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• W2025275840 abstract "We analyzed the G-actin-regulated transcriptome by gene expression analysis using previously characterized actin-binding drugs. We found many known MAL/MRTF-dependent target genes of serum response factor (SRF), as well as additional directly regulated genes. Surprisingly, several putative antiproliferative target genes were identified, including mig6/errfi-1, a negative regulator of the EGFR family. Mig6 induction occurred through actin-MAL-SRF signaling, and MAL was inducibly recruited to and activated a mig6 promoter element. Upregulation of Mig6 by lipid agonists such as LPA and S1P or actin drugs involved MAL and correlated with decreased activation of EGFR, MAPK/Erk, and c-fos. Mig6 depletion restored EGFR signaling and provided a proliferative advantage. Overexpression of MAL exhibited strong antiproliferative effects requiring the domains for SRF binding and transactivation, which supports antagonistic functions of MAL on growth-promoting signals. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signaling pathways toward SRF. We analyzed the G-actin-regulated transcriptome by gene expression analysis using previously characterized actin-binding drugs. We found many known MAL/MRTF-dependent target genes of serum response factor (SRF), as well as additional directly regulated genes. Surprisingly, several putative antiproliferative target genes were identified, including mig6/errfi-1, a negative regulator of the EGFR family. Mig6 induction occurred through actin-MAL-SRF signaling, and MAL was inducibly recruited to and activated a mig6 promoter element. Upregulation of Mig6 by lipid agonists such as LPA and S1P or actin drugs involved MAL and correlated with decreased activation of EGFR, MAPK/Erk, and c-fos. Mig6 depletion restored EGFR signaling and provided a proliferative advantage. Overexpression of MAL exhibited strong antiproliferative effects requiring the domains for SRF binding and transactivation, which supports antagonistic functions of MAL on growth-promoting signals. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signaling pathways toward SRF. Gene expression through the transcription factor serum response factor (SRF) is regulated by at least two signaling cascades controlling SRF-specific coactivators (Posern and Treisman, 2006Posern G. Treisman R. Actin' together: serum response factor, its cofactors and the link to signal transduction.Trends Cell Biol. 2006; 16: 588-596Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Ternary complex factors (TCFs) such as Elk-1, SAP-1, and Net/SAP-2 are activated by mitogen-activated protein kinase (MAPK) phosphorylation. In a complex with SRF, these proteins make sequence-specific DNA contacts on some immediate early genes, e.g., the growth-promoting genes c-fos and egr-1 (Buchwalter et al., 2004Buchwalter G. Gross C. Wasylyk B. Ets ternary complex transcription factors.Gene. 2004; 324: 1-14Crossref PubMed Scopus (273) Google Scholar). G protein-coupled receptors (GPCRs) and receptor tyrosine kinases such as the EGFR transmit extracellular signals via Ras to the intracellular mitogenic cascade, ultimately resulting in TCF-dependent gene expression (Treisman, 1995Treisman R. Journey to the surface of the cell: Fos regulation and the SRE.EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (340) Google Scholar). Negative regulators such as Mig6/Errfi-1 (also known as RALT or gene 33), which inhibits the kinase of the EGFR family, are important modifiers of the mitogenic cascade (Fiorentino et al., 2000Fiorentino L. Pertica C. Fiorini M. Talora C. Crescenzi M. Castellani L. Alemà S. Benedetti P. Segatto O. Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain.Mol. Cell. Biol. 2000; 20: 7735-7750Crossref PubMed Scopus (114) Google Scholar, Hackel et al., 2001Hackel P.O. Gishizky M. Ullrich A. Mig-6 is a negative regulator of the epidermal growth factor receptor signal.Biol. Chem. 2001; 382: 1649-1662Crossref PubMed Scopus (115) Google Scholar, Xu et al., 2006Xu D. Patten R.D. Force T. Kyriakis J.M. Gene 33/RALT is induced by hypoxia in cardiomyocytes, where it promotes cell death by suppressing phosphatidylinositol 3-kinase and extracellular signal-regulated kinase survival signaling.Mol. Cell. Biol. 2006; 26: 5043-5054Crossref PubMed Scopus (29) Google Scholar, Zhang et al., 2007aZhang X. Pickin K.A. Bose R. Jura N. Cole P.A. Kuriyan J. Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface.Nature. 2007; 450: 741-744Crossref PubMed Scopus (261) Google Scholar). Mig6 provides an autoinhibitory feedback loop and is required to prevent EGFR-dependent carcinogenesis (Ferby et al., 2006Ferby I. Reschke M. Kudlacek O. Knyazev P. Pantè G. Amann K. Sommergruber W. Kraut N. Ullrich A. Fässler R. Klein R. Mig6 is a negative regulator of EGF receptor-mediated skin morphogenesis and tumor formation.Nat. Med. 2006; 12: 568-573Crossref PubMed Scopus (195) Google Scholar, Hackel et al., 2001Hackel P.O. Gishizky M. Ullrich A. Mig-6 is a negative regulator of the epidermal growth factor receptor signal.Biol. Chem. 2001; 382: 1649-1662Crossref PubMed Scopus (115) Google Scholar, Zhang et al., 2007bZhang Y.W. Staal B. Su Y. Swiatek P. Zhao P. Cao B. Resau J. Sigler R. Bronson R. Vande Woude G.F. Evidence that MIG-6 is a tumor-suppressor gene.Oncogene. 2007; 26: 269-276Crossref PubMed Scopus (97) Google Scholar). Recent work has established a novel signaling pathway regulating another subset of SRF target genes via coactivators of the myocardin-related transcription factor (MRTF) family, MAL/MRTF-A/MKL1/BSAC and MRTF-B/MKL2 (Pipes et al., 2006Pipes G.C. Creemers E.E. Olson E.N. The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis.Genes Dev. 2006; 20: 1545-1556Crossref PubMed Scopus (362) Google Scholar, Posern and Treisman, 2006Posern G. Treisman R. Actin' together: serum response factor, its cofactors and the link to signal transduction.Trends Cell Biol. 2006; 16: 588-596Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Their activation requires the Rho family GTPases RhoA or Rac and changes in actin dynamics (Busche et al., 2008Busche S. Descot A. Julien S. Genth H. Posern G. Epithelial cell-cell contacts regulate SRF-mediated transcription via Rac-actin-MAL signalling.J. Cell Sci. 2008; 121: 1025-1035Crossref PubMed Scopus (70) Google Scholar, Du et al., 2004Du K.L. Chen M. Li J. Lepore J.J. Mericko P. Parmacek M.S. Megakaryoblastic leukemia factor-1 transduces cytoskeletal signals and induces smooth muscle cell differentiation from undifferentiated embryonic stem cells.J. Biol. Chem. 2004; 279: 17578-17586Crossref PubMed Scopus (132) Google Scholar, Kuwahara et al., 2005Kuwahara K. Barrientos T. Pipes G.C. Li S. Olson E.N. Muscle-specific signaling mechanism that links actin dynamics to serum response factor.Mol. Cell. Biol. 2005; 25: 3173-3181Crossref PubMed Scopus (172) Google Scholar, Posern et al., 2002Posern G. Sotiropoulos A. Treisman R. Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor.Mol. Biol. Cell. 2002; 13: 4167-4178Crossref PubMed Scopus (184) Google Scholar, Sotiropoulos et al., 1999Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics.Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). These changes are sensed by MAL, which harbors G-actin-binding RPEL motifs at the N terminus: Upon stimulation with Rho activators such as serum, lysophosphatidic acid (LPA), or sphingosine 1-phosphate (S1P), MAL is released from an inhibitory complex with monomeric, globular (G-) actin and strongly activates SRF-controlled transcription (Lockman et al., 2004Lockman K. Hinson J.S. Medlin M.D. Morris D. Taylor J.M. Mack C.P. Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors.J. Biol. Chem. 2004; 279: 42422-42430Crossref PubMed Scopus (133) Google Scholar, Miralles et al., 2003Miralles F. Posern G. Zaromytidou A.I. Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, Posern et al., 2004Posern G. Miralles F. Guettler S. Treisman R. Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL.EMBO J. 2004; 23: 3973-3983Crossref PubMed Scopus (112) Google Scholar, Vartiainen et al., 2007Vartiainen M.K. Guettler S. Larijani B. Treisman R. Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL.Science. 2007; 316: 1749-1752Crossref PubMed Scopus (441) Google Scholar). Among the few known actin/MAL-dependent target genes are vcl, srf, cyr61, and several cytoskeletal genes whose activation is largely independent of MAPK signaling (Gineitis and Treisman, 2001Gineitis D. Treisman R. Differential usage of signal transduction pathways defines two types of serum response factor target gene.J. Biol. Chem. 2001; 276: 24531-24539Crossref PubMed Scopus (174) Google Scholar, Miralles et al., 2003Miralles F. Posern G. Zaromytidou A.I. Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, Philippar et al., 2004Philippar U. Schratt G. Dieterich C. Muller J.M. Galgoczy P. Engel F.B. Keating M.T. Gertler F. Schule R. Vingron M. Nordheim A. The SRF target gene Fhl2 antagonizes RhoA/MAL-dependent activation of SRF.Mol. Cell. 2004; 16: 867-880Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). These genes and their promoters respond differentially to actin-binding drugs: Treatment with cytochalasin D activates MAL-dependent transcription by releasing MAL from G-actin, while latrunculin B blocks the dissociation of the G-actin:MAL complex (Gineitis and Treisman, 2001Gineitis D. Treisman R. Differential usage of signal transduction pathways defines two types of serum response factor target gene.J. Biol. Chem. 2001; 276: 24531-24539Crossref PubMed Scopus (174) Google Scholar, Miralles et al., 2003Miralles F. Posern G. Zaromytidou A.I. Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, Posern et al., 2002Posern G. Sotiropoulos A. Treisman R. Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor.Mol. Biol. Cell. 2002; 13: 4167-4178Crossref PubMed Scopus (184) Google Scholar, Sotiropoulos et al., 1999Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics.Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, Vartiainen et al., 2007Vartiainen M.K. Guettler S. Larijani B. Treisman R. Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL.Science. 2007; 316: 1749-1752Crossref PubMed Scopus (441) Google Scholar). The subset of TCF-independent SRF target genes is also thought to play important roles in the phenotype of SRF-depleted mice, which show severe perturbations in cell adhesion, motility, and differentiation in various tissues, resulting in embryonic lethality (Arsenian et al., 1998Arsenian S. Weinhold B. Oelgeschläger M. Rüther U. Nordheim A. Serum response factor is essential for mesoderm formation during mouse embryogenesis.EMBO J. 1998; 17: 6289-6299Crossref PubMed Scopus (296) Google Scholar, Balza and Misra, 2006Balza Jr., R.O. Misra R.P. Role of the serum response factor in regulating contractile apparatus gene expression and sarcomeric integrity in cardiomyocytes.J. Biol. Chem. 2006; 281: 6498-6510Crossref PubMed Scopus (76) Google Scholar, Knöll et al., 2006Knöll B. Kretz O. Fiedler C. Alberti S. Schütz G. Frotscher M. Nordheim A. Serum response factor controls neuronal circuit assembly in the hippocampus.Nat. Neurosci. 2006; 9: 195-204Crossref PubMed Scopus (127) Google Scholar, Koegel et al., 2009Koegel H. von Tobel L. Schäfer M. Alberti S. Kremmer E. Mauch C. Hohl D. Wang X.J. Beer H.D. Bloch W. et al.Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice.J. Clin. Invest. 2009; 119: 899-910Crossref PubMed Scopus (43) Google Scholar, Schratt et al., 2002Schratt G. Philippar U. Berger J. Schwarz H. Heidenreich O. Nordheim A. Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells.J. Cell Biol. 2002; 156: 737-750Crossref PubMed Scopus (143) Google Scholar). However, depletion of MAL or MRTF-B leads to relatively mild and tissue-restricted phenotypes in myoepithelial and vascular smooth muscle cells, respectively (Li et al., 2005Li J. Zhu X. Chen M. Cheng L. Zhou D. Lu M.M. Du K. Epstein J.A. Parmacek M.S. Myocardin-related transcription factor B is required in cardiac neural crest for smooth muscle differentiation and cardiovascular development.Proc. Natl. Acad. Sci. USA. 2005; 102: 8916-8921Crossref PubMed Scopus (115) Google Scholar, Li et al., 2006Li S. Chang S. Qi X. Richardson J.A. Olson E.N. Requirement of a myocardin-related transcription factor for development of mammary myoepithelial cells.Mol. Cell. Biol. 2006; 26: 5797-5808Crossref PubMed Scopus (137) Google Scholar, Oh et al., 2005Oh J. Richardson J.A. Olson E.N. Requirement of myocardin-related transcription factor-B for remodeling of branchial arch arteries and smooth muscle differentiation.Proc. Natl. Acad. Sci. USA. 2005; 102: 15122-15127Crossref PubMed Scopus (117) Google Scholar, Sun et al., 2006bSun Y. Boyd K. Xu W. Ma J. Jackson C.W. Fu A. Shillingford J.M. Robinson G.W. Hennighausen L. Hitzler J.K. et al.Acute myeloid leukemia-associated Mkl1 (Mrtf-a) is a key regulator of mammary gland function.Mol. Cell. Biol. 2006; 26: 5809-5826Crossref PubMed Scopus (117) Google Scholar). Transcription profiling of MAL-depleted mammary glands and cell lines revealed only partially overlapping gene expression patterns (Medjkane et al., 2009Medjkane S. Perez-Sanchez C. Gaggioli C. Sahai E. Treisman R. Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis.Nat. Cell Biol. 2009; 11: 257-268Crossref PubMed Scopus (288) Google Scholar, Selvaraj and Prywes, 2004Selvaraj A. Prywes R. Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent.BMC Mol. Biol. 2004; 5: 13Crossref PubMed Scopus (118) Google Scholar, Sun et al., 2006bSun Y. Boyd K. Xu W. Ma J. Jackson C.W. Fu A. Shillingford J.M. Robinson G.W. Hennighausen L. Hitzler J.K. et al.Acute myeloid leukemia-associated Mkl1 (Mrtf-a) is a key regulator of mammary gland function.Mol. Cell. Biol. 2006; 26: 5809-5826Crossref PubMed Scopus (117) Google Scholar). Here, we searched for genes that are directly regulated by G-actin, using the well-characterized actin drugs. We expected to find MAL-dependent SRF targets and potentially other G-actin-regulated genes. We found several known MAL/SRF targets and also unknown genes, many of which are involved in negative control of proliferation. Among those, we further characterized mig6 and showed that it is regulated via the G-actin-MAL-SRF signaling axis through a MAL- and SRF-binding promoter element. Induction of mig6 by actin drugs or lipid agonists attenuated the response to EGF signaling, demonstrating a negative crosstalk between the MAL and MAPK signaling modules. MAL exhibited strong antiproliferative effects through transcription, and we propose the existence of negatively acting transcriptional circuits to regulate signaling toward SRF. We and others have previously shown that G-actin regulates a transcriptional coactivator called MAL/MRTF-A/MKL1/BSAC (referred to as MAL) (Busche et al., 2008Busche S. Descot A. Julien S. Genth H. Posern G. Epithelial cell-cell contacts regulate SRF-mediated transcription via Rac-actin-MAL signalling.J. Cell Sci. 2008; 121: 1025-1035Crossref PubMed Scopus (70) Google Scholar, Du et al., 2004Du K.L. Chen M. Li J. Lepore J.J. Mericko P. Parmacek M.S. Megakaryoblastic leukemia factor-1 transduces cytoskeletal signals and induces smooth muscle cell differentiation from undifferentiated embryonic stem cells.J. Biol. Chem. 2004; 279: 17578-17586Crossref PubMed Scopus (132) Google Scholar, Kuwahara et al., 2005Kuwahara K. Barrientos T. Pipes G.C. Li S. Olson E.N. Muscle-specific signaling mechanism that links actin dynamics to serum response factor.Mol. Cell. Biol. 2005; 25: 3173-3181Crossref PubMed Scopus (172) Google Scholar, Miralles et al., 2003Miralles F. Posern G. Zaromytidou A.I. Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar). This coactivator is involved in transcription via SRF and regulates a subset of known SRF target genes (Gineitis and Treisman, 2001Gineitis D. Treisman R. Differential usage of signal transduction pathways defines two types of serum response factor target gene.J. Biol. Chem. 2001; 276: 24531-24539Crossref PubMed Scopus (174) Google Scholar, Lockman et al., 2004Lockman K. Hinson J.S. Medlin M.D. Morris D. Taylor J.M. Mack C.P. Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors.J. Biol. Chem. 2004; 279: 42422-42430Crossref PubMed Scopus (133) Google Scholar). However, the plethora of G-actin-regulated genes remained unclear. Thus, we aimed at identifying G-actin target genes on a genome-wide basis. For this, we utilized actin-binding drugs that we recently characterized as differential activators or repressors of the actin-MAL pathway: Treatment with cytochalasin D activates MAL-dependent transcription by releasing MAL from G-actin, while latrunculin B blocks the dissociation of the G-actin:MAL complex. We reasoned that both drugs depolymerize F-actin, however: Genes depending on an intact cytoskeleton rather than on the G-actin switch should therefore not score as differentially expressed. Thus, three conditions were used for the microarray analysis: control NIH 3T3 cells, cells treated with cytochalasin D, and cells treated with latrunculin B and cytochalasin D (Figure 1A). Optimal concentrations and times were established by reporter assays and validated by quantitative RT-PCR of known MAL-regulated SRF targets (Figure S1). In addition, translation was blocked in all samples by cycloheximide to enrich primarily regulated targets and further reduce indirect effects from transcriptional circuits. Statistical analysis of three independent experiments per condition resulted in only 225 differentially regulated probe sets with a maximum false discovery rate (FDR) of 3.75; when this restriction was extended to a maximum FDR of 5.43, the number of differentially regulated probe sets increased marginally to 255 (Table S1). Given that 45,000 probe sets were analyzed, this result suggested that a rather specific group of genes was differentially regulated by the combination of the three drugs. The subsequent analysis of differentially regulated probe sets revealed that only 39 showed a downregulation by cytochalasin D; however, all of them were repressed by latrunculin B as well and thus failed our criteria for G-actin-regulated genes. Apparently, no inversely regulated group of genes exists that can respond to the same G-actin switch that activates MAL. The remaining 216 probe sets were upregulated by cytochalasin D and revealed a considerable reduction in the presence of latrunculin B (Table S1). Such differential regulation is not explainable by stress induction, since both drugs equally affect, e.g., the stress kinases p38 and JNK (Figure S2). Further analysis showed a striking accumulation of known MAL-dependent SRF targets among the genes strongly upregulated by cytochalasin D, serving as a positive control for the suitability of our approach (Figure 1B). Negative controls such as typical “housekeeping” genes did not show any significant regulation. More importantly, SRF targets such as c-fos and egr-1, which are mainly regulated by TCFs, failed to show statistically significant patterns of regulation by the actin drugs used (Figure 1B). Beside many cytoskeletal target genes, we found, surprisingly, a large number of genes with presumed antiproliferative or proapoptotic function. These included the proapoptotic factors Pmaip/NOXA and Bok. We also identified Errfi-1, Dusp5, and Zfp36, which are negative regulators of the mitogenic cascade, at the level of the receptor kinases, the MAP kinase module, and the stability of target mRNAs, respectively (Figure 1B). For Errfi-1 (also known as RALT, gene 33, and mitogen-inducible gene 6; referred to as mig6), two independent probe sets were found. Their induction rates by cytochalasin D were 7.3- and 5-fold, and simultaneous treatment with latrunculin B reduced this value to 37% and 46%, respectively; calculated q values (i.e., the lowest FDR at which this particular probe set would be detected as differentially expressed) were zero (Figure 1B). To this end, we choose this gene for further analysis of its connection to G-actin-MAL signaling. We first sought to validate mig6 induction in the absence of cycloheximide. Using quantitative real-time RT-PCR, we observed a 6-fold induction of mig6 mRNA by cytochalasin D within 90 min, which was reduced to less than 2-fold by pretreatment with latrunculin B (Figure 2A). This pattern was comparable to ctgf, a previously characterized MAL/SRF target gene (Muehlich et al., 2007Muehlich S. Cicha I. Garlichs C.D. Krueger B. Posern G. Goppelt-Struebe M. Actin-dependent regulation of connective tissue growth factor.Am. J. Physiol. Cell Physiol. 2007; 292: C1732-C1738Crossref PubMed Scopus (55) Google Scholar, Philippar et al., 2004Philippar U. Schratt G. Dieterich C. Muller J.M. Galgoczy P. Engel F.B. Keating M.T. Gertler F. Schule R. Vingron M. Nordheim A. The SRF target gene Fhl2 antagonizes RhoA/MAL-dependent activation of SRF.Mol. Cell. 2004; 16: 867-880Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Next, we tested the accumulation of the Mig6 protein following treatment with various actin-binding drugs in several cell lines, including mouse embryo fibroblasts (MEFs) and hepatocellular carcinoma cells. Mig6 was induced by the two depolymerizing agents, cytochalasin D and swinholide A, as well as by the F-actin-stabilizing drug jasplakinolide (Figure 2B). In contrast, latrunculin B alone did not elevate Mig6, but partially blocked induction by cytochalasin D. This pattern of regulation by actin-binding drugs is known to be typical for MAL-dependent transcription (Miralles et al., 2003Miralles F. Posern G. Zaromytidou A.I. Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar). The dose-response curve revealed that around 2 μM of cytochalasin D is sufficient for full Mig6 protein induction (Figure 2C). Time course analysis showed an observably increased Mig6 protein level after 3 hr of stimulation with cytochalasin or jasplakinolide, which was maintained for at least 7 hr (Figure 2D). Since MAPK Erk signaling is also known to stimulate Mig6 expression, we analyzed EGFR and Erk phosphorylation during this time course. However, we failed to observe pronounced induction of EGFR or Erk phosphorylation, in contrast to control cells treated with EGF (Figure 2D), suggesting that MAPK signaling is not causative for Mig6 induction by actin-binding drugs. Together, Mig6 regulation on both mRNA and protein level occurred through G-actin, and the regulatory pattern is highly reminiscent of MAL-dependent SRF targets. To directly test Mig6 regulation, we transiently expressed activated constructs of MAL, its close relative myocardin, and SRF. An increased level of Mig6 protein was observed in the total lysates of MEFs (Figure 3A), despite a limited transfection rate of approximately 20%. Similarly, MAL ΔN and SRF-VP16 upregulated Mig6 in NIH 3T3 cells (Figure 3A). Full-length MAL was not sufficient to observably induce Mig6 in this assay, however, which is consistent with its weaker transcriptional effects due to tight regulation by upstream signaling. Concomitantly, the mRNA level for mig6 as well as the MAL and SRF target genes ctgf and integrin alpha 5 was upregulated by SRF-VP16, MAL ΔN, and activated RhoA Q63L, a potent inducer of MAL-SRF-controlled gene expression (Figure 3B). This suggests that activation of RhoA, MAL, and SRF correlates with mig6 transcription, similar to previously characterized MAL-dependent target genes. The promoter of mig6 is thought to be 5′ of an untranslated exon. We constructed various promoter-reporter constructs, ranging from −1635 to +1, and analyzed the luciferase activity in the presence or absence of activated MAL ΔN and SRF-VP16. A promoter element covering at least the 330 bp in front of the putative transcription start site contained a MAL-responsive element, while a further truncation to −147 abolished reporter induction by MAL ΔN (Figure 3C). Deletion of a putative CArG-like element at position −260 in the murine promoter rendered this reporter unresponsive to MAL (Figure 3C). This proximal element of the mig6 promoter is therefore required for regulation through MAL. A fragment of the mig6 promoter, including the response element at −260, permitted MAL- and SRF-induced reporter activity when fused in front of a basal TATA box, whereas a mutated sequence showed significantly diminished inducibility (Figure 3D). Despite the lack of considerable induction by MAL ΔN, some responsiveness to SRF-VP16 was maintained; however, this was not mediated through the only other potential SRF-binding site at position −125 (Figure S3). Finally, we directly analyzed MAL and SRF recruitment to the mig6 promoter using chromatin immunoprecipitation (ChIP). The mig6 promoter covering the position −260 as well as the known target promoters of the vcl and srf genes were amplified from MAL immunoprecipitates upon serum stimulation, demonstrating an inducible recruitment (Figure 3E). Moreover, mig6, vcl, and srf promoters were also bound by the SRF protein. We note that SRF recruitment to mig6 and, to a lesser extent, to vcl and srf, appears to be slightly elevated following serum stimulation, but the significance of this remains to be tested. In contrast, a fragment within the first intron of mig6, or the gapdh gene, was not amplified from either MAL or SRF chromatin immunoprecipitates (Figure 3E). Together, our results suggest that mig6 is directly regulated by MAL through binding and activating a promoter region at −260 of the murine mig6 gene. Mig6 is a negative regulator of receptor tyrosine kinases, and Mig6 depletion in mice results in elevated phosphorylation and activity of EGFR and MAPK Erk (Ferby et al., 2006Ferby I. Reschke M. Kudlacek O. Knyazev P. Pantè G. Amann K. Sommergruber W. Kraut N. Ullrich A. Fässler R. Klein R. Mig6 is a negative regulator of EGF receptor-mediated skin morphogenesis and tumor formation.Nat. Med. 2006; 12: 568-573Crossref PubMed Scopus (195) Google Scholar). Structural and biochemical evidence suggested that Mig6 blocks the formation of asymmetric EGFR dimers, thereby inhibiting the tyrosine kinase activity and the phosphorylation of critical C-terminal residues, including Y1173 (Anastasi et al., 2007Anastasi S. Baietti M.F. Frosi Y. Alemà S. Segatto O. The evolutionarily conserved EBR module of RALT/MIG6 mediates suppression of the EGFR catalytic activity.Oncogene. 2007; 26: 7833-7846Crossref PubMed Scopus (59) Google Scholar, Zhang et al., 2007aZhang X. Pickin K.A. Bose R. Jura N. Cole P.A. Kuriyan J. Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface.Nature. 2007; 450: 741-744Crossref PubMed Scopus (261) Google Scholar). We therefore investigated whether the observed upregulation of Mig6 has implications on the EGFR signaling axis. EGFR phosphorylation was analyzed first in HepG2 cells, which harbor low basal mig6 and high EGFR expression (M.R., unpublished data). Cells were pretreated with the actin-binding drugs cytochalasin or swinholide, allowing the accumulation of Mig6 (Figure 4A). Subsequent short-term activation by EGF showed a pronounced reduction in the Y1173 phosphorylation of the EGFR (Figure 4A), suggesting that the Mig6 induction by actin drugs downregulates signaling through EGFR. The EGFR is also activated by amphiregulin and results in the stimulation of the MAP kinase Erk. We tested whether our Mig6 induction affects the level of activated phospho-Erk. Stimulation with amphiregulin for 30 min increased Erk phosphorylation without strongly stimulating Mig6 protein levels (Figure 4B). In contrast, cells with cytochalasin-induced Mig6 levels showed a decreased Erk phosphorylation after amphiregulin stimulation, suggesting that Mig6 induction though actin signaling interferes with the mitogenic cascade. To test whether Mig6 is critical for reduced EGFR signaling after actin drug treatment, we generated a stable Mig6 knockdown in HepG2 cells. Although the knockdown of Mig6 protein appeared to be efficient in untreated control" @default.
• W2025275840 created "2016-06-24" @default.
• W2025275840 creator A5011132701 @default.
• W2025275840 creator A5028900149 @default.
• W2025275840 creator A5066747407 @default.
• W2025275840 creator A5073271517 @default.
• W2025275840 creator A5076478873 @default.
• W2025275840 creator A5090328045 @default.
• W2025275840 date "2009-08-01" @default.
• W2025275840 modified "2023-10-18" @default.
• W2025275840 title "Negative Regulation of the EGFR-MAPK Cascade by Actin-MAL-Mediated Mig6/Errfi-1 Induction" @default.
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