Regulation of the ETS Transcription Factor ER81 by the 90-kDa Ribosomal S6 Kinase 1 and Protein Kinase A
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m205501200
ISSN1083-351X
Autores Tópico(s)Cell death mechanisms and regulation
ResumoThe ETS transcription factor ER81 is activated in response to many signals via mitogen-activated protein kinases (MAPKs). However, ER81 is not only phosphorylated on MAPK sites but also at other sites that impact on its transactivation potential. Here we describe that the 90-kDa ribosomal S6 kinase 1 (RSK1), a protein kinase downstream of the extracellular signal-regulated kinase (ERK) subclass of MAPKs, binds to ER81, phosphorylates it, and enhances ER81-dependent transcription. Two in vivo RSK1 phosphorylation sites within ER81, Ser191 and Ser216, were identified, whose mutation to alanine reduces ER81 activity upon ERK-MAPK stimulation. Furthermore, RSK1 activates the ER81 cofactor CREB-binding protein and may thereby augment ER81-dependent transcription. Similar to RSK1, the cAMP-dependent protein kinase A (PKA) phosphorylates ER81 on Ser191/Ser216. Additionally, PKA targets ER81 on Ser334 in vivo. Surprisingly, phosphorylation of Ser334 severely reduces the DNA-binding ability of ER81 but also enhances the transactivation potential of ER81. These counteractive effects of PKA phosphorylation on ER81-dependent transcription may cause the selective up-regulation of promoters with high but not low affinity for ER81. Collectively, we have identified mechanisms for how two distinct signaling pathways with different effector protein kinases, RSK1 and PKA, converge on ER81, which may regulate ER81 function during development and tumorigenesis. The ETS transcription factor ER81 is activated in response to many signals via mitogen-activated protein kinases (MAPKs). However, ER81 is not only phosphorylated on MAPK sites but also at other sites that impact on its transactivation potential. Here we describe that the 90-kDa ribosomal S6 kinase 1 (RSK1), a protein kinase downstream of the extracellular signal-regulated kinase (ERK) subclass of MAPKs, binds to ER81, phosphorylates it, and enhances ER81-dependent transcription. Two in vivo RSK1 phosphorylation sites within ER81, Ser191 and Ser216, were identified, whose mutation to alanine reduces ER81 activity upon ERK-MAPK stimulation. Furthermore, RSK1 activates the ER81 cofactor CREB-binding protein and may thereby augment ER81-dependent transcription. Similar to RSK1, the cAMP-dependent protein kinase A (PKA) phosphorylates ER81 on Ser191/Ser216. Additionally, PKA targets ER81 on Ser334 in vivo. Surprisingly, phosphorylation of Ser334 severely reduces the DNA-binding ability of ER81 but also enhances the transactivation potential of ER81. These counteractive effects of PKA phosphorylation on ER81-dependent transcription may cause the selective up-regulation of promoters with high but not low affinity for ER81. Collectively, we have identified mechanisms for how two distinct signaling pathways with different effector protein kinases, RSK1 and PKA, converge on ER81, which may regulate ER81 function during development and tumorigenesis. mitogen-activated protein kinase S94A/T139A/T143A/S146A mutant of ER81 glutathione S-transferase protein kinase A 90-kDa ribosomal S6 kinase hemagglutinin phenylmethylsulfonyl fluoride dithiothreitol CREB-binding protein cAMP-response element-binding protein The transcription factor ER81 belongs to the family of ETS proteins that are characterized by a conserved, ∼85-amino acid-long, DNA-binding ETS domain and perform many essential functions in homeostasis, signaling response, and development (1Brown T.A. McKnight S.L. Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (234) Google Scholar, 2Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 3Sharrocks A.D. Nat. Rev Mol. Cell. Biol. 2001; 2: 827-837Crossref PubMed Scopus (811) Google Scholar, 4Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar). In addition to its ETS domain, ER81 possesses three further functional domains, an N-terminal and a C-terminal transactivation domain as well as a central inhibitory domain that regulates both transactivation domains (5Janknecht R. Mol. Cell. Biol. 1996; 16: 1550-1556Crossref PubMed Scopus (115) Google Scholar). Gene transcription mediated by ER81 is strongly stimulated by mitogen-activated protein kinases (MAPKs),1 which involves phosphorylation of ER81 at multiple sites (5Janknecht R. Mol. Cell. Biol. 1996; 16: 1550-1556Crossref PubMed Scopus (115) Google Scholar, 6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar). Furthermore, ER81 is stimulated by the cAMP-dependent protein kinase A (PKA), although it is at present unknown whether ER81 is directly phosphorylated by PKA in vivo and how this may affect its ability to up-regulate gene transcription (7Coutte L. Monte D. Imai K. Pouilly L. Dewitte F. Vidaud M. Adamski J. Baert J.L. de Launoit Y. Oncogene. 1999; 18: 6278-6286Crossref PubMed Scopus (22) Google Scholar).Expression of ER81 is both developmentally and tissue-specifically regulated. For instance, high levels ofER81 expression are detectable in the adult brain, heart, or lung, whereas very little is found in liver or skeletal muscle (1Brown T.A. McKnight S.L. Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (234) Google Scholar, 8Monte D. Coutte L. Baert J.L. Angeli I. Stehelin D. de Launoit Y. Oncogene. 1995; 11: 771-779PubMed Google Scholar). Moreover, ER81 expression is undetectable during early embryogenesis yet at later stages is observable at various sites in the embryo, including the central nervous system, eye, heart, lung, salivary gland, kidney, and pituitary gland (9Chotteau-Lelievre A. Desbiens X. Pelczar H. Defossez P.A. de Launoit Y. Oncogene. 1997; 15: 937-952Crossref PubMed Scopus (131) Google Scholar, 10Chotteau-Lelievre A. Dolle P. Peronne V. Coutte L. de Launoit Y. Desbiens X. Mech. Dev. 2001; 108: 191-195Crossref PubMed Scopus (49) Google Scholar). An ER81knock-out mouse revealed that the ER81 protein is required for the proper connection of sensory and motor neurons in the developing spinal cord, and its absence causes severe motor discoordination (11Arber S. Ladle D.R. Lin J.H. Frank E. Jessell T.M. Cell. 2000; 101: 485-498Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar).ER81 expression has also been noted in breast tissue, and a subset of breast cancer cell lines even overexpresses ER81. In addition, ER81 expression is up-regulated in mammaries of mice specifically overexpressing the HER2/Neuproto-oncogene in this organ (6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar, 12Baert J.L. Monte D. Musgrove E.A. Albagli O. Sutherland R.L. de Launoit Y. Int. J. Cancer. 1997; 70: 590-597Crossref PubMed Scopus (88) Google Scholar, 13Shepherd T.G. Kockeritz L. Szrajber M.R. Muller W.J. Hassell J.A. Curr. Biol. 2001; 11: 1739-1748Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and ER81 itself enhances the transcription of the HER2/Neu gene (14Bosc D.G. Janknecht R. J. Cell. Biochem. 2002; 86: 174-183Crossref PubMed Scopus (36) Google Scholar). These observations suggest that the ER81 protein may be involved in breast tumor formation. Indeed, ER81 is a downstream target of the transmembrane receptor tyrosine kinase HER2/Neu, whose overexpression in ∼30% of human breast cancers is correlated to a poor prognosis, and ER81 may thus execute the deleterious effects of HER2/Neu in breast as well as in ovarian, gastric, and lung cancer (6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar, 15Hung M.C. Lau Y.K. Semin. Oncol. 1999; 26: 51-59PubMed Google Scholar, 16Hynes N.E. Stern D.F. Biochim. Biophys. Acta. 1994; 1198: 165-184Crossref PubMed Scopus (998) Google Scholar). Furthermore, ER81 has been shown to up-regulate transcription of two matrix metalloproteinase genes, interstitial collagenase andmatrilysin, which may contribute to the high metastatic potential of HER2/Neu-overexpressing cancer cells through an enhanced ability to remodel the extracellular matrix (6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar,17Crawford H.C. Fingleton B. Gustavson M.D. Kurpios N. Wagenaar R.A. Hassell J.A. Matrisian L.M. Mol. Cell. Biol. 2001; 21: 1370-1383Crossref PubMed Scopus (159) Google Scholar).HER2/Neu elicits phosphorylation of ER81 via MAPKs, but not all ER81 phosphorylation sites are directly targeted by MAPKs (6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar). This raises the possibility that protein kinases downstream of MAPKs may also phosphorylate ER81. One protein kinase family downstream of the ERK subclass of MAPKs is the 90-kDa ribosomal S6 kinase (RSK) family. This family of serine/threonine protein kinases includes three isoforms (RSK1, RSK2, RSK3), all of which are activated through direct phosphorylation by ERK-MAPKs. RSKs contain an N-terminal and a C-terminal kinase domain, which are separated by a linker region. The activation of RSKs is thought to be a complex process; ERK-MAPK, which is constitutively bound to the C terminus of RSKs, phosphorylates RSKs upon mitogenic stimulation at several sites, including the activation domain of their C-terminal kinase domain. Then the activated C-terminal kinase phosphorylates the RSK linker region, thereby generating a docking site for 3-phosphoinositide-dependent protein kinase-1 that in turn phosphorylates the activation loop of the N-terminal kinase domain. This generates a fully activated RSK molecule, which then, at least in part, translocates to the cell nucleus (18Richards S.A. Dreisbach V.C. Murphy L.O. Blenis J. Mol. Cell. Biol. 2001; 21: 7470-7480Crossref PubMed Scopus (77) Google Scholar, 19Frödin M. Gammeltoft S. Mol. Cell. Endocrinol. 1999; 151: 65-77Crossref PubMed Scopus (614) Google Scholar, 20Frödin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (254) Google Scholar, 21Jensen C.J. Buch M.B. Krag T.O. Hemmings B.A. Gammeltoft S. Frödin M. J. Biol. Chem. 1999; 274: 27168-27176Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). There, the RSK N-terminal kinase domain can phosphorylate and thus regulate transcription factors such as c-Fos, the cAMP-response element-binding protein, or the estrogen receptor (22Chen R.H. Abate C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10952-10956Crossref PubMed Scopus (257) Google Scholar, 23Joel P.B. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (310) Google Scholar, 24Xing J. Ginty D.D. Greenberg M.E. Science. 1996; 273: 959-963Crossref PubMed Scopus (1081) Google Scholar, 25Xing J. Kornhauser J.M. Xia Z. Thiele E.A. Greenberg M.E. Mol. Cell. Biol. 1998; 18: 1946-1955Crossref PubMed Google Scholar). In addition, RSKs may impact on chromatin structure by inducing the phosphorylation of histone H3 (26Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. Hanauer A. Allis C.D. Science. 1999; 285: 886-891Crossref PubMed Scopus (424) Google Scholar).The N-terminal kinase domain of RSKs is closely related to the catalytic subunit of PKA. Accordingly, RSKs and PKA may phosphorylate and thereby regulate the same substrates, most notably demonstrated for the transcription factor cAMP- response element-binding protein and its phosphorylation on serine 133 (24Xing J. Ginty D.D. Greenberg M.E. Science. 1996; 273: 959-963Crossref PubMed Scopus (1081) Google Scholar, 27Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2041) Google Scholar). However, the signal transduction pathways leading to the activation of PKA are different from those activating RSKs. Briefly, PKA is activated by many neurotransmitters and hormones, which bind to G-protein-coupled transmembrane receptors. This in turn leads to the activation of an adenylyl cyclase and the production of cAMP. cAMP binds to the regulatory subunits of PKA, which then dissociate from and thereby lead to the activation of the catalytic PKA subunits and also enable them to translocate to the cell nucleus (28Daniel P.B. Walker W.H. Habener J.F. Annu. Rev. Nutr. 1998; 18: 353-383Crossref PubMed Scopus (212) Google Scholar).In this report, we describe that ER81-dependent transcription can be regulated by RSK1 and PKA. Furthermore, we have uncovered mechanisms how these two protein kinases distinctively affect ER81 function.DISCUSSIONIn this report, we have identified RSK1 as an ER81-associated protein kinase that phosphorylates ER81 and enhances the transactivation potential of both ER81 and its cofactors CBP/p300. Furthermore, we have uncovered PKA phosphorylation sites in ER81 and unraveled how this affects DNA binding as well as transactivation mediated by ER81.RSK1 and PKA are related serine/threonine protein kinases (19Frödin M. Gammeltoft S. Mol. Cell. Endocrinol. 1999; 151: 65-77Crossref PubMed Scopus (614) Google Scholar). Both kinases phosphorylate basophilic motifs, characterized by an arginine at position n-3 and an arginine or lysine at positionn-5 in the case of RSK1, and in the case of PKA by arginines at positions n-3 and n-2 with respect to the phosphorylated serine/threonine residue (39Leighton I.A. Dalby K.N. Caudwell F.B. Cohen P.T. Cohen P. FEBS Lett. 1995; 375: 289-293Crossref PubMed Scopus (109) Google Scholar, 45Kennelly P.J. Krebs E.G. J. Biol. Chem. 1991; 266: 15555-15558Abstract Full Text PDF PubMed Google Scholar). However, deviations from these strict consensus motifs have often been observed; in particular, many PKA sites have only one arginine at positionn-3, and the arginine/lysine residue at n-5 is not essential for RSK1 phosphorylation. As such, the ER81 amino acids Phe-Arg-Arg-Gln-Leu-Ser191, Tyr-Gln-Arg-Gln-Met-Ser216, and Tyr-Gln-Arg-Arg-Gly-Ser334 all conform to the minimal requirements for being both RSK1 and PKA phosphorylation sites. However, whereas RSK1 readily phosphorylates Ser191, Ser216, and Ser334 in vitro, it appears to only phosphorylate Ser191 and Ser216 in vivo, indicating that access to Ser334 is restricted in full-length ER81 in vivo. One potential reason for this could be ER81-interacting proteins, including CBP/p300 or RSK1 itself, which prevent the N-terminal kinase domain of RSK1 from phosphorylating Ser334. In contrast, maybe due to its smaller size, PKA has access to Ser334 and can phosphorylate Ser334 both in vitro and in vivo. Further, Ser334 is even more efficiently phosphorylated by PKA in vivo than Ser191 and Ser216, possibly because only Ser334 is part of an optimal PKA target site, Arg-Arg-Xaa-Ser (45Kennelly P.J. Krebs E.G. J. Biol. Chem. 1991; 266: 15555-15558Abstract Full Text PDF PubMed Google Scholar).Phosphorylation of ER81 solely confined to Ser191/Ser216 has been shown to inhibit ER81 function (32Papoutsopoulou S. Janknecht R. Mol. Cell. Biol. 2000; 20: 7300-7310Crossref PubMed Scopus (73) Google Scholar), explaining why the S191A/S216A mutant is more active than the wild type upon PKA phosphorylation. However, when additional phosphorylation occurs at the MAPK sites (Ser94, Thr139, Thr143, and Ser146), phosphorylation of Ser191/Ser216 actually promotes ER81-dependent transcription (6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar), which is why the S191A/S216A mutant of ER81 is less active than the wild type in the presence of RSK1 and/or BXB. Consistent with RSKs' inability to phosphorylate Ser334 in vivo, mutation of Ser334 to alanine has no effect on the transactivation potential of ER81 upon stimulation of the ERK-MAPK pathway.In contrast to RSK1, PKA readily phosphorylates Ser334 in vivo, thereby reducing ER81's DNA binding activity; a similar reduction of DNA binding activity has been observed upon PKA phosphorylation of hepatocyte nuclear factor-4 and the ER81-related protein ERM within their DNA-binding domains (46Baert J.L. Beaudoin C. Coutte L. de Launoit Y. J. Biol. Chem. 2002; 277: 1002-1012Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 47Viollet B. Kahn A. Raymondjean M. Mol. Cell. Biol. 1997; 17: 4208-4219Crossref PubMed Scopus (147) Google Scholar). Consequently, at low intracellular concentrations of ER81, promoter occupancy by the S334A mutant of ER81 is much higher than for wild-type ER81, resulting in more transcription with the S334A mutant than with wild-type ER81. However, we observed that this does not hold true at high intracellular concentrations of ER81. If the intracellular concentration of ER81 is very high, the law of mass action predicts that even ER81 phosphorylated on Ser334 will effect a high degree of promoter occupancy. Therefore, at high concentrations of ER81, the difference in the abilities of wild-type ER81 and of the S334A mutant to bind to DNA becomes functionally irrelevant. As such, one will observe only a difference in transactivation potential, and this is smaller for the S334A mutant than for wild-type ER81. Thus, phosphorylation of ER81 on Ser334 has a dual function: reducing the DNA-binding ability of ER81 but enhancing its transactivation potential.Normal intracellular ER81 concentrations will probably never be as high as achieved upon transfection of 1000 ng of ER81 expression plasmid as utilized in Fig. 10 A. Therefore, PKA phosphorylation will effectively block DNA binding of ER81 to low affinity sites under physiological conditions, yet at high affinity sites, this reduction in DNA binding affinity may still be irrelevant, as long as the intracellular concentration of ER81 significantly exceeds the dissociation constant (K d) for such sites. Thus, PKA phosphorylation at Ser334 may enforce that ER81 only stimulates transcription from gene promoters containing high affinity DNA-binding sites, a means to ensure specificity of gene activation.CBP and p300 are cofactors that are essential during development and important for the function of a plethora of transcription factors, including ER81 (32Papoutsopoulou S. Janknecht R. Mol. Cell. Biol. 2000; 20: 7300-7310Crossref PubMed Scopus (73) Google Scholar, 41Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar, 42Janknecht R. Histol. Histopathol. 2002; 17: 657-668PubMed Google Scholar). Our data indicate that RSK1 can stimulate the transactivation potential of CBP/p300, thereby potentially affecting gene activation mediated by many different transcription factors. Similar to RSK1, PKA can also stimulate the transactivation potential of CBP/p300 (48Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 49Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar). As such, RSK1 and PKA may not only regulate ER81-dependent transcription by direct phosphorylation of ER81 but also by affecting the cofactors CBP/p300; to answer the question of whether this involves direct phosphorylation of CBP/p300, future studies should be directed at the mapping and mutation of respective phosphorylation sites in CBP/p300. Another way how RSK, potentially in conjunction with the acetyltransferases CBP/p300, can facilitate ER81-dependent transcription is by inducing histone acetylation, thereby loosening up the chromatin structure (26Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. Hanauer A. Allis C.D. Science. 1999; 285: 886-891Crossref PubMed Scopus (424) Google Scholar). Interestingly, regulation of transcription factor activity may not be the sole effect of RSK1-mediated phosphorylation; phosphorylation of the transcription factor CCAAT/enhancer-binding protein β by RSK1 has been reported to promote its association with procaspases 1 and 8, thereby inhibiting their activation in response to proapoptotic stimuli (50Buck M. Poli V. Hunter T. Chojkier M. Mol. Cell. 2001; 8: 807-816Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).PKA can cross-talk with the ERK-MAPK pathway (Fig.11) (51Stork P.J. Schmitt J.M. Trends Cell Biol. 2002; 12: 258-266Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Depending on the cell line studied, PKA may inhibit signaling via the MAPK pathway (52Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (818) Google Scholar, 53Cook S.J. McCormick F. 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Thus, PKA may impact on ERK-MAPK-triggered, ER81-dependent transcription in several ways: by reducing ER81 DNA binding through Ser334 phosphorylation, by enhancing ER81 transactivation potential through Ser334phosphorylation, by acting in concert with RSK1 to phosphorylate Ser191/Ser216, and by either stimulating or inhibiting the ERK-MAPK signaling pathway. How these different mechanisms of action will integrate to a net effect on ER81-dependent transcription is probably cell type-specific. This shows a novel way how neurotransmitters and hormones that activate PKA may attenuate or enhance the response of ER81 target genes to ERK-MAPK-activating growth factors.ER81 is highly expressed in the brain and also in the spinal cord, where it is required for the proper establishment of contacts between sensory and motor neurons (1Brown T.A. McKnight S.L. Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (234) Google Scholar, 8Monte D. Coutte L. Baert J.L. Angeli I. Stehelin D. de Launoit Y. Oncogene. 1995; 11: 771-779PubMed Google Scholar, 11Arber S. Ladle D.R. Lin J.H. Frank E. Jessell T.M. Cell. 2000; 101: 485-498Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 59Lin J.H. Saito T. Anderson D.J. Lance-Jones C. Jessell T.M. Arber S. Cell. 1998; 95: 393-407Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). In neuronal cells, Ca2+ influx through ion channels is a prominent way of relaying signals and memory formation. Ca2+ influx can funnel into both the ERK-MAPK and the PKA signaling pathways (60Bading H. Greenberg M.E. Science. 1991; 253: 912-914Crossref PubMed Scopus (412) Google Scholar, 61Chawla S. Hardingham G.E. Quinn D.R. Bading H. Science. 1998; 281: 1505-1509Crossref PubMed Scopus (375) Google Scholar, 62Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (595) Google Scholar, 63Rusanescu G., Qi, H. Thomas S.M. Brugge J.S. Halegoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar, 64Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Crossref PubMed Scopus (1760) Google Scholar). Thereby, ER81 activity could be regulated by Ca2+ in neuronal cells, which may be important for neuronal function and development.ER81 has been implicated in the development of breast cancer, since it is a downstream target of HER2/Neu (6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar), a proto-oncoprotein known to be capable of inducing breast cancer (15Hung M.C. Lau Y.K. Semin. Oncol. 1999; 26: 51-59PubMed Google Scholar, 16Hynes N.E. Stern D.F. Biochim. Biophys. Acta. 1994; 1198: 165-184Crossref PubMed Scopus (998) Google Scholar), and since ER81 expression is even up-regulated in HER2/Neu-overexpressing mammary tumors (13Shepherd T.G. Kockeritz L. Szrajber M.R. Muller W.J. Hassell J.A. Curr. Biol. 2001; 11: 1739-1748Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). HER2/Neu is a receptor tyrosine kinase that can activate the Ras → Raf → MEK1/2 → ERK-MAPK pathway and therefore also RSKs. As such, RSK phosphorylation of ER81 may contribute to breast tumor formation upon HER2/Neuoverexpression. Notably, ER81 may also be activated through RSKs in tumors characterized by oncogenic Ras mutations, one of the most commonly observed mutations in cancer cells. On the other hand, activation of PKA by cAMP may act either positively or negatively on cell proliferation (65Cho-Chung Y.S. Pepe S. Clair T. Budillon A. Nesterova M. Crit. Rev. Oncol. Hematol. 1995; 21: 33-61Crossref PubMed Scopus (131) Google Scholar). In MDA-MB231 breast cancer cells, for instance, the PKA stimulator oxytocin inhibits cell proliferation (66Cassoni P. Sapino A. Fortunati N. Munaron L. Chini B. Bussolati G. Int. J. Cancer. 1997; 72: 340-344Crossref PubMed Scopus (82) Google Scholar). It is tempting to speculate that one reason for this could be the reduction of ER81 DNA binding activity induced by Ser334phosphorylation and accordingly the suppression of ER81 target gene activity.In conclusion, we have unraveled how two distinct signaling pathways, the growth factor-activated ERK-MAPK pathway and the neurotransmitter/hormone-activated PKA pathway, converge on the transcription factor ER81 and modulate its activity in a complex manner. This knowledge may help us to understand how ER81 functions during ontogenesis and carcinogenesis. The transcription factor ER81 belongs to the family of ETS proteins that are characterized by a conserved, ∼85-amino acid-long, DNA-binding ETS domain and perform many essential functions in homeostasis, signaling response, and development (1Brown T.A. McKnight S.L. Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (234) Google Scholar, 2Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 3Sharrocks A.D. Nat. Rev Mol. Cell. Biol. 2001; 2: 827-837Crossref PubMed Scopus (811) Google Scholar, 4Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar). In addition to its ETS domain, ER81 possesses three further functional domains, an N-terminal and a C-terminal transactivation domain as well as a central inhibitory domain that regulates both transactivation domains (5Janknecht R. Mol. Cell. Biol. 1996; 16: 1550-1556Crossref PubMed Scopus (115) Google Scholar). Gene transcription mediated by ER81 is strongly stimulated by mitogen-activated protein kinases (MAPKs),1 which involves phosphorylation of ER81 at multiple sites (5Janknecht R. Mol. Cell. Biol. 1996; 16: 1550-1556Crossref PubMed Scopus (115) Google Scholar, 6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar). Furthermore, ER81 is stimulated by the cAMP-dependent protein kinase A (PKA), although it is at present unknown whether ER81 is directly phosphorylated by PKA in vivo and how this may affect its ability to up-regulate gene transcription (7Coutte L. Monte D. Imai K. Pouilly L. Dewitte F. Vidaud M. Adamski J. Baert J.L. de Launoit Y. Oncogene. 1999; 18: 6278-6286Crossref PubMed Scopus (22) Google Scholar). Expression of ER81 is both developmentally and tissue-specifically regulated. For instance, high levels ofER81 expression are detectable in the adult brain, heart, or lung, whereas very little is found in liver or skeletal muscle (1Brown T.A. McKnight S.L. Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (234) Google Scholar, 8Monte D. Coutte L. Baert J.L. Angeli I. Stehelin D. de Launoit Y. Oncogene. 1995; 11: 771-779PubMed Google Scholar). Moreover, ER81 expression is undetectable during early embryogenesis yet at later stages is observable at various sites in the embryo, including the central nervous system, eye, heart, lung, salivary gland, kidney, and pituitary gland (9Chotteau-Lelievre A. Desbiens X. Pelczar H. Defossez P.A. de Launoit Y. Oncogene. 1997; 15: 937-952Crossref PubMed Scopus (131) Google Scholar, 10Chotteau-Lelievre A. Dolle P. Peronne V. Coutte L. de Launoit Y. Desbiens X. Mech. Dev. 2001; 108: 191-195Crossref PubMed Scopus (49) Google Scholar). An ER81knock-out mouse revealed that the ER81 protein is required for the proper connection of sensory and motor neurons in the developing spinal cord, and its absence causes severe motor discoordination (11Arber S. Ladle D.R. Lin J.H. Frank E. Jessell T.M. Cell. 2000; 101: 485-498Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). ER81 expression has also been noted in breast tissue, and a subset of breast cancer cell lines even overexpresses ER81. In addition, ER81 expression is up-regulated in mammaries of mice specifically overexpressing the HER2/Neuproto-oncogene in this organ (6Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (96) Google Scholar, 12Baert J.L. Monte D. Musgrove E.A. Albagli O. Sutherland R.L. de Launoit Y. Int. J. Cancer. 1997; 70: 590-597Crossref PubMed Scopus (88) Google Scholar, 13Shepherd T.G. Kockeritz L. Szrajber M.R. Muller W.J. Hassell J.A. Curr. Biol. 2001; 11: 1739-1748Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and ER81 itself enhances the transcription of the HER2/Neu gene (14Bosc D.G. Janknecht R. J. Cell. Biochem. 2002; 86: 174-183Crossref PubMed Scopus (36) Google Scholar). These observations suggest that the ER81 protein may be involved in breast tumor formation. Indeed, ER81 is a downstream target of the transmembrane receptor
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