ERK8 Down-regulates Transactivation of the Glucocorticoid Receptor through Hic-5

Extracellular signal-regulated kinase 8 (ERK8) is the most recently identified member of the ERK subfamily of MAPKs. Although other members of the ERK subfamily are established regulators of signaling pathways involved in cell growth and/or differentiation, less is known about ERK8. To understand the cellular function of ERK8, a yeast two-hybrid screen of a human lung library was performed to identify binding partners. One binding partner identified was Hic-5 (also known as ARA55), a multiple LIM domain containing protein implicated in focal adhesion signaling and the regulation of specific nuclear receptors, including the androgen receptor and the glucocorticoid receptor (GR). Co-immunoprecipitation experiments in mammalian cells confirmed the interaction between Hic-5 and both ERK8 and its rodent ortholog ERK7. The C-terminal region of ERK8 was not required for the interaction. Although the LIM3 and LIM4 domains of Hic-5 were sufficient and required for this interaction, the specific zinc finger motifs in these domains were not. Transcriptional activation reporter assays revealed that ERK8 can negatively regulate transcriptional co-activation of androgen receptor and GRα by Hic-5 in a kinase-independent manner. Knockdown of endogenous ERK8 in human airway epithelial cells enhanced dexamethasone-stimulated transcriptional activity of endogenous GR. Transcriptional regulation of GRα and interaction with its ligand binding domain by ERK8 were dependent on the presence of Hic-5. These results provide the first physiological function for human ERK8 as a negative regulator of human GRα, acting through Hic-5, and suggest a broader role for ERK8 in the regulation of nuclear receptors beyond estrogen receptor α. Extracellular signal-regulated kinase 8 (ERK8) is the most recently identified member of the ERK subfamily of MAPKs. Although other members of the ERK subfamily are established regulators of signaling pathways involved in cell growth and/or differentiation, less is known about ERK8. To understand the cellular function of ERK8, a yeast two-hybrid screen of a human lung library was performed to identify binding partners. One binding partner identified was Hic-5 (also known as ARA55), a multiple LIM domain containing protein implicated in focal adhesion signaling and the regulation of specific nuclear receptors, including the androgen receptor and the glucocorticoid receptor (GR). Co-immunoprecipitation experiments in mammalian cells confirmed the interaction between Hic-5 and both ERK8 and its rodent ortholog ERK7. The C-terminal region of ERK8 was not required for the interaction. Although the LIM3 and LIM4 domains of Hic-5 were sufficient and required for this interaction, the specific zinc finger motifs in these domains were not. Transcriptional activation reporter assays revealed that ERK8 can negatively regulate transcriptional co-activation of androgen receptor and GRα by Hic-5 in a kinase-independent manner. Knockdown of endogenous ERK8 in human airway epithelial cells enhanced dexamethasone-stimulated transcriptional activity of endogenous GR. Transcriptional regulation of GRα and interaction with its ligand binding domain by ERK8 were dependent on the presence of Hic-5. These results provide the first physiological function for human ERK8 as a negative regulator of human GRα, acting through Hic-5, and suggest a broader role for ERK8 in the regulation of nuclear receptors beyond estrogen receptor α. Since the identification of ERK1 and ERK2, additional ERK 5The abbreviations used are: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ARA, androgen receptor activator; AR, androgen receptor; ERα, estrogen receptor α; GR, glucocorticoid receptor; PPARγ, peroxisome proliferator-activated receptor-γ; LBD, ligand binding domain; DHT, dihydrotestosterone; Hic-5, hydrogen peroxide inducible clone-5; FAK, focal adhesion kinase; Pyk2, proline-rich tyrosine kinase; MMTV, mouse mammary tumor virus; ARE, androgen receptor response element; GST, glutathione S-transferase; αENaC, α subunit epithelial sodium channel; HA, hemagglutinin; FBS, fetal bovine serum; siRNA, short interfering RNA; RT, reverse transcription; h, human; UAS, upstream activating sequence.5The abbreviations used are: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ARA, androgen receptor activator; AR, androgen receptor; ERα, estrogen receptor α; GR, glucocorticoid receptor; PPARγ, peroxisome proliferator-activated receptor-γ; LBD, ligand binding domain; DHT, dihydrotestosterone; Hic-5, hydrogen peroxide inducible clone-5; FAK, focal adhesion kinase; Pyk2, proline-rich tyrosine kinase; MMTV, mouse mammary tumor virus; ARE, androgen receptor response element; GST, glutathione S-transferase; αENaC, α subunit epithelial sodium channel; HA, hemagglutinin; FBS, fetal bovine serum; siRNA, short interfering RNA; RT, reverse transcription; h, human; UAS, upstream activating sequence. family members have been identified and extensively studied. ERK1 and ERK2 are classically activated by growth factors and mediate signals leading to proliferation or differentiation of most cell types (1Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 2Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2278) Google Scholar). These founding members of the ERK subfamily of MAPKs have a characteristic threonine-glutamine-tyrosine (TEY) motif that requires dual phosphorylation of the threonine and tyrosine residues for full kinase activity. ERK3 is an atypical member of the ERK/MAPK family having an SEG activation motif. Recently, it has been found to interact with and facilitate MAPK-activated protein kinase 5 (MK5) autoactivation (3Seternes O.M. Mikalsen T. Johansen B. Michaelsen E. Armstrong C.G. Morrice N.A. Turgeon B. Meloche S. Moens U. Keyse S.M. EMBO J. 2004; 23: 4780-4791Crossref PubMed Scopus (110) Google Scholar, 4Schumacher S. Laass K. Kant S. Shi Y. Visel A. Gruber A.D. Kotlyarov A. Gaestel M. EMBO J. 2004; 23: 4770-4779Crossref PubMed Scopus (99) Google Scholar). This interaction may play a role in embryonic development. These proteins have overlapping expression patterns in the mouse and is of ERK3 in an mouse in embryonic (3Seternes O.M. Mikalsen T. Johansen B. Michaelsen E. Armstrong C.G. Morrice N.A. Turgeon B. Meloche S. Moens U. Keyse S.M. EMBO J. 2004; 23: 4780-4791Crossref PubMed Scopus (110) Google Scholar, 4Schumacher S. Laass K. Kant S. Shi Y. Visel A. Gruber A.D. Kotlyarov A. Gaestel M. EMBO J. 2004; 23: 4770-4779Crossref PubMed Scopus (99) Google Scholar). was identified by with an ERK1 J. PubMed Scopus Google Scholar). is known about including its 2004; PubMed Scopus Google Scholar). has a activation motif and was identified as an ERK J. M. J. Biol. PubMed Scopus Google Scholar). it has been found to activated a growth signaling and to play an role in and development. has a activation motif and is a member of the family of MAPKs known as Y. J. Res. PubMed Scopus Google Scholar, A. U. S. A. PubMed Scopus Google Scholar). has a activation motif is an atypical member of the ERK family in that it kinase activity of a known upstream K. J. Biol. PubMed Scopus Google Scholar). ERK8 is the member of this subfamily to identified M. B. M. J. Biol. PubMed Scopus Google Scholar). Although a of and of human ERK8 that it rodent revealed that these are PubMed Scopus Google Scholar, U. S. A. PubMed Scopus Google Scholar). are in with on human M. B. M. J. Biol. PubMed Scopus Google and on mouse are The of and for ERK8 and ERK3 and are the founding ERK family ERK1 and ERK2, of C-terminal that have of was found to cell growth of kinase activity dependent on its C-terminal region Biol. PubMed Scopus Google Scholar). of was found to of the human estrogen receptor α a in a B. Biol. 23: PubMed Scopus Google Scholar). of a human ERK8, with in human cell and human B. Biol. 23: PubMed Scopus Google a role for and ERK8 in is a member of the nuclear receptor of members of this family the androgen receptor the glucocorticoid receptor α the the the the receptors, and the peroxisome proliferator-activated of expression by nuclear receptors is by binding of the specific ligand or of on the nuclear and binding to specific on the or with the nuclear receptors to regulate transcriptional activity. the yeast two-hybrid to screen a human lung identified nuclear receptor hydrogen peroxide inducible known as androgen receptor Hic-5 was identified as a hydrogen peroxide or growth inducible in a mouse cell of Hic-5 in a M. J. T. K. J. Biol. PubMed Google Scholar). Hic-5 was found to to focal and interact with focal adhesion kinase through its N-terminal region K. M. K. J. 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These that the the of and/or transcriptional activity by ERK8 that of transcriptional results suggest a of a is with the of LIM domain containing proteins to facilitate Cell Biol. 2004; PubMed Scopus Google Scholar). the of Hic-5 and GRα, LIM3 and LIM4 have been to required for binding of Hic-5 to GRα a yeast two-hybrid J. J. 2004; PubMed Scopus Google Scholar). These LIM domains or with the N-terminal region of Hic-5, were not sufficient for GRα binding that the other LIM domains to the binding J. J. 2004; PubMed Scopus Google Scholar). the of Hic-5 and ERK8, both the LIM3 and LIM4 domains are required for the zinc finger these are not required for this interaction. in the of GRα domain is sufficient for binding of ERK8. 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Biol. 1997; PubMed Scopus Google Scholar). is by ERK and in on of ERK1 and ERK2 or pathways has been to transcriptional activity in mammalian of ERK1 and ERK2 or pathways transcriptional activity. the to the of to that ERK1 and/or ERK2 phosphorylation of was not required for the of by these M.J. U. S. A. 1998; PubMed Scopus Google Scholar). Although of by to on phosphorylation of GR, ERK2 of phosphorylation of a a role for ERK8 as a negative regulator of is with that of other family This ERK8 as a regulator of the transcriptional activity of GRα and other nuclear receptors, as AR, through its to interact with Hic-5. ERK8 and Hic-5 are in lung with other M. B. M. J. Biol. PubMed Scopus Google Scholar, M. J. T. K. J. Biol. PubMed Google Scholar). identified expression of both ERK8 and Hic-5 in human airway epithelial cells and found that ERK8 can transcriptional activity of of endogenous ERK8 enhanced GRα and expression of in these cells a GRα is known to play an role in lung by the response K. 2004; PubMed Scopus Google and in and lung PubMed Scopus Google Scholar). human GRα is in of airway M.H. J. PubMed Scopus Google Scholar). human lung is in the of the airway and as as the of T. J. PubMed Scopus Google Scholar). The subunit is of a that sodium in lung as as other J. 1997; Google Scholar). Although is in human lung to E. M. U. S. A. PubMed Scopus Google its expression the The of this and the of subunit of lung E. J. A. PubMed Scopus Google this sodium as a regulator of lung Recently, Hic-5 was implicated in the differentiation of S. E. K. PubMed Scopus Google Scholar). Hic-5, as a of PPARγ, was found to regulate expression of characteristic of as to other with development. are to ERK8 and Hic-5 have a role in the differentiation of airway acting through nuclear and for and for of this the of Cancer and in the of