BMP-2 decreases Mash1 stability by increasing Id1 expression
2004; Springer Nature; Volume: 23; Issue: 17 Linguagem: Inglês
10.1038/sj.emboj.7600360
ISSN1460-2075
AutoresFrancesc Viñals, J. Reiriz, Santiago Ambrosio, Ramón Bartrons, José Luís Rosa, Francesc Ventura,
Tópico(s)NF-κB Signaling Pathways
ResumoArticle19 August 2004free access BMP-2 decreases Mash1 stability by increasing Id1 expression Francesc Viñals Francesc Viñals Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Julia Reiriz Julia Reiriz Departament d'Infermeria Fonamental i Medicoquirúrgica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Santiago Ambrosio Santiago Ambrosio Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ramon Bartrons Ramon Bartrons Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Jose Luis Rosa Jose Luis Rosa Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Francesc Ventura Corresponding Author Francesc Ventura Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Francesc Viñals Francesc Viñals Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Julia Reiriz Julia Reiriz Departament d'Infermeria Fonamental i Medicoquirúrgica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Santiago Ambrosio Santiago Ambrosio Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ramon Bartrons Ramon Bartrons Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Jose Luis Rosa Jose Luis Rosa Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Francesc Ventura Corresponding Author Francesc Ventura Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Author Information Francesc Viñals1, Julia Reiriz2, Santiago Ambrosio1, Ramon Bartrons1, Jose Luis Rosa1 and Francesc Ventura 1 1Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain 2Departament d'Infermeria Fonamental i Medicoquirúrgica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain *Corresponding author. Departament de Ciències Fisiològiques II, Unitat de Bioquímica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, 08907 L'Hospitalet de Llobregat, Spain. Tel.: +34 93 402 4281; Fax: +34 93 402 4268; E-mail: [email protected] The EMBO Journal (2004)23:3527-3537https://doi.org/10.1038/sj.emboj.7600360 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In neural development, bone morphogenetic proteins (BMPs) restrict neuronal differentiation, thereby promoting the maintenance of progenitor cells or even inducing astrocytogenesis. We report that exposure of neuroendocrine lung carcinoma cells to BMP-2 leads to a rapid decline in steady-state levels of Mash1 protein and some neuron-specific markers. BMP-2 induces a post-transcriptional decrease in Mash1 levels through enhanced degradation. We demonstrate that Mash1 protein stability is tightly regulated by the E47/Id1 expression ratio. Transient induction of Id1 by BMP-2 negatively correlates with Mash1 levels. Furthermore, an ectopic increase in Id1 levels is sufficient to induce degradation of either ectopic or endogenous Mash1, whereas expression of Mash1 in Id1-deficient cells or overexpression of E47 makes Mash1 levels refractory to the addition of BMP-2. Furthermore, we show that the E47/Id1 expression ratio also regulates CK2-mediated phosphorylation of Mash1 on Ser152, which increases interaction of Mash1–E47 heterodimers. We propose a novel mechanism in which the balance between Id and E protein levels regulates not only the transcriptional function but also protein stability of the neurogenic bHLH transcription factor Mash1. Introduction During differentiation, progenitor cells develop into more specialized phenotypes by highly specific changes in gene expression. The developmental maturation of precursor cells can be divided into at least two stages: the commitment of undifferentiated cells to a particular lineage, followed by terminal differentiation into a specific phenotype. Generally, the determination of cell fate depends on the activity of transcription factors. One of the best-known regulators of cell differentiation is the family of basic helix–loop–helix (bHLH) transcription factors. Most members of this family play a central role in the development of distinct mammalian cell lineages, including muscle and nerve cells. There is strong evidence that sequential expression of bHLH factors represents regulatory cascades in which early expressed factors modulate the appearance of others that are expressed later. For example, two members of the bHLH family, MyoD and myf-5, execute myogenic lineage determination, while myogenin and MRF4 appear to regulate the differentiation program (Rawls and Olson, 1997). Similarly, the bHLH factors Mash1, Math and neurogenin control neurogenesis in the central (CNS) and peripheral (PNS) nervous systems (Lee, 1997; Cai et al, 2000). Mash1 is expressed in proliferating neural precursors in restricted regions of the CNS and PNS, olfactory epithelium and neuroendocrine cells (Lo et al, 1991; Guillemot et al, 1993; Borges et al, 1997; Casarosa et al, 1999). Mice that are homozygous for targeted disruption of the Mash1 gene show fewer autonomic, enteric and olfactory neurons as well as neuroendocrine cells in the lung (Guillemot et al, 1993; Borges et al, 1997). A variety of neuroendocrine marker genes, such as Phox2a, tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH), are directly or indirectly regulated by Mash1 (Lo et al, 1998, 1999). For instance, disrupting Mash1 function by antisense treatment of small cell lung carcinoma (SCLC) cells results in a strong decrease in neuroendocrine markers, whereas overexpression of this gene in transgenic mice induces the appearance of neuroendocrine tumors from pulmonary epithelium (Linnoila et al, 2000). Mash1 and all the members of the bHLH family heterodimerize with ubiquitously expressed bHLH E proteins, such as E2A gene products, E12 and E47, through their HLH domain. Heterodimers bind to DNA through their basic domain and activate the transcription of genes that have a CANNTG sequence (E box) in their promoter region (Lee, 1997). Only one subfamily of HLH factors, known as Id proteins, lacks this basic region. Heterodimerization of Id proteins with bHLH is sufficient to block their DNA binding and function (Norton et al, 1998; Norton, 2000). Thus, Id family members are mainly known as negative regulators of the commitment and/or differentiation that the bHLH factors promote in muscle, lymphoid and neurogenic precursors (Sun, 1994; Spits et al, 2000; Kee et al, 2001). Four mammalian Id proteins, Id1 to Id4, that display partially overlapping expression patterns and certain levels of functional redundancy have been identified (Norton et al, 1998; Norton, 2000). This network of expression and function of transcription factors and their antagonists is ultimately controlled by cell-intrinsic programs and by extracellular signaling factors. Although many studies have focused on positive signals, negative molecular signals are equally important in balancing both the timing of differentiation and cell specification (Ross et al, 2003). Bone morphogenetic proteins (BMPs) are good candidates for molecules that act as negative regulators of neurogenesis (reviewed in Mehler et al, 1997). BMPs belong to the TGF-β superfamily of cytokines (Derynck and Zhang, 2003; Shi and Massagué, 2003). Each ligand of this family exerts its biological function by inducing the formation of heteromeric complexes of specific type I and II serine/threonine kinase receptors, which in turn propagate the signal inside the cell through phosphorylation of the Smad family of transcription factors. Smads1, 5 and 8 are mainly involved in BMP signaling, whereas Smads 2 and 3 function in TGF-β/activin signaling. Following phosphorylation, receptor-specific Smads hetero-oligomerize with Smad4, the only co-Smad isolated from mammals to date, and translocate into the nucleus. The third subfamily includes Smads 6 and 7, which are named I-Smads because of their ability to inhibit receptor-mediated signaling. Upon entry to the nucleus, Smads usually form complexes that contain sequence-specific DNA-binding factors and transcriptional coactivators or corepressors in order to achieve stable binding and transcriptional regulation (ten Dijke et al, 2000; Shi and Massagué, 2003; Derynck and Zhang, 2003). Interestingly, BMP-2 and leukemia inhibitory factor (LIF) synergistically promote neuroepithelial cells to become astrocytes through interaction between Smad1 and STAT3 (Nakashima et al, 1999). During embryonic neural induction, BMPs promote epidermal fate and suppress neural fate in the developing ectoderm (Wilson and Hemmati-Brivanlou, 1995). These cytokines promote glial differentiation in late embryonic or adult CNS precursors and simultaneously inhibit neurogenesis (Gross et al, 1996; Shou et al, 1999; Nakashima et al, 1999, 2001; Lim et al, 2000). However, BMPs also cause apoptosis in CNS precursor cells and induce neurogenesis in neural crest stem cells or cortical progenitors precursors (Shah et al, 1996). The mechanism by which BMPs mediate this wide variety of responses depending on the time of development is unclear. However, it may result from a diversity of signal transduction components that allow distinct cell populations to respond differentially to gradients of BMP concentration. In addition, compared with this relatively strong phenomenological evidence, little is known about the molecular mechanisms that mediate the antineurogenic effects of BMPs. Therefore, we studied the molecular mechanisms involved in the antineurogenic function of BMPs in SCLC cells with a neuroendocrine phenotype. We show that BMP-2 decreases the expression of neuroendocrine markers and causes a post-transcriptional decrease in Mash1 protein levels. Our data indicate that induction of Id proteins by BMP-2 is necessary and sufficient to block stabilization of Mash1 by means of direct competition between Mash1 and Id1 for binding to ubiquitous E proteins. We propose a novel mechanism where the balance between Id and E protein levels regulates not only function but also protein stability of tissue-specific bHLH transcription factors. Results BMP-2 downregulates neuroendocrine markers in small cell lung carcinoma cells To assess the effects of BMP-2 on the maintenance of the neuroendocrine phenotype, we first confirmed relatively high expression levels of several known neural markers in the SCLC cell line DMS53 (Borges et al, 1997). Addition of BMP-2 (5 nM) reduced the expression of TH and synaptophysin and did not affect other neural markers such as β-tubulin III or SNAP-25 (Figure 1A). Dose–response analysis of BMP-2 showed that TH protein levels were strongly decreased by BMP-2 stimulation at 5–10 nM. Similarly, to assess the temporal pattern of this response more accurately, we performed time-course assays indicating that both TH protein and mRNA reached minimal levels after 4–8 h and increased thereafter (Figure 1B). Interestingly, as previously described (Gómez-Santos et al, 2002), the related cytokine TGF-β had the opposite effects, and strongly increased TH expression. To further examine whether the regulation of TH by BMP-2 is mediated at the level of gene transcription, we transfected C17.2 cells with a reporter construct containing 4.3 kb of mouse TH promoter-enhancer region (Suzuki et al, 2002). Reporter activity was significantly reduced (up to 45% decrease with respect to control cells) in BMP-2-treated cells, indicating that the BMP-2 effects on TH protein levels were mainly due to a decreased transcriptional rate (Figure 1C). Figure 1.(A) DMS53 cells were treated with 5 nM BMP-2 for 6 or 12 h. Total extracts were obtained and analyzed by immunoblotting for the neural markers indicated. (B) DMS53 cells were treated for 4 h with the indicated amounts of BMP-2 or TGF-β (upper panel) or with 10 nM BMP-2 for the times indicated (lower panel). TH and actin expressions were analyzed by immunoblotting and/or semiquantitative RT–PCR. (C) C17.2 cells were cotransfected with a TH promoter-driven reporter construct and the combinations of Mash1 and/or E47 expression vectors indicated. Luciferase assay was performed after treatment for 16 h with 5 nM BMP-2 (filled bars) or no addition (empty bars). The results are expressed as mean±s.e.m. of triplicates from four independent transfections. (D) Northern blot analysis was performed with total RNA from DMS53 cells treated for the indicated times with 10 nM BMP-2 as described in Materials and methods. Download figure Download PowerPoint SCLC exhibits a neuroendocrine phenotype in tight association with Mash1 and Phox2 expression (Borges et al, 1997; Lo et al, 1999; Ito et al, 2000; Brosenitsch and Katz, 2002). Thus, we explored the transcriptional regulation of TH transcription by the Mash1/E47 bHLH protein complex and their response to BMP-2 (Figure 1C). As expected, overexpression of Mash1 increased reporter responses, which were further augmented by coexpression of E47. Addition of 5 nM BMP-2 decreased transcriptional responses in both cases, indicating that BMP-2 interferes with the function of bHLH transcriptional complexes. Post-transcriptional regulation of Mash1 expression by BMP-2 We analyzed modulation of endogenous levels of Mash1 mRNAs by BMP-2. We also analyzed the levels of Id1, a negative regulator of neurogenic bHLH transcription factors, which is induced by BMP-2 in several differentiation models (Nakashima et al, 2001; López-Rovira et al, 2002). BMP-2 induced a transient increase in Id1 mRNA levels. However, Mash1 mRNA levels showed no significant differences up to 6 h after BMP-2 addition (Figure 1D). BMP-2 inhibits neurogenesis in olfactory epithelium cultures through enhanced proteolytic degradation of Mash1. These effects are very rapid (2 h) and require new transcription of an early induced gene by BMP (Shou et al, 1999). Immunoblot analysis showed a strong decrease in Mash1 protein levels (up to 60% decrease at 2 h) followed by a recovery after 6 h (Figure 2A). Dose–response assays also indicate that concentrations of BMP-2 higher than 5 nM were required to produce a significant decrease (Figure 2C). These profiles provide a temporal link that indicates that the decrease in Mash1 levels precede those of TH. Interestingly, immunoblot analysis of Id1 protein levels showed a marked negative correlation between Id1 and Mash1 protein levels (Figure 2A and B). To further confirm that the decrease in Mash1 protein levels arose from a post-transcriptional mechanism, we generated DMS53 cells stably overexpressing an epitope-tagged version of Mash1 under a heterologous CMV promoter. Transcription from the CMV promoter is not modulated by BMP-2 up to 6 h (data not shown). BMP-2 effects were analyzed in two independent clones. Similar to endogenous Mash1, ectopic Mash1 expression was downregulated 2 h after BMP-2 addition (Figure 2D). Figure 2.(A) Cell extracts were obtained from DMS53 cells treated with 5 nM BMP-2 for the times indicated and analyzed by immunoblotting. (B) Quantifications of Mash1 (filled circles) and Id1 (filled squares) protein levels from five separate assays were performed using α-tubulin as normalization and were expressed as fold-change over control samples at time zero and expressed as mean±s.e.m. (C) DMS53 cells were treated for 2 h with the indicated amounts of BMP-2 and Mash1 expression was analyzed by immunoblotting. (D) Parental and two independent myc-Mash1 overexpressing clones were treated for 2 h with 5 nM BMP-2 and expressions of ectopic Mash1 (upper panel) or endogenous Mash1 (lower panel) were analyzed by immunoblotting. Download figure Download PowerPoint We then assayed N-Acetyl-Leu-Leu-Norleucinal (LLnL), which inhibits the activity of the 26S proteasome, for its ability to abolish BMP-induced Mash1 degradation. Addition of LLnL blocked the degradation of Mash1 after 2 h incubation with BMP-2 (Figure 3A). In order to confirm the ability of the ubiquitin system to target Mash1 for proteolytic degradation, HEK-293 cells were transfected with distinct combinations of myc-tagged Mash1 and His-tagged ubiquitin. Immunoblotting of Ni2+-NTA-purified complexes from cells cotransfected with these two constructs allowed us to detect higher molecular weight complexes containing ubiquitinated Mash1 (Figure 3B). Figure 3.(A) DMS53 cells were treated with 50 μM LLnL and/or 5 nM BMP-2 for 2 h and expression of Mash1 was analyzed by immunoblotting. (B) Extracts from HEK-293 cells transfected with the indicated expression constructs and treated overnight with 50 μM LLnL were subjected to immunoblotting to detect myc-Mash1 expression before (left panel) or after Ni2+-NTA-agarose purification (right panel). Download figure Download PowerPoint The ratio between Id1 and E47 levels modulates Mash1 protein stability These previous data led us to hypothesize that, in addition to inhibiting Mash1/E47 transcriptional complexes and function, the induction of Id proteins is responsible for the BMP-2-increased degradation of Mash1. To test this, HEK-293 cells were transfected with distinct combinations of myc-tagged Mash1, E47 and Id1 expression vectors. As previously described (Bounpheng et al, 1999; Shou et al, 1999; Sriuranpong et al, 2002), Mash1 and Id1 levels were strongly enhanced by the addition of LLnL, indicating that these proteins are quickly turned over in the cell (Figure 4A). More importantly, coexpression of Mash1 and E47 resulted in a profound stabilization of Mash1 in the absence of the proteasome inhibitor. Furthermore, Mash1 stabilization by E47 was partially lost by coexpression of Id1. We confirmed that E47 heterodimerizes with either Id1 or Mash1, whereas Mash1 and Id1 do not (Norton, 2000; Jögi et al, 2002; data not shown). To analyze the importance of Mash1–E47 heterodimer formation in Mash1 stabilization, we made similar assays using two E47 deletion mutants (E47 HLH, which contains bHLH, and E47 ΔHLH, defective in heterodimer formation; Voronova and Baltimore, 1990). The E47 mutant, which was unable to heterodimerize with Mash1, was also defective in Mash1 stabilization (Figure 4B). Interestingly, expression of a mutant form of Mash1, devoid of the bHLH region, was much less enhanced by the addition of LLnL, suggesting that bHLH might contain determinants for the rapid turnover of Mash1 (Figure 4B). We further confirmed the decreased Mash1 protein turnover in the heterodimeric state by pulse-chase assays in HEK-293 cells. Overexpression of E47 markedly enhanced the half-life of exogenous Mash1, which was about 3 h compared with only 50 min in cells that overexpressed Id1 either in combination with Mash1 alone or with both Mash1 and E47 (Figure 4C). These data indicate that Mash1 protein stability is strongly enhanced when Mash1 forms heterodimers with E proteins, which is competed by the presence of Id1. Figure 4.(A, B) Extracts from HEK-293 cells transfected with the expression constructs indicated, treated or not with 50 μM LLnL for 8 h, were analyzed by immunoblotting. (C) HEK-293 cells, transfected with the constructs indicated, were pulsed with [35S]methionine for 3 h and labeled proteins were chased in unlabeled media for different times. Labeled Mash1 was immunoprecipitated and visualized by SDS–PAGE. Quantification of Mash1 levels from three to four separate assays is expressed as mean±s.e.m. of percentage values of samples at time zero. Download figure Download PowerPoint To modulate Id1 protein levels independently of cytokine addition, we generated DMS53 clones that expressed murine Id1 under a tetracycline-responsive promoter (Tet off) (Chambard and Pognonec, 1998). Several clones resulted positive for tetracycline-regulated expression of exogenous Id1. Exogenous murine Id1 has a slightly higher electrophoretic mobility compared with human Id1, which allows us to discriminate easily between ectopic and endogenous Id1 expression (Figure 5A). We then performed time-course and dose–response analysis of Id1 induction and analyzed endogenous levels of Mash1 expression. When two independent clones were incubated with decreasing amounts of tetracycline, they displayed increasing levels of ectopic Id1 that correlated with decreasing amounts of Mash1 protein (Figure 5B). Similarly, parental and two Id1-overexpressing clones were incubated overnight with 30 ng/ml tetracycline in order to repress ectopic Id1 expression, and then depleted of tetracycline for a range of times. The induction profile of Id1 protein levels correlated with a decrease in Mash1 expression (Figure 5C). Figure 5.(A) Immunoblot analysis of tetracycline (Tet) responsiveness of several stable DMS53-Id1 clones. (B) Id1-overexpressing clones (Id16 and Id17) were incubated in the presence of 30 ng/ml of tetracycline overnight, and then incubated for 8 h in the presence of different concentrations of tetracycline. Expressions of Mash1 and Id1 were analyzed by immunoblotting. (C) Parental and two Id1-overexpressing clones (Id16 and Id17) were incubated overnight with 30 ng/ml of tetracycline in order to block ectopic Id1 expression. Then cells were deprived of tetracycline for the times indicated and expressions of Mash1 and Id1 were analyzed by immunoblotting. Download figure Download PowerPoint DMS53 cell clones, which stably overexpressed E47, were generated and analyzed (Figure 6A). Clones 10 and 17 showed an increase in the steady-state levels of endogenous Mash1 compared with wild-type cells (Figure 6A). More importantly, after addition of BMP-2 for 2 h, overexpressed E47 partially prevented the degradation of Mash1. Figure 6.(A) E47 expression of DMS53 cell clones, stably overexpressing E47, was analyzed by immunoblotting and is shown in the upper panel. Parental and two independent clones (E10 and E17) were treated for 2 or 4 h with 5 nM BMP-2 and expressions of endogenous Mash1 and Id1 were analyzed by immunoblotting (lower panel). (B) Wild-type or Id1−/− MEFs were transfected with the indicated expression constructs. After 24 h, cells were splitted and treated with 10 nM BMP-2 for 3 h. Expressions of Mash1, Id1 and tubulin were analyzed by immunoblotting. Download figure Download PowerPoint To examine the requirement of Id1 for the BMP-induced effects on Mash1 levels, we analyzed BMP-induced Mash1 degradation in Id1−/− mouse embryonic fibroblasts (MEFs). Wild-type as well as Id1−/− MEFs were able to respond to BMP-2. This was observed using a reporter containing the BMP-specific enhancer of the Id1 gene promoter (López-Rovira et al, 2002) (data not shown). When Mash1 was ectopically expressed in wild-type or Id1−/− MEFs, BMP-2-induced degradation of Mash1 was lost in Id1−/− MEFs (Figure 6B). These results indicate that the induction of Id1 is necessary and sufficient to mediate the BMP effects on Mash1 levels. E47-dependent phosphorylation on Ser152 increases Mash1 protein stability SDS–PAGE analysis of cell extracts from HEK-293 cells cotransfected with Mash1 and E47 showed that Mash1 appeared as two major species compared with a single band in extracts from cells transfected with Mash1 alone (Figure 4C). To determine whether the retarded band is due to phosphorylation events, we immunoprecipitated 35S-labeled Mash1 from cells cotransfected with E47 and tested the immunoprecipitates with alkaline phosphatase. Phosphatase treatment converted the upper form into the lower Mash1 species, indicating that the upper band was caused by phosphorylation events (data not shown). We next examined the effects of cotransfection of E47 and Id1 on Mash1 phosphorylation and E47 interaction. Cotransfection of E47 led to increased levels of phosphorylated forms of Mash1 and the appearance of co-immunoprecipitated E47, compared with cells transfected with Mash1 alone (Figure 7A). In contrast, cotransfection of E47 plus Id1 partially reverted the appearance of phosphorylated forms of Mash1 and levels of co-immunoprecipitated E47, which further supports a correlation between the amounts of E47 that can interact with Mash1 and levels of hyperphosphorylated Mash1. Similar to Id1, when a constitutively active form of BMP receptor type I was cotransfected with E47, we also observed a decrease in the hyperphosphorylated forms of Mash1 (Figure 7B). To analyze the requirement of Mash1–E47 heterodimer formation in Mash1 phosphorylation, we made similar assays using two E47 deletion mutants (E47 HLH, which contains bHLH, and E47 ΔHLH, defective in heterodimer formation). The E47 mutant, which was unable to heterodimerize with Mash1, was also unable to induce Mash1 phosphorylation (Figure 7C). Similarly, expression of a mutant form of Mash1, devoid of the bHLH region, was not phosphorylated in the presence of E47. We also analyzed extracts from transfected cells labeled with 32Pi and [35S]methionine and compared the ratios of the major labeled forms of Mash1. Mash1 was present as two main phosphorylated bands and a higher mobility unphosphorylated one. While coexpression of E47 induced the appearance of the higher phosphorylated band, coexpression of Id1 increased the amount of Mash1 in the unphosphorylated form (Figure 7D). Phosphoamino acid analysis revealed that all phosphorylated residues were phosphoserine (data not shown). To determine E47-induced phosphorylation sites on Mash1, tryptic peptides were generated from the two phosphorylated bands, and the resulting peptides were analyzed by Tris–Tricine SDS–PAGE. Comparison of 32P-labeled peptides from these bands indicated that differential phosphorylation was located in a peptide with a molecular mass of about 400–800 Da (data not shown). Sequence analysis revealed that only three distinct peptides of about that mass contain serines conserved in Mash1 orthologs. We generated site-directed mutants corresponding to these sites and confirmed them by sequencing. Analysis of these mutants demonstrated that the mutant in which Ser152 was changed to Ala (S152A) lost E47-dependent phosphorylation (Figure 8A). Examination of the structure of bHLH transcription factors revealed that Ser152 is located in the solvent-exposed loop that joins the two α-helices involved in transcription factor heterodimerization (Chavali et al, 2001). It is noteworthy that Mash1(S152A) co-immunoprecipitated significantly lower levels of E47. To assess the relevance of Ser152 phosphorylation on transcriptional activity, mutants were transfected with the TH reporter construct. The Ser152 mutant showed higher transcriptional activity than wild type or the other mutants when transfected alone, whereas no significant differences were observed when cotransfected with E47 (Figure 8B). On the basis of our previous results, we tested whether Ser152 phosphorylation was involved in the stabilization of Mash1 protein levels by heterodimerization with E47. Pulse-chase assays indicated that, as previously shown, wild-type Mash1 levels were stabilized by coexpression of E47. In contrast, Mash1(S152A) levels were much less stabilized (Figure 8C and D). Figure 7.HEK-293 cells, transfected with the constructs indicated, were labeled with [35S]methionine or 32Pi for 3 h as described in Materials and methods. Labeled Mash1 or E47 was immunoprecipitated and visualized by SDS–PAGE (see text). Download figure Download PowerPoint Figure 8.(A) HEK-293 cells, transfected with the wild-type or mutant Mash1 constructs, with or without E47 expression construct as indicated, were labeled with [35S]methionine and Mash1 was immunoprecipitated as above. (B) C17.2 cells were cotransfected with a TH promoter-driven reporter construct and the indicated combinations of wild-type and mutant Mash1 with (filled bars) or without (empty bars) E47 expression vectors. The results are expressed as mean±s.e.m. of triplicates from five independent transfections. (C, D) Pulse-chase assays and quantification were performed in HEK-293 cells, transfected with the constructs indicated, as described in Figure 4B. Download figure Download PowerPoint Involvement of CK2 in E47-dependent phosphorylation of Mash1 Analysis of the sequence flanking Ser152 of Mash1 from several species revealed consensus sites for PKA (KKx Ser152) and CK2 (Ser152xxE). To assess the involvement of distinct
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