Mona, a novel hematopoietic-specific adaptor interacting with the macrophage colony-stimulating factor receptor, is implicated in monocyte/macrophage development
1998; Springer Nature; Volume: 17; Issue: 24 Linguagem: Inglês
10.1093/emboj/17.24.7273
ISSN1460-2075
AutoresRoland P. Bourette, Sylvie Arnaud, Gary M. Myles, J. P. Blanchet, Larry R. Rohrschneider, Guy Mouchiroud,
Tópico(s)Signaling Pathways in Disease
ResumoArticle15 December 1998free access Mona, a novel hematopoietic-specific adaptor interacting with the macrophage colony-stimulating factor receptor, is implicated in monocyte/macrophage development Roland P. Bourette Roland P. Bourette Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France Search for more papers by this author Sylvie Arnaud Sylvie Arnaud Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France Search for more papers by this author Gary M. Myles Gary M. Myles Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA Search for more papers by this author Jean-Paul Blanchet Jean-Paul Blanchet Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France Search for more papers by this author Larry R. Rohrschneider Larry R. Rohrschneider Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA Search for more papers by this author Guy Mouchiroud Corresponding Author Guy Mouchiroud Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France Search for more papers by this author Roland P. Bourette Roland P. Bourette Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France Search for more papers by this author Sylvie Arnaud Sylvie Arnaud Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France Search for more papers by this author Gary M. Myles Gary M. Myles Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA Search for more papers by this author Jean-Paul Blanchet Jean-Paul Blanchet Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France Search for more papers by this author Larry R. Rohrschneider Larry R. Rohrschneider Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA Search for more papers by this author Guy Mouchiroud Corresponding Author Guy Mouchiroud Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France Search for more papers by this author Author Information Roland P. Bourette1, Sylvie Arnaud1, Gary M. Myles2, Jean-Paul Blanchet1, Larry R. Rohrschneider2 and Guy Mouchiroud 1 1Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne, Cedex, France 2Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:7273-7281https://doi.org/10.1093/emboj/17.24.7273 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The production, survival and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF results in Fms autophosphorylation on specific tyrosines that act as docking sites for intracellular signaling molecules containing SH2 domains. Using a yeast two-hybrid screen, we cloned a novel adaptor protein which we called ‘Mona’ for monocytic adaptor. Mona contains one SH2 domain and two SH3 domains related to the Grb2 adaptor. Accordingly, Mona interacts with activated Fms on phosphorylated Tyr697, which is also the Grb2-binding site. Furthermore, Mona contains a unique proline-rich region located between the SH2 domain and the C-terminal SH3 domain, and is apparently devoid of any catalytic domain. Mona expression is restricted to two hematopoietic tissues: the spleen and the peripheral blood mononuclear cells, and is induced rapidly during monocytic differentiation of the myeloid NFS-60 cell line in response to M-CSF. Strikingly, overexpression of Mona in bone marrow cells results in strong reduction of M-CSF-dependent macrophage production in vitro. Taken together, our results suggest an important role for Mona in the regulation of monocyte/macrophage development as controlled by M-CSF. Introduction Signaling by receptor tyrosine kinases plays a major role in regulation of developmental processes. Once activated by their ligands, these transmembrane receptors are the starting point of a cascade of events initiated by recruitment and/or activation of intracellular molecules into multiprotein signaling complexes (Ullrich and Schlessinger, 1990; Fantl et al., 1993). In such a context, the specificity of receptor expression and the innate responsiveness of the cell, including the nature and concentration of the signaling molecules, will govern cell fate (Pawson and Scott, 1997). Defining all components of the initial signaling complexes is thus necessary in order to understand how a receptor controls specific biological responses at different stages of development. Activation of macrophage colony-stimulating factor (M-CSF, also called CSF-1) receptor offers an interesting example of the inherent complexity of regulating opposing processes such as cell proliferation and terminal differentiation. During hematopoiesis, M-CSF is the principal regulator of the production of monocytes and macrophages both in vivo and in vitro (Metcalf, 1989). Osteopetrotic mutant mice lack functional M-CSF and are deficient in osteoclasts and macrophages, but can be cured by injection of M-CSF (Wiktor-Jedrzejczak et al., 1991). In vitro, M-CSF enables proliferation and differentiation of committed bone marrow progenitors, the colonyforming unit macrophage (CFU-M), into macroscopic colonies of macrophages (Stanley et al., 1978). M-CSF also stimulates survival of monocytes and macrophages, and enhances their specialized functions (Stanley, 1981; Tushinsky et al., 1982). M-CSF receptor, encoded by the c-fms proto-oncogene (Sherr et al., 1985; Woolford et al., 1985), is a member of the type III receptor tyrosine kinase family, which includes α and β platelet-derived growth factor (PDGF), stem cell factor (SCF) and Flt3/FLK2 receptors. M-CSF binding to its receptor (Fms) induces dimerization of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of the cytoplasmic domain on specific tyrosine residues used as binding sites for Src homology 2 (SH2) domain-containing molecules that will initiate various signaling cascades (Rohrschneider, 1995). Four such proteins interacting directly with Fms have been described. Src family members can associate with phosphotyrosine 559 in the juxtamembrane Fms region (Alonso et al., 1995). Three molecules interact with the Fms kinase insert (KI): Grb2, the p85 subunit of phosphatidylinositol 3′ kinase (p85-PI3K) and phospholipase C-γ2 (PLCγ2). Grb2 binds to phosphotyrosine 697 (van der Geer and Hunter, 1993; Lioubin et al., 1994) and translocates the nucleotide exchange factor mSOS to the plasma membrane (Egan et al., 1993; Li et al., 1993). Both p85-PI3K and PLCγ2 bind to phosphotyrosine 721 (Reedijk et al., 1992; Bourette et al., 1997). In addition, Tyr706, another autophosphorylation site located in the KI, is necessary for STAT1 activation, although its direct interaction with STAT1 remains to be demonstrated (Novak et al., 1996). Other molecules not binding directly to Fms participate in early signaling complexes whose interactions are not well understood. Such molecules include Shc and Crk-II adaptors, SH2-containing inositol phosphatase SHIP and c-Cbl (Lioubin et al., 1994, 1996; Husson et al., 1997). Based on the numerous biological effects of M-CSF in monocytic development, we speculated that additional signal transduction proteins interact with activated Fms. We previously performed a yeast two-hybrid screen using the phosphorylated cytoplasmic domain of murine Fms as a bait, resulting in the demonstration of PLCγ2 interaction with phosphotyrosine 721 and its role in the differentiation signal (Bourette et al., 1997). In the same two-hybrid screen, we also identified a new SH2 domain related to Grb2. We have now cloned the full-length cDNA and characterized a novel signaling molecule of 38 kDa, whose expression is restricted to hematopoietic tissues in adult mouse (spleen and peripheral blood mononuclear cells). Furthermore, its expression was induced during monocytic differentiation of the bipotential hematopoietic cell line NFS-60. Due to the presence of an SH2, two SH3 domains and the absence of any catalytic domain, we postulate that this protein is an adaptor molecule involved in protein–protein interaction during M-CSF-dependent monocytic development. We have termed this protein ‘Mona’ for monocytic adaptor. Our results indicate that Mona interacts with Fms and participates in monocyte differentiation. Results Isolation of Mona, a novel adaptor interacting with the M-CSF receptor The yeast two-hybrid screen (Bourette et al., 1997) used a LexA–Fms cytoplasmic domain fusion protein as a bait and two different VP16 target libraries containing cDNAs derived from mouse embryos (Hollenberg et al., 1995) or from the murine hematopoietic cell line EML (Tsai et al., 1994). From this screen, four independent clones encoding a new SH2 domain with significant sequence similarity to the Grb2 SH2 domain were isolated from both libraries. Whereas three of these cDNAs encoded only the SH2 domain and short flanking sequences, a larger clone contained an extended open reading frame (ORF) 3′ to the SH2 domain followed by a large 3′-untranslated region. Since all four clones lacked the starting methionine, 5′ RACE-PCR was carried out on total RNA isolated from the NFS-60/MAC monocyte/macrophage cell line (Bourette et al., 1993) and mouse peripheral blood mononuclear cells (PBMNCs), two cell types expressing Fms, using a specific oligonucleotide primer corresponding to the N-terminal end of the SH2 domain. A 400 bp cDNA was then recovered with an ATG codon in an accurate context for translation initiation. Combining all sequence information led to a cDNA of 2929 nucleotides (accession No. AF055465) with a single ORF encoding a 322 amino acid protein (Figure 1A) with a calculated mol. wt of 36 787 Da. This protein contains an N-terminal SH3 domain (amino acids 1–56), an SH2 domain (amino acids 59–150), a proline-rich region (amino acids 167–216) with two P-XX-P putative SH3-binding sites, and a C-terminal SH3 domain (amino acids 264–322). No known catalytic domain was identified. This protein is most closely related to the human Grb2-related adaptor Grap (Feng et al., 1996) and the murine Grb2 adaptor (Suen et al., 1993), with 47 and 49% amino acid identities, respectively (Figure 1B). Interestingly, homologies concerned only the SH2 and both SH3 domains, whereas the proline-rich region is specific to our new protein. Based on structural features characteristic of adaptor proteins, its expression in a monocytic cell line and its functional properties as described below, this new protein was named Mona. During review of the manuscript, a DDBJ/EMBL/GenBank database homology search revealed a new cDNA sequence (accession No. AF053405) encoding a protein named Gads. Reported Mona and Gads cDNAs have different lengths (2929 and 1362 bp, respectively) but contain highly homologous (>99.5%) 1.3 kb sequences encoding identical proteins. Thus, Mona and Gads are probably products of the same gene. Figure 1.Amino acid sequence analysis of Mona. (A) Translation of the Mona-coding sequence with a schematic representation of the protein. Underlined bold characters represent the two putative SH3 domain-binding motifs (PxxP). (B) Alignment of the Mona sequence with murine Grb2 and human Grap proteins was performed using the ClustalX program. Regions of identity are outlined by gray boxes. Note that alignment is interrupted between the SH2 and C-terminal SH3 domains due to the proline-rich domain that is unique to Mona. Download figure Download PowerPoint In order to characterize Mona further, an antiserum was generated after injecting rabbits with a bacterial glutathione S-transferase (GST)–Mona SH2 fusion protein, while the entire Mona-coding sequence was inserted into the LXSN retroviral vector (Miller and Rosman, 1989) and transfected into ψ-2 packaging cells. By Western blotting with the antiserum, a protein of the expected size (38 kDa) was detected in ψ-2 cells transfected with L(Mona)SN vector but not in cells transfected with the empty vector (Figure 2). Furthermore, supernatants of ψ-2 cells were used to infect various hematopoietic cell lines, including the SCF-dependent pluripotent cell line EML (Tsai et al., 1994), interleukin-3 (IL-3)-dependent FDC-P1 cells overexpressing wild-type Fms (FD/wtFms cells; Bourette et al., 1995) and the M-CSF-dependent BAC1.2F5 cell line (Morgan et al., 1987). Western blotting analysis indicated that Mona could be expressed efficiently as a 38 kDa protein in hematopoietic cells using retroviral gene transfer (Figure 2). Figure 2.Immunoblot identification of Mona cDNA product in fibroblasts and various hematopoietic cell lines. Lysates of cells transfected with LXSN or L(Mona)SN vectors were run on a 12% polyacrylamide gel, blotted and probed with anti-Mona antiserum. Download figure Download PowerPoint Mona SH2 domain interacts in vitro with activated Fms via phosphorylated Tyr697 Using the GST–Mona SH2 domain fusion protein, we investigated the interaction between Mona and Fms. FD/wtFms cells were starved of growth factors and then either stimulated with M-CSF or left unstimulated. Cell lysates were mixed with the GST–Mona SH2 domain fusion protein coupled to glutathione–Sepharose beads, and interacting proteins were precipitated and probed with an anti-Fms antibody (Figure 3A). Under these conditions, the mature form of Fms was detected only when the GST fusion protein was mixed with lysate of M-CSF-stimulated FD/wtFms cells, showing that the Mona SH2 domain interacts in vitro with activated Fms. Accordingly, activated Fms was detected in Mona immunoprecipitates obtained from lysates of FD/wtFms cells overexpressing Mona (not shown). Figure 3.The Mona SH2 domain interacts with phosphorylated Tyr697 of the M-CSF receptor. (A) Lysates of FD/wtFms cells, stimulated (+) or not (−) for 1 h at 4°C with M-CSF, were mixed with GST–Mona SH2 fusion proteins immobilized on glutathione–Sepharose. Bound proteins were run on a 7.5% polyacrylamide gel, blotted and probed with anti-Fms antibody. (B) As in (A), except that FDC-P1 cells expressing different M-CSF receptors mutated on autophosphorylation sites were also used. For each cell lysate, immunodetection of Fms was performed on whole-cell lysate (upper panel) and on GST–Mona SH2-bound proteins (lower panel). (C) GST–KI Fms fusion proteins were produced as phosphoproteins in Epicurian cells, immobilized on glutathione–Sepharose and incubated with various cell lysates: FDC-P1 cells for PLCγ2 and p85 analysis, and Mona-overexpressing Ψ-2 cells for Grb2 and Mona analysis. Bound proteins were then subjected to Western blot analysis as specified in the figure. Download figure Download PowerPoint To determine which Fms autophosphorylation site is involved in this interaction, we performed similar experiments using lysates from M-CSF-stimulated FDC-P1 cells expressing various Fms autophosphorylation site mutants (Bourette et al., 1995). Cell lysates containing equal amounts of Fms protein were used (Figure 3B, upper panel). Mutations of tyrosines 706, 721 or 807 had no effect on Fms interaction with the Mona SH2 domain, but mutation of Tyr697 totally abolished the interaction (Figure 3B, lower panel), suggesting that Tyr697 is the Mona-binding site. In order to confirm this result, we performed the mirror experiment using GST fusion proteins containing either the wild-type Fms KI or KI mutated at different autophosphorylation sites (Y697, Y706 and Y721). Fusion proteins were expressed in the Epicurian TKX1 bacterial strain that enables tyrosine phosphorylation. Then, phosphorylated fusion proteins immobilized on glutathione–Sepharose were mixed with cell lysates of FDCP-1 cells or Mona-overexpressing ψ-2 cells. The presence of Mona and known Fms KI-binding molecules (PLCγ2, p85-PI3K and Grb2) among precipitated proteins was then examined by Western blotting using the relevant antibodies (Figure 3C). As expected, mutation of Tyr697 abolished Fms interaction with Grb2 adaptor (van der Geer and Hunter, 1993), and mutation of Tyr721 abolished Fms interaction with both p85 (Reedijk et al., 1992) and PLCγ2 (Bourette et al., 1997). In addition, consistent with the results shown in Figure 3B, mutation of Tyr697 abolished the interaction between Fms and Mona, demonstrating that Tyr697 is necessary and sufficient for binding of Mona to activated Fms. Mona expression is restricted to some hematopoietic cells Various tissues and cell lines were probed for Mona expression by Western blotting using anti-Mona SH2 antibody. Among all samples tested, only spleen and PBMNCs expressed the 38 kDa protein, whereas testis and liver unexpectedly expressed a 44 kDa protein (Figure 4A). In order to establish the relationship between this 44 kDa protein and Mona, RT–PCR analyses were performed on a selected set of tissues, using the primers specific to the Mona SH2 domain since our antiserum recognizes the same Mona protein region. Only those tissues expressing the 38 kDa protein (PBMNCs and spleen) gave a positive signal, whereas 44 kDa protein-expressing tissues (testis and liver) or non-expressing tissues (brain and kidney) were found to be negative (Figure 5A). We conclude that Mona is expressed in vivo as a single 38 kDa protein, and that the 44 kDa protein found in testis and liver is different from Mona, although both proteins are immunologically related. Figure 4.Western blotting analysis of Mona expression in various murine tissues and cell lines. (A) Expression in selected mouse tissues (PBMNCs, peripheral blood mononuclear cells). Other tissues tested that were negative for Mona expression included bone, heart, intestine, muscles, ovary, placenta, skin, stomach and uterus. (B) Expression in various cell lines, including multipotential cell lines (EML, FDCP-Mix and NFS-60), committed myeloid (FDC-P1, 32D), erythroid (E31, MEL), lymphoid (FL5.12, BaF/3 and CTLL-2) and monocyte/macrophage (NFS-60/MAC and BAC1-2F5) cell lines, fibroblasts (Rat-2 and NIH 3T3), pheochromocytoma cells (PC12), stroma cells (MS-5) and hepatocytoma cells (HepG2). (C) Expression in highly enriched T lymphocytes (CD4+ or CD8+ cells) and monocytes. All analyses were performed with 20 μg of protein loaded per lane, except for CD4+ cells (14 μg) and CD8+ cells (8 μg). Download figure Download PowerPoint Figure 5.Analysis of Mona mRNA expression in tissues and cell lines. (A) RT–PCR detection of transcripts was performed using primers specific to the Mona SH2 domain. HPRT was used as a control. (−) indicates analysis performed in the absence of RNA. (B) Northern blot analysis with 20 μg of total RNA loaded per lane. Membrane was hybridized with a 1.1 kb 32P-labeled probe corresponding to the entire Mona-coding sequence. The estimated size of the various transcripts is indicated on the right (in kb). The lower panel shows ethidium bromide staining of the gel before transfer onto nylon membrane and indicates that the PBMNC sample was underloaded. Download figure Download PowerPoint Since it is apparently restricted to some hematopoietic tissues, Mona expression was examined using a large panel of hematopoietic cell lines, including multipotent cells and committed erythroid, myeloid or lymphoid cells. Western blot (Figure 4B) and RT–PCR (Figure 5A) analyses showed that Mona is expressed in the T-lymphoid cell line, CTLL-2, and in the monocyte/macrophage cell line NFS-60/MAC, an M-CSF-dependent derivative of bipotential NFS-60 cells (Bourette et al., 1993). Accordingly, PBMNC sorting enabled us to confirm Mona expression in T cells (CD4+ cells and CD8+ cells) and monocytes (Figure 4C). Finally, Mona expression was assessed by Northern blotting using full-length Mona cDNA as a probe. Four mRNA species (5.0, 3.9, 3.3 and 1.5 kb) hybridized with this probe but exhibited a specific expression pattern (Figure 5B). The 3.9 and 1.5 kb species were found in all samples, whereas the 5.0 and 3.3 kb species were detected in Mona-expressing cells (PBMNCs, NFS-60/MAC and CTLL-2 cells), but not in Mona-negative cells (brain, testis, fibroblasts and NFS-60 cells). This clearly indicates that 5.0 and 3.3 kb mRNAs are products of Mona gene transcription. Moreover, since Mona and Grb2 cDNAs are strongly homologous (69% overall) and Grb2 is ubiquitously expressed as two mRNA species of 3.9 and 1.5 kb (Suen et al., 1993), these mRNAs may represent Grb2 mRNAs. Mona expression is induced during monocytic differentiation of NFS-60 cells Mona is expressed in the NFS-60/MAC cell line, an M-CSF-dependent variant of the NFS-60 line, which otherwise does not express Mona (Figures 4B and 5). Interestingly, whereas NFS-60 cells are IL-3-dependent bipotential progenitor cells and do not express Fms, NFS-60/MAC cells exhibit a monocyte/macrophage phenotype and strongly express Fms (Bourette et al., 1993), suggesting that Mona expression is induced during monocyte/macrophage differentiation. To test this hypothesis, we used NFS-60 cells transduced with murine Fms (N-Fms cells), which can differentiate to monocytes within 5 days of culture in the presence of M-CSF (Bourette et al, 1993; G.Pawlak, C.Valadoux-Delplanque, V.Revol, R.P.Bourette, J.P.Blanchet and G.Mouchiroud, in preparation). When maintained in the presence of IL-3, N-Fms cells, like parental NFS-60 cells, do not express Mona protein (Figure 6A, t0). However, Mona protein expression was induced in N-Fms cells after 1 day of culture in the presence of M-CSF, and then increased until day 5 (Figure 6A). RT–PCR showed that Mona expression was indeed induced very rapidly during the first day of culture, with a significant and reproducible expression signal after 8 h in the presence of M-CSF (Figure 6B). Induction of Mona in N-Fms cells was not a consequence of IL-3 withdrawal prior to M-CSF stimulation since it was also obtained in the response to both IL-3 and M-CSF (data not shown). Taken together, these observations suggest a role for Mona during development of the monocytic lineage as controlled by M-CSF. Figure 6.Mona expression is induced during monocytic differentiation of N-Fms cells. (A) Cells were starved of IL-3, cultivated in the presence of M-CSF (2500 U/ml) for different times as indicated, then lysed and analyzed for Mona or Grb2 expression by Western blotting (Grb2 was used as a control). (B) Cells were starved of IL-3, stimulated for different times at 37°C with M-CSF (2500 U/ml), and then total RNA was extracted. Mona transcripts were detected by RT–PCR performed using primers specific to the Mona SH2 domain. PBMNCs were used as a positive control for Mona expression. HPRT was used as a standard. Download figure Download PowerPoint Overexpression of Mona in mouse bone marrow cells strongly decreases macrophage production in vitro In order to look for its possible role during monocytic differentiation, Mona protein was first overexpressed in M-CSF-responsive cell lines that do not normally express the protein, such as BAC1.2F5 and FD/wtFms. Although high expression levels were obtained (Figure 2), Mona overexpression had no effect on cell response to M-CSF, whether it was proliferation or differentiation which was assessed (not shown). We then used the physiologically more relevant experimental system provided by bone marrow progenitor cells differentiating in vitro to macrophages in response to M-CSF. Differentiation of monocyte/macrophage progenitors (CFU-M) can then be assessed by macrophage colony formation in agar culture (Stanley et al., 1978) or by the production of macrophages in liquid cultures (Tushinsky et al., 1982). For these experiments, murine bone marrow cells (BMCs) infected by Mona-expressing vector [L(Mona)SN], or by empty LXSN vector as a control, were plated in agar cultures. Viral titers determined on NIH 3T3 cells were 5×105 and 2×105 viral particles/ml for ψ-2/LXSN or ψ-2/L(Mona)SN cells, respectively. This 2.5:1 ratio of LXSN and L(Mona)SN titers was also obtained using myeloid FDC-P1 cells as a target. Infected BMCs were plated in soft agar cultures containing G418 and M-CSF (1000 U/ml), and macrophage colonies were counted after 10 days. Colony numbers were significantly lower in L(Mona)SN cultures than in LXSN cultures. Taking into account the difference in virus titers between LXSN and L(Mona)SN Ψ-2 cell supernatants, we calculated an average 6.6-fold decrease in CFU-M development when Mona was overexpressed (Figure 7A). In liquid cultures containing G418 and M-CSF (1000 U/ml), infected BMCs proliferated and differentiated into adherent macrophages which were enumerated after 10 days of culture. Similarly to the agar assay results, we found an 8.5-fold reduction in macrophage number in L(Mona)SN cultures as compared with LXSN control cultures (Figure 7B). Examination of May–Grünwald–Giemsa-stained preparations revealed no significant morphological difference between macrophages obtained from these cultures (data not shown). These results demonstrate that overexpression of Mona in mouse bone marrow cells strongly decreases macrophage production in vitro. Figure 7.Overexpression of Mona in mouse bone marrow cells (BMCs) strongly decreased macrophage production in vitro. BMCs were infected with viral supernatant of ψ-2/LXSN or ψ-2/L(Mona)SN cells. Infected BMCs were washed and plated in agar (1–4×105 cells/ml) or liquid (1.5×106 cells/flask) cultures containing 10% FBS, G418 (1 mg/ml) and M-CSF (1000 U/ml). Macrophage colonies (A) and bone marrow-derived macrophages (B) were counted after 10 days. Colony or macrophage numbers were normalized based on the ratio between the viral titers of LXSN and L(Mona)SN. Data represent mean values ± standard error of the mean of three independent experiments. Download figure Download PowerPoint Discussion We have isolated a novel M-CSF receptor-binding molecule in a yeast two-hybrid screen. This 38 kDa protein was named Mona for monocytic adaptor because: (i) its SH3- and SH2-containing structure, closely related to that of Grb2, is typical of a molecular adaptor; (ii) its SH2 domain interacts with the M-CSF receptor which controls monocytic development; and (iii) its expression is induced during monocytic differentiation. Mona contains one SH2 domain and two SH3 domains closely related to the corresponding domains of Grb2 and Grap adaptors (Suen et al., 1993; Feng et al., 1996). No homology to known catalytic domains was found. However, an additional proline-rich region located between the SH2 and the C-terminal SH3 domain is unique to Mona. This region contains two PxxP motifs which represent the minimum binding site for an SH3 domain (Alexandropoulos et al., 1995). This would raise the interesting possibility that Mona may be able to establish more complex molecular interactions with signaling molecules than can Grb2 or Grap. Alternatively, recognition of these polyproline motifs by either Mona SH3 domain would provide a mechanism for regulating Mona activity by intramolecular interactions, as recently shown for the Hck kinase (Moarefi et al., 1997). Using GST fusion proteins, we demonstrated that Mona associates with Fms Tyr697, which is also the Grb2-binding site, suggesting that Mona may compete with Grb2 for binding to activated Fms. Due to their structural similarity, Mona and Grb2 might also compete for downstream signaling molecules, as previously demonstrated for the Grb2 isoform Grb3-3 that acts as an inhibitor of Grb2 and has a dominant-negative effect on the Ras pathway by forming inactive heterodimeric complexes with Sos (Fath et al., 1994). Since Grb2 binding to Fms results in translocation of Sos to the membrane and subsequent Ras activation (van der Geer and Hunter, 1993; Lioubin et al., 1994), our biochemical data raise the possibility that Mona functions as a Grb2 competitor thereby modulating the Ras pathway. It will now be important to determine the respective affinities of Grb2 and Mona for Fms and to analyze the interactions of Mona with known Grb2-binding partners to validate this competition model. Several hematopoietic-specific adaptor proteins have been reported recently, such as Lnk (Huang et al., 1995), LAT (Zhang et al., 1998) and TSad (Spurkland et al., 1998). Similarly, Mona is expressed only in spleen and PBMNCs of adult mice. Furthermore, using a representative selection of hematopoietic cell lines, we detected Mona in the monocyte/macrophage NFS-60-MAC cell line, but also in the T-lymphoid CTLL-2 cell line. Consistent with this observation, we found Mona in peripheral blood T cells and monocytes. Since it is expressed in circulating T cells, but not in thymocytes, Mona could be involved in T-lymphocyte development
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