signaling pathways, we examined whether Dab2 becomes phosphorylated during adhesion of cells to components of ECM. Serine phosphorylation was shown to be required for at least some functions of Dab2 in epithelial cells (Tseng et al., 1999). RAW cells overexpressing Dab2 were seeded on plates coated with laminin, or were kept in suspension. The appearance of phosphorylated forms of p96 Dab2 was monitored by western blotting (Figure 8A). The adhesion-induced phosphorylation of Dab2 was transient; it was clearly observed after 30 min and lasted for ∼2 h. Dab2 phosphorylation was observed also in RAW cells after their adhesion on other substrates, fibronectin and poly-D-lysine. Similar results were also obtained with BMM (Figure 8B and C). Figure 8.Dab2 is transiently phosphorylated and translocates into the cytoskeleton/membrane fraction in response to adhesion. (A) RAW macrophages overexpressing Dab2 were trypsinized and plated on laminin-coated culture dishes or were kept in suspension. At the time points indicated, total cell extracts of adherent cells and cells kept in suspension were analyzed by western blotting. RAW cells overexpressing Dab2 (B) or ICSBP−/− BMM (C) were treated as described in (A), and plated on culture dishes coated with fibronectin (FN), poly-D-lysine (pDL) or laminin (LA). After 30 min, total cell extracts of adherent cells and cells kept in suspension were prepared, and subjected to SDS–PAGE and western blotting with the anti-Dab2 antibody. (D) RAW cells overexpressing Dab2 were trypsinized and plated on culture dishes coated with fibronectin, or kept in suspension. After 60 min, cytosolic and particulate fractions were prepared as described in Materials and methods, and were analyzed by western blotting with anti-Dab2 antibody. Download figure Download PowerPoint We next investigated whether adhesion induces the tra