Dual Regulation of Phosphorylation and Dephosphorylation of C/EBPβ Modulate Its Transcriptional Activation and DNA Binding in Response to Growth Hormone
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m206886200
ISSN1083-351X
AutoresGraciela Piwien‐Pilipuk, Ormond A. MacDougald, Jessica Schwartz,
Tópico(s)ATP Synthase and ATPases Research
ResumoThe phosphorylation state of transcription factors is a critical determinant of their function. C/EBPβ occurs in cells as the transcriptional activator liver-enriched activating protein (LAP) and in the truncated form liver-enriched inhibitory protein (LIP) that inhibits transcription. Analysis of C/EBPβ phosphorylation by isoelectric focusing (IEF) shows that LAP is present in multiple forms, each with a different degree of phosphorylation in 3T3-F442A fibroblasts. Growth hormone (GH) treatment induces a new band near the negative pole, consistent with GH-promoted dephosphorylation of LAP. In addition, bands near the positive pole are rapidly and transiently induced, suggesting that GH also stimulates phosphorylation at some site(s) on LAP. C/EBPβ contains a highly conserved MAPK consensus site that corresponds to Thr188 in murine (m) LAP and Thr37 in mLIP. Immunoblotting with antiphosphopeptide antibodies specific for Thr188/37 of C/EBPβ (anti-P-C/EBPβ) shows that GH rapidly and transiently promotes phosphorylation of mLAP and mLIP on the MAPK site. MEK inhibitors prevent this GH-promoted phosphorylation of LAP and LIP, suggesting that such phosphorylation depends on GH-activated MAPK signaling. Mutation of Thr235 to Ala in the homologous MAPK site of human (h) LAP (hLAPT235A) inhibits transcription mediated by the c-fos promoter in response to GH, indicating that phosphorylation at the MAPK site is required for LAP to be transcriptionally active in the context of GH-stimulated activation of the c-fos promoter. Complexes bound to the c-fos C/EBP site transiently contain C/EBPβ phosphorylated at the MAPK site. As phosphorylation subsides, the binding of less transcriptionally active forms of LAP increases, consistent with the transient nature of c-fos stimulation by GH and other growth factors. Thus, both phosphorylation and dephosphorylation of C/EBPβ, in response to a single physiological stimulus such as GH, coordinately modulate the ability of C/EBPβ to activate transcription by modulating its DNA binding activity and its transactivation capacity. The phosphorylation state of transcription factors is a critical determinant of their function. C/EBPβ occurs in cells as the transcriptional activator liver-enriched activating protein (LAP) and in the truncated form liver-enriched inhibitory protein (LIP) that inhibits transcription. Analysis of C/EBPβ phosphorylation by isoelectric focusing (IEF) shows that LAP is present in multiple forms, each with a different degree of phosphorylation in 3T3-F442A fibroblasts. Growth hormone (GH) treatment induces a new band near the negative pole, consistent with GH-promoted dephosphorylation of LAP. In addition, bands near the positive pole are rapidly and transiently induced, suggesting that GH also stimulates phosphorylation at some site(s) on LAP. C/EBPβ contains a highly conserved MAPK consensus site that corresponds to Thr188 in murine (m) LAP and Thr37 in mLIP. Immunoblotting with antiphosphopeptide antibodies specific for Thr188/37 of C/EBPβ (anti-P-C/EBPβ) shows that GH rapidly and transiently promotes phosphorylation of mLAP and mLIP on the MAPK site. MEK inhibitors prevent this GH-promoted phosphorylation of LAP and LIP, suggesting that such phosphorylation depends on GH-activated MAPK signaling. Mutation of Thr235 to Ala in the homologous MAPK site of human (h) LAP (hLAPT235A) inhibits transcription mediated by the c-fos promoter in response to GH, indicating that phosphorylation at the MAPK site is required for LAP to be transcriptionally active in the context of GH-stimulated activation of the c-fos promoter. Complexes bound to the c-fos C/EBP site transiently contain C/EBPβ phosphorylated at the MAPK site. As phosphorylation subsides, the binding of less transcriptionally active forms of LAP increases, consistent with the transient nature of c-fos stimulation by GH and other growth factors. Thus, both phosphorylation and dephosphorylation of C/EBPβ, in response to a single physiological stimulus such as GH, coordinately modulate the ability of C/EBPβ to activate transcription by modulating its DNA binding activity and its transactivation capacity. Phosphorylation of transcription factors is a critical mode of regulation of gene expression (1Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1120) Google Scholar). Phosphorylation can modulate transcription factor activity by regulating subcellular localization (e.g. signal transducers and activators of transcription) (2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar, 3Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar), modulating DNA binding activity (e.g. c-Jun, CCAAT/enhancer-binding protein β (C/EBPβ) 1The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein β; LIP, liver-enriched inhibitory protein; LAP, liver-enriched activating protein; GSK-2, glycogen synthase kinase 3; PKC, protein kinase C; GH, growth hormone; IEF, isoelectric focusing; MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; GHR, growth hormone receptor; CMV, cytomegalovirus; mLAP, murine liver-enriched activating protein; RSV, Rous sarcoma virus; ERK, extracellular signal-regulated kinase; SRE, serum response element (4Boyle W.J. Smeal T. Delize L.H. Angel P. Woodgett J.R. Korin M. Hunter T. Cell. 1991; 64: 573-584Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 5Piwien-Pilipuk G. Van Mater D. Ross S.E. 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C/EBPβ, a member of the bZIP family of transcription factors, is present in cells as three alternate translation products: 35- and 32-kDa proteins in murine cells known as LAP (liver-enriched activating protein) and a 20-kDa protein known as LIP (liver-enriched inhibitory protein) (13Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar) (Fig. 2 A). The N-terminal region of LAP corresponds to the transactivation domain, whereas LIP lacks this transactivation domain and acts as an inhibitor of transcription (13Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar). C/EBPβ plays an important role during differentiation of a number of cell types, including adipocytes (14Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1350) Google Scholar, 15Lin F.-T. Lane M.D. Genes Dev. 1992; 6: 533-544Crossref PubMed Scopus (275) Google Scholar, 16Darlington G.J. 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Genes Dev. 1997; 11: 2153-2162Crossref PubMed Scopus (346) Google Scholar). C/EBPβ knockout mice have alterations in glucose homeostasis (23Croniger C. Trus M. Lysek-Stupp K. Cohen H. Liu Y. Darlington G.J. Poli V. Hanson R.W. Reshef L. J. Biol. Chem. 1997; 272: 26306-26312Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), show defects in macrophage-dependent antibacterial and antitumor defenses (19Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (472) Google Scholar), develop lymphoproliferative disorders (24Screpanti I. Romani L. Musiani P. Modesti A. Fattori E. Lazzaro D. Sellitto C. Scarpa S. Bellavia D. Lattanzio G. EMBO J. 1995; 14: 1932-1941Crossref PubMed Scopus (376) Google Scholar, 25Chen X. Liu W. Ambrosino C. Ruocco M.R. Poli V. Romani L. Quinto I. Barbieri S. Holmes K.L. Venuta S. Scala G. Blood. 1997; 90: 156-164PubMed Google Scholar), and have female infertility (22Sterneck E. Tessarollo L. Johnson P.F. Genes Dev. 1997; 11: 2153-2162Crossref PubMed Scopus (346) Google Scholar). The participation of C/EBPβ in such a wide variety of physiological events testifies to its versatility, and suggests that the multiple features of its regulation contribute to mediating varied biological outcomes. C/EBPβ contains phosphorylation sites for multiple protein kinases, including Ras-MAPK for human (h)LAP at Thr235 (7Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (518) Google Scholar), ERK-2 on Thr188 of rat (r) LAP (26Hanlon M. Sturgill T.W. Sealy L. J. Biol. Chem. 2001; 276: 38449-38456Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), glycogen-synthase kinase 3 (GSK-3) at a putative site on Ser184 of murine (m)LAP and Ser33 of mLIP (5Piwien-Pilipuk G. Van Mater D. Ross S.E. MacDougald O.A. Schwartz J. J. Biol. Chem. 2001; 276: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), calcium/calmodulin-dependent protein kinase on Ser276 of C/EBPβ (27Wegner M. Cao Z. Rosenfeld M.G. Science. 1992; 256: 370-373Crossref PubMed Scopus (308) Google Scholar), protein kinase C (PKC) on Ser105 and Ser240, protein kinase A (PKA) on Ser105, Ser299, Ser240 of LAP (nuclear factor-interleukin 6) (8Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar, 28Trautwein C. van der Geer P. Karin M. Hunter T. Chojkier M. J. Clin. Invest. 1994; 93: 2554-2561Crossref PubMed Scopus (129) Google Scholar, 29Chinery R. Brockman J.A. Dransfield D.T. Coffey R.J. J. Biol. 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Chem. 2001; 276: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Phosphorylation on Ser105 of rLAP or Thr235 of hLAP is also reported to be a key determinant of its transactivation capacity (7Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (518) Google Scholar, 8Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar, 30Buck M. Poli V. van der Geer P. Chojkier M. Hunter T. Mol. Cell. 1999; 4: 1087-1092Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). It has been postulated that C/EBPβ contains negative regulatory regions, and that phosphorylation(s) within these domains may regulate C/EBPβ function. Thus, C/EBPβ may be present in cells as a repressed transcription factor that becomes activated upon phosphorylation (31Kowenz-Leutz E. Twamley G. Ansieau S. Leutz A. Genes Dev. 1994; 8: 2781-2791Crossref PubMed Scopus (210) Google Scholar, 32Williams S.C. Baer M. Dillner A.J. Johnson P.F. EMBO J. 1995; 14: 3170-3183Crossref PubMed Scopus (200) Google Scholar). Regulation of C/EBPβ by growth hormone (GH) (5Piwien-Pilipuk G. Van Mater D. Ross S.E. MacDougald O.A. Schwartz J. J. Biol. Chem. 2001; 276: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 33Clarkson R.W.E. Chen C.M. Harrison S. Wells C. Muscat G.E.O. Waters M.J. Mol. Endocrinol. 1995; 9: 108-120Crossref PubMed Scopus (83) Google Scholar, 34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) contributes to GH-mediated regulation of transcription of the proto-oncogene c-fos (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), which participates in cellular growth pathways (35Muller R. Biochim. Biophys. Acta. 1986; 823: 207-225PubMed Google Scholar). Furthermore, GH has been found to promote dephosphorylation of mLAP and mLIP (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The GH-induced dephosphorylation of C/EBPβ is mediated, at least in part, by GH-stimulated phosphatidylinositol 3-kinase/Akt signaling, resulting in inhibition of GSK-3 (5Piwien-Pilipuk G. Van Mater D. Ross S.E. MacDougald O.A. Schwartz J. J. Biol. Chem. 2001; 276: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Dephosphorylation of LAP leads to an increase in binding of LAP-containing complexes to C/EBP sites in the c-fos andaP2 promoters. Conversely, GSK-3-induced phosphorylation of LAP decreases binding to the c-fos C/EBP site (5Piwien-Pilipuk G. Van Mater D. Ross S.E. MacDougald O.A. Schwartz J. J. Biol. Chem. 2001; 276: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, GH-promoted inhibition of GSK-3 is required for GH to stimulate c-fos promoter activity. The present study examines which among the multiple phosphorylation site(s) of LAP and LIP are functionally important for transcriptional activation, in the context of GH-stimulated c-fos promoter activation. By isoelectric focusing (IEF) LAP was found to be present in 3T3-F442A cells in several forms exhibiting different degrees of phosphorylation. IEF revealed that GH promotes not only dephosphorylation, but also a rapid and transient phosphorylation of murine C/EBPβ on Thr188 in mLAP and Thr37 in mLIP, sites that correspond to the same conserved mitogen-activated protein kinase (MAPK) consensus sequence in each form. Mutational analysis of hLAP at Thr235 (homologous to Thr188 in mLAP) indicates that this phosphorylation is critically important for LAP to be transcriptionally active in the context of the GH-stimulated c-fos promoter. Changes in DNA binding associated with changing levels of phosphorylated and dephosphorylated C/EBPβ may contribute to the transient pattern of GH-stimulated c-fos expression. Thus, C/EBPβ is a highly phosphorylated protein whose function is dynamically modulated by both phosphorylation and dephosphorylation in response to a single physiological regulator. Murine 3T3-F442A preadipocyte fibroblasts, which are highly responsive to GH through endogenous GH receptors (36Schwartz J. Carter-Su C. Endocrinology. 1988; 122: 2247-2256Crossref PubMed Scopus (40) Google Scholar), were provided by Dr. H. Green (Harvard University) and Dr. M. Sonenberg (Sloan-Kettering). Chinese hamster ovary cells expressing rat GHR containing the N-terminal half of the cytoplasmic domain (CHO-GHR) were provided by G. Norstedt (Karolinska Institute, Stockholm, Sweden) and N. Billestrup (Hagedorn Laboratory, Gentofte, Denmark) (37Billestrup N. Allevato G. Norstedt G. Moldrup A. Nielsen J.H. Proc. Soc. Exp. Biol. Med. 1994; 206: 205-209Crossref PubMed Scopus (14) Google Scholar). In CHO-GHR cells GH induces c-fos mRNA and stimulates transcriptional activation via the SRE-C/EBP site to the same extent as in CHO cells expressing full-length GHR (38Gong T.-W.L. Meyer D.J. Liao J. Hodge C.L. Campbell G.S. Wang X. Billestrup N. Carter-Su C. Schwartz J. Endocrinology. 1998; 139: 1863-1871Crossref PubMed Scopus (38) Google Scholar). Human embryonic kidney 293T cells were provided by Dr. M. Lazar (University of Pennsylvania). Recombinant human GH was provided by Eli Lilly, Inc. Culture media, calf serum, fetal calf serum, G418,l-glutamine, and antibiotic-antimycotic were purchased from Invitrogen. Bovine serum albumin (CRG7) was purchased from Intergen. The MEK inhibitor U0126 and luciferin were purchased from Promega. PD098059 was purchased from Sigma, and alkaline phosphatase and CompleteTM protease inhibitor mixture (EDTA-free) were purchased from Roche Molecular Biochemicals. Bradford reagent (Sigma) was used to determine the concentration of protein in cell or nuclear extracts for all experiments. [α-32P]dATP was purchased from PerkinElmer Life Sciences. β-Galactosidase chemiluminescence reagent was purchased from Tropix. The ECL detection system was purchased from Amersham Biosciences. 3T3-F442A preadipocyte fibroblasts and 293T human embryonic kidney cells were grown in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose and 8% calf serum in an atmosphere of 10% CO2, 90% air at 37 °C. CHO-GHR cells were grown in Ham's F-12 medium containing 10% fetal calf serum and 0.5 mg/ml G418 in an atmosphere of 5% CO2, 95% air at 37 °C. All media were supplemented with 1 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin. Prior to treatment, cells were deprived of serum by incubation overnight in the appropriate medium containing 1% bovine serum albumin instead of serum. Then cells were incubated with or without GH at 500 ng/ml (23 nm) or as indicated in figure legends. A plasmid encoding mLAP driven by the CMV promoter (CMV-mLAP) was kindly provided by U. Schibler (University of Geneva) and L. Sealy (Vanderbilt University). Plasmids encoding hLAP (also known as NF-IL-6) and a mutant hLAP where Thr235 was mutated to Ala (hLAP-T235A) were kindly provided by Dr. S. Akira (Osaka University, Japan) (7Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (518) Google Scholar) courtesy of L. Sealy. The plasmid fos-Luc, provided by Dr. W. Wharton (University of S. Florida) contains 379 bp of the mouse c-fos promoter immediately 5′ of the transcription start site, cloned upstream of the luciferase gene (39Harvat B.L. Wharton W. Cell Growth Differ. 1995; 6: 955-964PubMed Google Scholar). The plasmid RSV-β-galactosidase was provided by Dr. M. Uhler (University of Michigan). pcDNA3.1 was purchased from Clontech. Specific rabbit polyclonal antibodies against a synthetic phospho-Thr235 peptide (keyhole limpecthemocyanin coupled) corresponding to residues surrounding Thr235 of human C/EBPβ (identical to Thr188 in mC/EBPβ) (anti-P-C/EBPβ) were provided by Cell Signaling Technology, Inc. (Beverly, MA). Specific rabbit polyclonal antibodies against a peptide corresponding to amino acids 278–295 at the C terminus of C/EBPβ (anti-C/EBPβ) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against phosphorylated extracellular signal-regulated kinases (ERK)1/ERK2 (anti-P-ERK1/2) or antibodies against total ERK1/ERK2 (anti-ERK1/2) were purchased from Promega. Nuclei from 3T3-F442A fibroblasts were prepared as previously described (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Nuclei were lysed in urea buffer (9m urea, 1% Nonidet P-40, 1% dithiothreitol). Nuclear lysate was sonicated and centrifuged at 13,500 rpm at 4 °C for 15 min, and the supernatant was stored at −80 °C. For alkaline phosphatase treatment, nuclei, prepared as described (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), were incubated in the presence or absence of 200 units of alkaline phosphatase for 1 h at 37 °C; the reaction was stopped by adding an equivalent volume of 9 m urea buffer. Polyacrylamide-urea minigels were made according to the manufacturer's instructions (Bio-Rad) with a 1:4:1 mixture of pH 3 to 10, pH 5 to 8, and pH 8 to 10.5 ampholytes, respectively. Protein samples (15–20 μg) were loaded and focused for 15 min at 100 V, 15 min at 200 V, and 1.5 h at 450 V. Proteins were then transferred to ImmobilonTM membrane for immunoblot analysis using anti-C/EBPβ (1:1000) or anti-P-C/EBPβ (1:500). Whole cell lysates from 3T3-F442A fibroblasts were prepared and analyzed (35–40 μg) by immunoblotting as previously described (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) using anti-P-C/EBPβ (1:500) or anti-C/EBPβ (1:1000). In some experiments 3T3-F442A cells were preincubated 30 min with the indicated concentrations of the MEK inhibitors U0126 or PD098059 prior to addition of GH (5 min). Membranes were stripped and reprobed with anti-P-ERK 1/2 (1:5000) or anti-ERK 1/2 (1:5000), as previously described (12Hodge C. Liao J. Stofega M. Guan K. Carter-Su C. Schwartz J. J. Biol. Chem. 1998; 273: 31327-31336Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The apparentM r are based on prestained molecular weight standards (Invitrogen). Cell extracts enriched in mLAP, hLAP, or hLAP-T235A were obtained by transfection of 293T cells with 1 μg of CMV-mLAP, CMV-hLAP, or CMV-hLAP-T235A DNA, as previously described (5Piwien-Pilipuk G. Van Mater D. Ross S.E. MacDougald O.A. Schwartz J. J. Biol. Chem. 2001; 276: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Nuclear extracts from 3T3-F442A cells were prepared as previously described (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and a volume of nuclear extracts containing 8–10 μg of protein was used for EMSA. Binding reactions proceeded for 30 min at room temperature with 32P-labeled oligonucleotide containing the c-fos C/EBP site and flanking SRE (C/EBP-SRE) (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) or the C/EBP site of the aP2 promoter (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 40Christy R.J. Yang V.W. Ntambi J.M. Geiman D.E. Landschulz W.H. Friedman A.D. Nakabeppu Y. Kelly T.J. Lane M.D. Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (467) Google Scholar) as described (5Piwien-Pilipuk G. Van Mater D. Ross S.E. MacDougald O.A. Schwartz J. J. Biol. Chem. 2001; 276: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In some experiments, cell extracts were incubated for 20 min at room temperature with 1 μl of anti-CEBPβ (1:10 final dilution), anti-P-CEBPβ (1:10 final dilution), or nonimmune (1:10 final dilution) antiserum prior to EMSA, as indicated in the figure legends. Complexes were separated by nondenaturing 7% PAGE followed by autoradiography. CHO-GHR cells (1 × 105 cells/35 mm well) were transiently transfected by calcium phosphate coprecipitation (41Chen D. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar) with c-fos-Luc (0.4 μg) and RSV-β-galactosidase (0.1 μg) plasmids, in the presence or absence of CMV-hLAP (2 ng), CMV-hLAPT235A (2 ng), or corresponding amounts of pcDNA3.1 vector per well. Twenty-four h after transfection, cells were deprived of serum by incubation in medium containing 1% bovine serum albumin for 18 h prior to treatment. Cell lysates were prepared as described previously (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and luciferase or β-galactosidase activity was measured using an Opticomp Luminometer. The luciferase values were normalized to β-galactosidase activity. Each condition was tested in duplicate in each experiment. A two-sample t test was used (SigmaStat) to judge statistical significance. A value of p < 0.05 was considered to be statistically significant. GH promotes a rapid and transient dephosphorylation of endogenous mLAP and mLIP in 3T3-F442A preadipocytes (34Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Similar promotion of LAP and LIP dephosphorylation was also induced by insulin-like growth factor-1 and insulin. 2A. Sieh, A. Sachdeva, and J. Schwartz, unpublished data. However, in all cases, a considerable amount of phosphorylated C/EBPβ is still evident on immunoblots. Because both LAP and LIP contain phosphorylation sites for multiple kinases, C/EBPβ may be present as multiple phosphorylated species not resolved by conventional SDS-PAGE. IEF was used to analyze C/EBPβ, because IEF separates proteins according to charge density in a pH gradient generated in the gel. mLAP overexpressed in 293T cells is resolved into at least 8 bands by IEF (Fig.1 A, lane 1). This suggests that overexpressed mLAP is present in multiple forms with different degrees of phosphorylation. As expected, overexpressed mLIP is detected as a single band that runs close to the negative pole (Fig.1 A, lane 2), consistent with LIP being a basic protein (isoelectric point = 10.42), which is not further resolved under the conditions of IEF used. Endogenous LAP from untreated 3T3-F442A fibroblasts can be resolved into at least 4 bands (Fig. 1 B, lane 1, bands b-e), indicating that endogenous mLAP is present in quiescent cells in multiple forms with different degrees of phosphorylation. The dense uppermost band at the basic end (Fig. 1 B,arrowhead) in this and subsequent figures is thought to correspond to the migration of endogenous LIP because it coincides with migration of overexpressed mLIP. The pattern of migration of mLAP on IEF changes in several ways upon treatment of the cells with GH. Some bands that migrate closer to the acidic end (positive pole) increase in intensity over 15–60 min (Fig.1 B, lanes 2–4, bands b-e). Other bands that migrate toward the acidic end are induced (bands f andg) by 15–30 min after GH treatment. These more acidic bands may correspond to more phosphorylated forms of LAP, because upon alkaline phosphatase treatment of control 3T3-F442A nuclear extracts bands b, c, d, and e are reduced in intensity, shifting to band a (Fig. 1 B,lane 6 versus 5). The new band a close to the basic end (Fig. 1 B, lanes 3–4, band a) appears after 30–60 min of GH treatment. The GH-induced banda is thought to correspond to a less phosphorylated or dephosphorylated form of LAP. In support of this, band acomigrates with the band that appears upon alkaline phosphatase treatment of control extracts (Fig. 1 B, lane 6, band a), suggesting that band a corresponds to a dephosphorylated form of mLAP. Thus, IEF reveals multiple phosph
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