TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis
1998; Springer Nature; Volume: 17; Issue: 15 Linguagem: Inglês
10.1093/emboj/17.15.4442
ISSN1460-2075
Autores Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoArticle3 August 1998free access TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis Danilo Perrotti Corresponding Author Danilo Perrotti Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Silvia Bonatti Silvia Bonatti Department of Biomedical Sciences, University of Modena, Italy Search for more papers by this author Rossana Trotta Rossana Trotta Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Robert Martinez Robert Martinez Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Tomasz Skorski Tomasz Skorski Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Paolo Salomoni Paolo Salomoni Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Emanuela Grassilli Emanuela Grassilli Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Renato V. Iozzo Renato V. Iozzo Department of Pathology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, 19107 USA Search for more papers by this author Denise R. Cooper Denise R. Cooper Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine and the James A.Haley Veterans Hospital, Tampa, FL, 33612 USA Search for more papers by this author Bruno Calabretta Corresponding Author Bruno Calabretta Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Danilo Perrotti Corresponding Author Danilo Perrotti Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Silvia Bonatti Silvia Bonatti Department of Biomedical Sciences, University of Modena, Italy Search for more papers by this author Rossana Trotta Rossana Trotta Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Robert Martinez Robert Martinez Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Tomasz Skorski Tomasz Skorski Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Paolo Salomoni Paolo Salomoni Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Emanuela Grassilli Emanuela Grassilli Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Renato V. Iozzo Renato V. Iozzo Department of Pathology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, 19107 USA Search for more papers by this author Denise R. Cooper Denise R. Cooper Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine and the James A.Haley Veterans Hospital, Tampa, FL, 33612 USA Search for more papers by this author Bruno Calabretta Corresponding Author Bruno Calabretta Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA Search for more papers by this author Author Information Danilo Perrotti 1, Silvia Bonatti2, Rossana Trotta1, Robert Martinez1, Tomasz Skorski1, Paolo Salomoni1, Emanuela Grassilli1, Renato V. Iozzo3, Denise R. Cooper4 and Bruno Calabretta 1 1Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA, 19107 USA 2Department of Biomedical Sciences, University of Modena, Italy 3Department of Pathology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, 19107 USA 4Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine and the James A.Haley Veterans Hospital, Tampa, FL, 33612 USA *Corresponding authors. E-mail: per[email protected] or E-mail: [email protected] The EMBO Journal (1998)17:4442-4455https://doi.org/10.1093/emboj/17.15.4442 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCβII-dependent FUS phosphorylation. Moreover, suppression of PKCβII activity in BCR/ABL-expressing cells by treatment with the PKCβII inhibitor CGP53353, or by expression of a dominant-negative PKCβII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) β-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression. Introduction The BCR/ABL fusion genes of the Philadelphia chromosome (Ph1), encode the p210bcr/abl or p185bcr/abl oncoproteins that transform immature hematopoietic cells in vitro (McLaughlin et al., 1987) and cause chronic myelogenous leukemia (CML)-like syndromes in mice (Daley et al., 1990; Heisterkamp et al., 1990). The ability of BCR/ABL oncoproteins to transform hematopoietic cells depends on their tyrosine kinase activity (Lugo et al., 1990), which is essential to recruit and activate multiple biochemical pathways that transduce oncogenic signals (Cortez et al., 1995). Thus, the identification of signaling molecules regulated by BCR/ABL proteins is essential to elucidate the mechanism(s) underlying the leukemogenic process. While the role in leukemogenesis of certain cytoplasmic downstream effectors, such as RAS and PI-3K (Goga et al., 1995; Sawyers et al., 1995; Skorski et al., 1997), is understood in some detail, much less is known about the nuclear effectors activated by BCR/ABL and the mechanisms by which they contribute to the phenotype of BCR/ABL-transformed cells. Nuclear effectors of BCR/ABL might include myeloid-specific zinc finger proteins, because of their involvement in hematopoietic differentiation and leukemogenesis (Tenen et al., 1997). Using electrophoretic mobility shift assay (EMSA) on lysates from BCR/ABL-expressing cells and a double-stranded oligonucleotide containing a zinc finger consensus sequence recognized by fingers 1–4 of the myeloid zinc finger protein 1 (MZF-1) transcription factor (Morris et al., 1994), we detected a DNA–protein complex whose formation depends on the tyrosine kinase activity of the p210BCR/ABL oncoprotein. Additional studies led to the identification of FUS as the only protein of this BCR/ABL-regulated DNA–protein complex. FUS was first discovered as the N-terminal part of a fusion gene with CHOP in myxoid liposarcoma carrying the translocation t(12;16) (Crozat et al., 1993; Rabbitts et al., 1993). Homologies were found with the EWS oncogene, which is rearranged in Ewing sarcomas and other neoplasia (Aman et al., 1996). In the t(16;21) translocation, detected in different types of human myeloid leukemia (Shimizu et al., 1993), the C-terminal region of FUS is replaced by the DNA-binding domain of ERG (Panagopoulos et al., 1995). In other chromosomal translocations, the Nterminal region of FUS or of the homologous EWS gene is fused to the DNA-binding domain of one of several transcription factors such as FLI1, ERG, ATF1, CHOP and WT1, to generate oncogenes with high transforming potential (Ladanyi, 1995; Ron, 1997). While the oncogenic forms of FUS (fused to CHOP or ERG) have been the subject of intense investigation since their identification in myxoid liposarcoma and myeloid leukemias, much less is known about the function of its normal cellular form (Ron, 1997). The C-terminus of FUS contains a highly conserved region of 80 amino acids, the ribonucleoprotein consensus sequence (RNP-CS) (Crozat et al., 1993), an RNA recognition motif (RRM) flanked by Arg-Gly-Gly (RGG) repeats and a C2-C2 zinc finger domain homologous in structure to that found in the RNA binding proteins snRNP-associated protein 69KD and RBP56 (Hackl and Luhrmann, 1996; Morohoshi et al., 1996). The N-terminal domain of FUS contains instead a sequence of glutamine- and proline-rich degenerate hexapeptide repeats, which resembles the transcriptional activation domain of Sp-1 (Courey and Tjian, 1988). The C-terminal region is required, both in vitro and in vivo, for FUS binding to pre-mRNA and mRNA (Crozat et al., 1993; Zinszner et al., 1997a), while the FUS N-terminus functions as a potent transcriptional activation domain necessary for the oncogenic potential of FUS–CHOP or FUS–ERG proteins (Crozat et al., 1993; Prasad et al., 1994). FUS is expressed at high levels in hematopoietic and non-hematopoietic tissues (Aman et al., 1996; Morohoshi et al., 1996) and is primarily localized in the nucleus (Crozat et al., 1993), where it might be involved in nucleo-cytoplasmic shuttling (Zinszner et al., 1997b). Like the homologues hTAFII68, EWS and Drososphila SARFH protein, FUS might function as a basal transcription regulator, as suggested by its presence in RNA pol II transcription complexes (Zinszner et al., 1994; Immanuel et al., 1995; Bertolotti et al., 1996). The association of FUS with products of RNA pol II transcription is dependent on ongoing transcription and leads to the formation of large ternary complexes with other heterogeneous nuclear RNA-binding proteins (hnRNPs) such as hnRNP A1 and C1/C2 (Zinszner et al., 1994). Consistent with its involvement in pre-mRNA processing and mRNA export, FUS has been independently identified as the hnRNP P2 protein (Calvio et al., 1995). Despite the wealth of information on structure, RNA-binding or single-stranded (ss) DNA-binding specificity, and cellular distribution (Ron, 1997), little is known about the mechanisms regulating FUS expression and function. In this study, following the identification of FUS in the BCR/ABL-regulated DNA–protein complex, we have analyzed the BCR/ABL-dependent pathway(s) leading to FUS activation and assessed the functional consequences of interfering with FUS expression. We report here that a PKCβII-dependent pathway is required for the activation of FUS and that FUS activity is important for the growth factor independence and reduced propensity for differentiation of BCR/ABL-transformed cells. Results Characterization of two BCR/ABL-regulated DNA binding proteins (p55 and p40) The phenotype of hematopoietic cells expressing the BCR/ABL oncoproteins includes reduced susceptibility to apoptosis, growth factor-independent proliferation and differentiation arrest. To assess whether BCR/ABL regulates the activity of zinc finger transcription factors with a potential role in hematopoietic cell differentiation, a double-stranded oligodeoxynucleotide (dsODN ZnSab: 5′-ttttctccccacttttagatc-3′) containing a canonical zinc finger consensus sequence recognized by the MZF-1 transcription factor (Morris et al., 1994; Perrotti et al., 1995) was tested in EMSA for its ability to bind proteins in cell extracts from two interleukin-3 (IL-3)-dependent murine myeloid cell lines, 32Dcl3 and DAGM, expressing wild-type p210BCR/ABL or not. Two complexes, C1 and C2, were detected in lysate of both cell lines cultured with IL-3 or IL-3-starved 32Dcl3 cell lysate (Figure 1A, lanes 2–5 and lanes 6 and 7, respectively). Formation of the C2 complex was specifically induced in BCR/ABL-expressing cells, while the C1 complex was more clearly detectable in parental cells. Both complexes were resistant to sodium deoxycholate concentrations (0.2–0.8%) that disrupt protein–protein interaction or to formamide treatment (20–30%) (not shown), suggesting that a complex of multiple proteins in which only one interacts with the DNA is not involved in the binding to dsODN ZnSab. Moreover, the presence of bivalent cations in the binding reaction was required for C2 complex formation (not shown). Figure 1.Characterization and purification of the dsODN ZnSab binding proteins. (A) EMSA performed with 32P-labeled dsODN ZnSab and extracts from parental and BCR/ABL-expressing DAGM and 32Dcl3 cells. (B) Left panel, in situ UV cross-linking analysis of C1 and C2 complexes; middle panel, Southwestern assay with 32P-labeled dsODN ZnSab on extracts from IL-3-deprived parental and BCR/ABL-expressing 32Dcl3 cells; right panels, EMSA with 32P-labeled dsODN ZnSab and 30 μg cell extracts (T) from 32Dp210BCR/ABL cells (lane 5) or 25 ng of ZnSab affinity-purified proteins (E2.3) (lane 6); silver staining of the affinity-purified fraction E2.3 (lane 7). Download figure Download PowerPoint In in situ UV cross-linking experiments using the ZnSab probe and cell extracts from parental and BCR/ABL-expressing 32Dcl3 cells, the two complexes resolved as two distinct species migrating with apparent molecular masses of 40 and 55 kDa, respectively (Figure 1B, lanes 1 and 2). Southwestern analysis indicated that the two complexes derived from the interaction of the dsODN ZnSab with proteins migrating in SDS–PAGE at ∼40 and 68 kDa, respectively (Figure 1B, lanes 3 and 4). Purification of total lysate from 32Dp210BCR/ABL cells by size-fractionation and DNA-affinity chromatography using a column prepared with the multimerized dsODN ZnSab, but not with a 35mer unrelated double-stranded ODN (5′-ggggtccccccttactggactcaggttgccccctg-3′), yielded two DNA binding proteins which were enzymatically cleaved and sequenced by mass spectrometry (Figure 1B, lanes 5–7). Computer search for homology to the peptide sequences p55(1): LKGEATVSFDDPPSAK; p55(2): AAIDWFDGK; and p40(1): IFVGGLSPDTPEEK, identified p55 as the murine homologue of FUS, while p40 was identical to the hnRNP C1/C2. Direct proof that the C2 complex detected in EMSA with lysates of parental and BCR/ABL-expressing 32Dcl3 cells (Figure 2A, lanes 1 and 2) was due to the interaction of FUS with dsODN ZnSab was obtained by observing a supershift with a polyclonal FUS antiserum (Figure 2A, lanes 3 and 4), but not with an unrelated polyclonal antiserum (Figure 2A, lane 5). Figure 2.Sequence specificity of FUS DNA-binding activity. (A) EMSA with 32P-labeled dsODN ZnSab and lysates from parental (lanes 1 and 3) and BCR/ABL-expressing 32Dcl3 cells (lanes 2 and 4–9). Assays were performed in the presence of a polyclonal FUS antiserum (lanes 3, 4 and 7–9) or in the presence of an irrelevant polyclonal antiserum (lane 5). Cold ssODN ZnSa (lane 8) or ssODN ZnSb (lane 9) was added (100-fold molar excess) to the reaction to determine binding specificity. s.s. indicates the supershifted complex. (B) EMSA performed on extracts from BCR/ABL-expressing 32Dcl3 cells and wild-type 32P-labeled ZnSab or mutated (ZnMut1, ZnMut2 and ZnMut3) dsODNs used as probes or as competitors. Representative of three different experiments with similar results. (C) EMSA performed on extracts from parental and BCR/ABL-expressing 32Dcl3 cells with 32P-labeled ZnSa or ZnSb, or with 32P-labeled ZnSab in the absence (lane 7) or in the presence of increasing concentration (5-, 10-, 50- and 100-fold molar excess) of cold ZnSa or ZnSb used as competitor. (D) EMSA performed with 32P-labeled dsODN ZnSab or ssODN ZnSb, and increasing amount of lysates (1, 2.5, 5, 7.5 and 15 μg) from BCR/ABL-expressing 32Dcl3 cells (lanes 1–5 and 10–14, respectively). Assays using 15 μg of lysates were also performed in the presence of increasing concentrations (5-, 10-, 50- and 100-fold molar excess) of cold ZnSb (lanes 6–9) or ZnSab (lanes 15–18). Download figure Download PowerPoint To investigate sequence requirements for the dsODN ZnSab–FUS interaction, EMSA were performed with dsODN ZnSab mutated in the zinc finger motif (ZnMut1) or in the nucleotides flanking it (ZnMut2 and ZnMut3) used as probes or as competitor of 32P-labeled wild-type dsODN ZnSab. The C2 complex was competed by ZnMut1, but not by ZnMut2 or ZnMut3 (Figure 2B); conversely, only the 32P-labeled ZnMut1 formed the C2 complex upon incubation with lysate from BCR/ABL-expressing 32Dcl3 cells (Figure 2B). Since FUS reportedly binds mRNA and ssDNA (Prasad et al., 1994; Zinszner et al., 1997b), we investigated whether FUS binding to ssDNA is also sequencedependent. By EMSA, a complex similar to that formed with dsODN ZnSab was detected in extracts of BCR/ABL-expressing 32Dcl3 cells with the 32P-labeled ssODN ZnSb (Figure 2C, lane 4), but not with the complementary ZnSa (Figure 2C, lane 2). Specificity of the ZnSb–FUS interaction was confirmed in competition experiments in which an excess of ssODN ZnSb, but not ssODN ZnSa, abrogated the detection of the ZnSab–FUS–FUSAb or the ZnSab–FUS complex (Figure 2A, lanes 6–9 and 2C, lanes 7–15, respectively). The relative affinities of FUS for ZnSb and ZnSab were investigated by EMSA in which the ZnSab or the ZnSb probes were incubated with increasing amounts of lysate from BCR/ABL-expressing 32Dcl3 cells (Figure 2D, lanes 1–5 and 10–14, respectively), or with increasing amounts of cold ssODN ZnSb or dsODN ZnSab used as competitor (Figure 2D, lanes 6–9 and 15–18, respectively). The results of these experiments indicate that FUS has a higher affinity for ssODN ZnSb than for dsODN ZnSab. FUS expression and DNA binding activity in BCR/ABL-expressing cells To assess whether induction of FUS binding activity in BCR/ABL-expressing 32Dcl3 cells was associated with enhanced expression of FUS, Western blot analysis was performed on lysates from parental 32Dcl3 cells and from cells stably or transiently expressing p210BCR/ABL. Transient expression of p210BCR/ABL was achieved in retrovirus-infected cells (72 h post-infection) in experiments designed to determine whether enhanced FUS expression is an early change in BCR/ABL-expressing cells. FUS was readily detectable in IL-3-starved BCR/ABL-expressing 32Dcl3 cells, while in parental 32Dcl3 cells it was detected exclusively when cells were maintained in the presence of IL-3 (Figure 3A), suggesting that BCR/ABL expression circumvents the requirement for signals generated in non-transformed cells by IL-3–interleukin-3 receptor (IL-3R) interaction. Figure 3.FUS expression and binding activity in murine and human BCR/ABL-expressing cells. (A) Left panel, Western blot shows FUS expression in total cell lysate (25 μg) from parental and BCR/ABL-expressing 32Dcl3 cells cultured in IL-3-containing medium (lanes 3 and 4) or after IL-3 starvation (lanes 1 and 2); right panel, FUS expression (second row) and binding activity (bottom row) in non-infected parental 32Dcl3 cells (lane 1), and in cells infected with wild-type BCR/ABL (lane 2) or an insert-less retrovirus (lane 3). (B) FUS expression and binding activity in lysates from IL-3-starved 32Dcl3 cells, normal and CML marrow cells, and from the Ph1-positive K562 and BV173 cell lines, as indicated. Actin and BCR/ABL were detected using an anti-Actin (Santa Cruz) or an anti-ABL (Ab3, Oncogene Science) antibody. Representative of three different experiments. Download figure Download PowerPoint FUS expression and its ability to bind the dsODN ZnSab correlated with BCR/ABL levels also in the Ph1-positive cell lines K562 and BV173 and in primary cells from patients with chronic phase- or blast crisis-CML (Figure 3B). Phosphorylation-dependence of FUS DNA binding activity in cells expressing a functional BCR/ABL tyrosine kinase EMSA of cell lysates from IL-3-starved 32Dcl3 cells stably or transiently expressing wild-type or kinase-deficient (p210K1172R) BCR/ABL revealed the C2 complex only in lysates from cells expressing wild-type p210BCR/ABL (Figure 4A, lanes 2 and 4). The absence of C2 complex in EMSA of 32Dp210K1172R extracts was not due to lack of BCR/ABL expression (Figure 4A, lanes 3 and 5), indicating that a tyrosine kinase-dependent pathway is required to maintain elevated levels of FUS expression and activity (Figure 4A). Indeed, FUS binding to dsZnSab was identical in IL-3-deprived 32Dcl3 cells expressing any of several BCR/ABL mutants (32Dp210ΔSH3, 32Dp210ΔSH2 and 32Dp210ΔBCR) lacking different portions of the BCR/ABL chimeric protein but retaining the tyrosine kinase activity and in cells transfected with wild-type BCR/ABL (not shown). Figure 4.Phosphorylation-dependent DNA binding activity of FUS. (A) EMSA with 32P-labeled dsODN ZnSab (top) and Western blots with anti-FUS, anti-ABL, and anti-Actin antibodies, on lysates from parental 32Dcl3 cells (lane 1), cells stably expressing wild-type p210BCR/ABL (lane 2) or kinase-deficient BCR/ABL protein (K1172R) (lane 3), and from 32Dcl3 cells transiently expressing wild-type p210BCR/ABL (lane 4) or the kinase-deficient BCR/ABL (K1172R) protein (lane 5). Cells were IL-3-starved for 8 h prior to lysis. (B) EMSA (upper panel) with 32P-labeled dsODN ZnSab and Western blot (middle panel) with the anti-FUS serum on IL-3-starved parental and BCR/ABL-expressing 32Dcl3 cells. When indicated, lysates were treated with AP in the presence or absence of phosphatase inhibitors. (C) Southwestern analysis with 32P-labeled dsODN ZnSab on 50 μg lysate from parental (lane 1) and BCR/ABL-expressing cells (lane 2), and on immunoprecipitates from BCR/ABL-expressing 32Dcl3 cells using the indicated anti-phosphoprotein Abs (lanes 4–6). Immunoprecipitates with an unrelated IgG were used as control (lane 3). (D) Lower panel, Western blots with anti-phosphoserine/ threonine (PSR-45 and PTR-8; Sigma Chemical Co.) antibody mixtures on total lysates and on FUS immunoprecipitates from IL-3-starved parental and BCR/ABL (wild-type or kinase deficient)-expressing 32Dcl3 cells; upper panel, Western blots with FUS antiserum on total lysates, FUS immunoprecipitates and protein G-preclearing performed as control. Immunoprecipitates with an irrelevant IgG were also used as control (not shown). Autoradiograms are representative of three experiments. Download figure Download PowerPoint Binding activity of FUS to dsODN ZnSab was abolished by treating the cell extracts with alkaline phosphatase (AP), and was restored by adding phosphatase inhibitors (see Materials and methods) to the binding reaction prior to AP treatment (Figure 4B). In agreement with these data, Southwestern analysis with the 32P-labeled dsODN ZnSab as probe on phosphoproteins immunoprecipitated from 32Dp210BCR/ABL cells using anti-Ptyr, anti-PSer and anti-PThr monoclonal antibodies detected the ∼68 kDa protein identified as FUS, which is overexpressed in 32Dp210BCR/ABL cells (Figure 4C, compare lanes 1 and 2). FUS binding activity was readily detected in the anti-phosphoserine immunoprecipitates (Figure 4C, lane 5), present at lower levels in the anti-phosphotyrosine and anti-phosphothreonine immunoprecipitates (Figure 4C, lanes 4 and 6), and undetectable in immunoprecipitates using an isotype-matched irrelevant antibody (Figure 4C, lane 3). Western blots with anti-phosphoserine/threonine antibody mixtures on FUS immunoprecipitates from IL-3-deprived parental and BCR/ABL (wild-type or kinase deficient)-expressing 32Dcl3 cells revealed a phosphorylated FUS protein (∼68 kDa) in 32Dp210BCR/ABL cells (Figure 4D, lane 5) but not in parental 32Dcl3 or 32Dp210K1172R cells (Figure 4D, lanes 4 and 6, respectively). In addition to FUS, a phosphoprotein of ∼80 kDa was detected in the anti-phosphoserine/threonine blot (Figure 4D, lane 5, lower panel). Dependence of FUS activation on a BCR/ABL-regulated PKCβII-kinase pathway To investigate mechanism(s) whereby BCR/ABL regulates FUS DNA-binding activity, EMSA were performed using dsODN ZnSab as probe and whole-cell extracts from IL-3-starved 32Dp210BCR/ABL cells treated with kinase or phosphatase inhibitors, some of which interfere with BCR/ABL-dependent pathways involved in cell survival and proliferation. The levels of the C2 complex (Figure 5A, lane 1) were markedly reduced after treatment with the serine-threonine kinase inhibitor staurosporine (1 μM) (Figure 5A, lane 6), or the specific protein kinase C (PKC) inhibitor calphostin C (200 ng/ml) (Tamaoki et al., 1991) (Figure 5A, lane 5), suggesting that a PKC-dependent pathway is involved in the regulation of FUS binding activity. The DNA binding activity of FUS was suppressed by treatment of BCR/ABL-expressing cells with the specific phospholipase C-γ (PLC-γ) inhibitor U73122 (1 μM), but not with its inactive U73343 (1 μM) derivative (Chen et al., 1996) (Figure 5A, lanes 4 and 3, respectively), and upon treatment with the Ca2+ chelator EGTA (1 mM), (Figure 5A, lane 8). Since PLC-γ activates certain PKC isoforms via an increase in the intracellular levels of Ca2+ and diacylglycerol (DAG), Western blots were performed on subcellular extracts from IL-3-cultured parental and BCR/ABL-expressing 32Dcl3 cells with an anti-cPKC antibody that recognizes the DAG- and Ca2+-dependent α, βI, βII and γ isoforms. Compared with parental cells or cells expressing the kinase-deficient BCR/ABL, expression of the nuclear PKC isoforms was enhanced in wild-type BCR/ABL-expressing cells, upon culture in the absence of IL-3 (not shown). In contrast, the levels of other conventional PKC isoforms present in the cytosolic/membrane cell fraction were unchanged (not shown). In light of these observations and the fact that FUS is primarily localized in the nucleus, 32Dcl3 cells expressing wild-type BCR/ABL were treated with CGP53353 (10 μM), a specific inhibitor of the nuclear PKCβII isoform (Chalfant et al., 1996), to determine whether this kinase regulates the DNA binding activity of FUS. Indeed, EMSA assays revealed that the levels of the C2 complex were reduced after treatment of BCR/ABL-expressing cells with CGP53353 (Figure 5A, lane 7); moreover, a marked inhibition of C2 complex formation was also observed in EMSA performed with extracts from 32Dp210BCR/ABL cells transiently expressing a PKCβII dominant negative mutant (Chalfant et al., 1996) (Figure 5A, lane 10). Instead, FUS binding to dsODN ZnSab was not affected by treatment of 32Dp210BCR/ABL cells with other inhibitors such as the serine-threonine phosphatase inhibitor okadaic acid (50 nM), the p70S6kinase inhibitor rapamycin (15 nM) (Downward, 1994), the mitogen-activated protein (MAP) kinase inhibitor PD098059 (50 μM) (Dudley et al., 1995) and the phosphatidylinositol-3 kinase (PI-3K) inhibitor wortmannin (50 nM) (Powis et al., 1994) (Figure 4A, lanes 2 and 11–13). As expected, the levels of FUS binding to dsODN ZnSab in extracts from cells treated with each of these compounds correlated with that of PKCβII (Figure 5A, black bars), but not casein kinase II (CKII) (Figure 5A, gray bars) activity. Figure 5.Identification of a BCR/ABL-dependent PKCβII pathway involved in FUS activation. (A) Upper panel, FUS DNA binding activity in IL-3-deprived, untreated BCR/ABL-expressing 32Dcl3 cells (lane 1), or cells treated with okadaic acid (5 h) (lane 2), U73343 (3.5 h) (lane 3), U73122 (3.5 h) (lane 4), calphostin (5 h) (lane 5), staurosporine (5 h) (lane 6), CGP53353 (8 h) (lane 7), EGTA/MgCl2 (8 h) (lane 8), rapamicyn (5 h) (lane 11), PD098059 (5 h) (lane 12) and wortmannin (5 h) (lane 13). FUS binding activity was also assessed in IL-3-deprived BCR/ABL-expressing 32Dcl3 cells infected with an insert-less retrovirus (lane 9) or with a retrovirus carrying a dominant negative PKCβII (lane 10). Middle panel, PKCβII (black bars) and casein kinase II (CKII) (gray bars) activity in untreated or inhibitor-treated BCR/ABL-expressing 32Dcl3 cells. PKCβII was immunoprecipitated using a specific anti-PKCβII polyclonal serum (Santa Cruz). Representative of two experiments. Lower panels, FUS and actin expression (Western blotting) in untreated or inhibitor-treated BCR/ABL-expressing 32Dcl3 cells. (B) Left, PKCβII expression in parental, wild-type and kinase-deficient BCR/ABL-expressing 32Dcl3 cells. Right, PKCβII expression (Western blotting) in 32Dcl3 cells retrovirally infected with the insert-less vector or with wild-type (lane 8) or kinase deficient (K1172R) BCR/ABL. Seventy-two hours post-infection, cells were IL-3-deprived for 8 h and lysates were prepared as described (Perrotti et al., 1996). (C) Upper panel, detection of PKCβII (Western blotting) in FUS-immunoprecipitates (IP) from IL-3-deprived parental, wild-type, or kinase-deficient (K1172R) BCR/ABL-expressing 32Dcl3 cells; lower panel, FUS was detected (Western blotting) as control. (D) Upper panel, PKCβII kinase as
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