Activation of the Human DNA Polymerase β Promoter by a DNA-alkylating Agent through Induced Phosphorylation of cAMP Response Element-binding Protein-1
1996; Elsevier BV; Volume: 271; Issue: 31 Linguagem: Inglês
10.1074/jbc.271.31.18508
ISSN1083-351X
AutoresSatya Narayan, Feng He, Samuel H. Wilson,
Tópico(s)HIV/AIDS drug development and treatment
ResumoTreatment of cells with the DNA-alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces expression of the endogenous mammalian DNA polymerase β (β-pol) gene and of the cloned promoter in transient expression studies. The lone cAMP response element (CRE) in the core promoter, along with functional protein kinase A, is critical for the MNNG-induced up-regulation. Recently, we described a kinetic mechanism for transcriptional regulation of the β-pol promoter in vitro and found that CRE-binding protein (CREB) from MNNG-treated cells differentially up-regulates the promoter by stimulating formation of closed preinitiation complex (RPc). Here, using a CRE-dependent chimeric β-pol promoter, we purified the RPc assembled with nuclear extract from MNNG-treated and control HeLa cells. Comparison of proteins in the purified RPc samples revealed that the MNNG induction is associated with a strong increase in the Ser133-phosphorylated form of recombinant CREB (CREB-1). CREB depletion of the nuclear extracts diminished transcriptional activity, and addition of purified Ser133-phosphorylated CREB-1 restored activity, whereas unphosphorylated CREB-1 did not. Addition of phosphorylated CREB-1 to the control cell extract mimicked the MNNG-induced up-regulation of transcriptional activity. These results indicate that phosphorylation of CREB-1 is the probable mechanism of activation of the β-pol promoter after treatment of cells with the DNA-alkylating agent MNNG. Treatment of cells with the DNA-alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces expression of the endogenous mammalian DNA polymerase β (β-pol) gene and of the cloned promoter in transient expression studies. The lone cAMP response element (CRE) in the core promoter, along with functional protein kinase A, is critical for the MNNG-induced up-regulation. Recently, we described a kinetic mechanism for transcriptional regulation of the β-pol promoter in vitro and found that CRE-binding protein (CREB) from MNNG-treated cells differentially up-regulates the promoter by stimulating formation of closed preinitiation complex (RPc). Here, using a CRE-dependent chimeric β-pol promoter, we purified the RPc assembled with nuclear extract from MNNG-treated and control HeLa cells. Comparison of proteins in the purified RPc samples revealed that the MNNG induction is associated with a strong increase in the Ser133-phosphorylated form of recombinant CREB (CREB-1). CREB depletion of the nuclear extracts diminished transcriptional activity, and addition of purified Ser133-phosphorylated CREB-1 restored activity, whereas unphosphorylated CREB-1 did not. Addition of phosphorylated CREB-1 to the control cell extract mimicked the MNNG-induced up-regulation of transcriptional activity. These results indicate that phosphorylation of CREB-1 is the probable mechanism of activation of the β-pol promoter after treatment of cells with the DNA-alkylating agent MNNG. INTRODUCTIONDNA polymerase β (β-pol) 1The abbreviations used are: β-polDNA polymerase βATFactivating transcription factorCREcAMP response elementCREBCRE-binding proteinMNNGN-methyl-N′-nitro-N-nitrosoguanidinePKAprotein kinase ANEHeLa nuclear extractNEdCREB-depleted HeLa nuclear extractRPcclosed preinitiation complexPAGEpolyacrylamide gel electrophoresisphospho-CREB-1protein kinase A-phosphorylated recombinant CREB-1CBPCREB-binding protein. is a eukaryotic cellular polymerase involved in "gap-filling" synthesis during base excision repair (for review, see Ref. 1Singhal R.K. Wilson S.H. J. Biol. Chem. 1993; 268: 15906-15911Abstract Full Text PDF PubMed Google Scholar). Accordingly, β-pol has been found to promote repair of a G:U base pair (2Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), a monofunctional DNA adduct (3Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (260) Google Scholar, 4Jenkins T.M. Saxena J.K. Kumar A. Wilson S.H. Ackerman E.J. Science. 1992; 258: 475-478Crossref PubMed Scopus (47) Google Scholar), and an abasic site in DNA (5Matsumoto Y. Bogenhagen D.F. Mol. Cell. Biol. 1989; 9: 3750-3757Crossref PubMed Scopus (52) Google Scholar). Embryonic mouse fibroblasts with a homozygous β-pol gene deletion are deficient in base excision repair activity and are hypersensitive to monofunctional DNA-alkylating agents (6Sobol R.W. Horton J.K. Kühn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar). The core promoters of the human, bovine, and rodent β-pol genes have been described (7Widen S.G. Kedar P. Wilson S.H. J. Biol. Chem. 1988; 263: 16992-16998Abstract Full Text PDF PubMed Google Scholar, 8Yamaguchi M. Hayashi Y. Matsuoka S. Takahashi T. Matsukage A. Eur. J. Biochem. 1994; 221: 227-237Crossref PubMed Scopus (34) Google Scholar, 9Chen K.-H. Wood T. He F. Narayan S. Wilson S.H. Gene (Amst.). 1995; 164: 323-327Crossref PubMed Scopus (14) Google Scholar). These cloned β-pol promoters lack typical TATA and CCAAT elements, are G+C-rich, and have distinct binding elements for Sp1 and activating transcription factor (ATF)/cAMP response element-binding protein, referred to as CREB throughout. The CRE site in the β-pol promoters is required for full promoter activity (7Widen S.G. Kedar P. Wilson S.H. J. Biol. Chem. 1988; 263: 16992-16998Abstract Full Text PDF PubMed Google Scholar), and a purified CREB from bovine testis binds specifically to the conserved CRE site of the β-pol promoters and stimulates promoter activity in vitro (7Widen S.G. Kedar P. Wilson S.H. J. Biol. Chem. 1988; 263: 16992-16998Abstract Full Text PDF PubMed Google Scholar, 10Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar). The human and bovine β-pol promoters also are known to have a functional binding element for the YY1 family of initiation site binding proteins (11He F. Narayan S. Wilson S.H. Biochemistry. 1996; 35: 1775-1782Crossref PubMed Scopus (12) Google Scholar).β-Pol gene expression is induced after exposure of cells to the DNA-alkylating agent MNNG. This induction required transcription (12Fornace A.J. Zmudzka B. Hollander C. Wilson S.H. Mol. Cell. Biol. 1989; 9: 851-853Crossref PubMed Scopus (147) Google Scholar), and use of a transfected β-pol core promoter fusion gene revealed transcriptional up-regulation of the β-pol promoter after MNNG treatment. This response is mediated through the CRE of the β-pol promoter (13Kedar P.S. Widen S.G. Englander E.W. Fornace A.J. Wilson S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3729-3733Crossref PubMed Scopus (43) Google Scholar), and up-regulation of promoter activity is dependent upon the protein kinase A (PKA) signal transduction pathway (14Englander E.W. Wilson S.H. DNA Cell Biol. 1992; 11: 61-69Crossref PubMed Scopus (13) Google Scholar, 15Englander E.W. Wilson S.H. Nucleic Acids Res. 1992; 20: 5527-5531Crossref PubMed Scopus (18) Google Scholar).Using an in vitro transcription assay system, we found that the rate of transcript formation from a chimeric human β-pol promoter was much higher with nuclear extract (NE) from MNNG-treated HeLa cells than with extract from control HeLa cells. Further results indicated that this up-regulation was dependent upon CREB. The role of CREB from normal and MNNG-treated cells on the transcription initiation process has been described; CREB from MNNG-treated cells supports recruitment of more RPc than CREB from control cells (16Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (35) Google Scholar). The present investigation was conducted to isolate RPc assembled from control and MNNG-treated HeLa cells and to characterize the CREB family member(s) present and its possible modification as a function of cellular MNNG treatment.DISCUSSIONThe present study describes characterization of a CREB family member that is involved in β-pol gene regulation, as well as a mechanism for transcriptional activation of β-pol gene expression by an alkylating agent in vivo. Previous work, with the cloned human β-pol promoter, indicated that the PKA signal transduction pathway plays a required role in transcriptional activation after exposure of cells to MNNG (12Fornace A.J. Zmudzka B. Hollander C. Wilson S.H. Mol. Cell. Biol. 1989; 9: 851-853Crossref PubMed Scopus (147) Google Scholar, 13Kedar P.S. Widen S.G. Englander E.W. Fornace A.J. Wilson S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3729-3733Crossref PubMed Scopus (43) Google Scholar, 15Englander E.W. Wilson S.H. Nucleic Acids Res. 1992; 20: 5527-5531Crossref PubMed Scopus (18) Google Scholar) and that the CRE site of the β-pol promoter is required for the MNNG response (15Englander E.W. Wilson S.H. Nucleic Acids Res. 1992; 20: 5527-5531Crossref PubMed Scopus (18) Google Scholar). Recently, we extended these observations using an in vitro transcription assay with HeLa NE and a chimeric β-pol promoter (10Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar) and found that NE from cells exposed to MNNG is transcriptionally more active than that from control cells. This transcriptional activation is mediated by CREB (16Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (35) Google Scholar). Using a kinetic model of transcriptional initiation, it was shown that CREB from MNNG-treated cells stimulates formation of more RPc than CREB from control cells. The present studies were designed to purify RPc assembled with pSH15 promoter with NE from control and MNNG-treated cells and then to characterize CREB in the two complexes. We first confirmed that the amount of RPc assembled, instead of the rate of assembly, was increased after MNNG treatment. The apparent rate of RPc assembly was not altered by MNNG treatment. Immobilized promoter DNA was used to purify RPc. A larger amount of single cycle run-off transcript was formed by purified RPc from MNNG-treated cells than from the same number of control cells, indicating that more RPc was formed from MNNG-treated cells. Purified RPc was used as starting material to characterize the CREB family members present. RPc assembled from both NEs had a 43-kDa protein, similar to CREB-1 in mass. The identity of this protein was confirmed by DNA binding analysis and Western blotting (Fig. 2). We found that the amount of CREB in RPc from MNNG-treated cells was similar to that in RPc from control cells. These results are consistent with previous findings (15Englander E.W. Wilson S.H. Nucleic Acids Res. 1992; 20: 5527-5531Crossref PubMed Scopus (18) Google Scholar, 16Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (35) Google Scholar) and suggested that a physical modification of CREB secondary to MNNG treatment may account for the transcriptional up-regulation.Phosphorylation of CREB-1 by PKA may be indispensable for CREB transcriptional activation (23Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2041) Google Scholar, 26Yamamoto K.K. Gonzalez G.A. Briggs W.H. Montminy M.R. Nature. 1988; 334: 494-498Crossref PubMed Scopus (968) Google Scholar, 27Lee C.Q. Yun Y.D. Hoeffler J.P. Habener J.F. EMBO J. 1990; 9: 4455-4465Crossref PubMed Scopus (183) Google Scholar). The phosphorylation of CREB-1 by PKA does not affect the protein's dimerization or DNA binding affinity for strong CRE sites (26Yamamoto K.K. Gonzalez G.A. Briggs W.H. Montminy M.R. Nature. 1988; 334: 494-498Crossref PubMed Scopus (968) Google Scholar). However, in some cases PKA phosphorylation enhances binding of CREB-1 to asymmetrical CREs, such as that in the tyrosine aminotransferase gene (TGACGCAG) (28Nichols M. Weih F. Schmid W. DeVack C. Kowenz-Leutz E. Luckow B. Boshart M. Shutz G. EMBO J. 1992; 11: 3337-3346Crossref PubMed Scopus (274) Google Scholar; for reviews, see Refs. 29Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar and 30Delmas V. Molina C.A. Lalli E. de Groot R. Foulkes N.S. Masquilier D. Sassone-Corsi P. Rev. Physiol. Biochem. Pharmacol. 1994; 124: 1-28Crossref PubMed Google Scholar). Since the MNNG transcriptional response is mediated through the PKA pathway (15Englander E.W. Wilson S.H. Nucleic Acids Res. 1992; 20: 5527-5531Crossref PubMed Scopus (18) Google Scholar), and CREB purified from MNNG-treated cells is transcriptionally activated (16Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (35) Google Scholar), it was reasonable to propose that the transcriptional activation is due to increased phosphorylation. We examined this possibility by metabolically labeling cells with [32P]orthophosphate and simultaneously treating them with MNNG. 32P-Labeled CREB was purified from RPc and resolved by SDS-PAGE. A 43-kDa protein identical in mass to CREB-1 was much more strongly phosphorylated after exposure of cells to MNNG than in control cells. Using an anti-phospho-CREB polyclonal antibody that is specific for the Ser133-phosphorylated form of CREB-1, we further confirmed that phosphorylation of NE CREB-1 at Ser133 was strongly increased by MNNG treatment.To test the functional activity of CREB and to attempt to mimic the MNNG response, we used a mixed activator approach in which PKA-phosphorylated recombinant CREB-1 was added to the NE from control cells. A very small amount of added phosphorylated CREB-1 (10 ng) (but not unphosphorylated CREB-1) was able to stimulate promoter activity. The role of phosphorylated CREB-1 in transcriptional up-regulation of the β-pol promoter was examined further with NEd. Phosphorylated CREB-1 could restore transcriptional activity of NEd, but unphosphorylated CREB-1 could not. The results indicate that exposure of cells to the alkylating agent MNNG induces phosphorylation of CREB-1, which in turn recruits more RPc onto the promoter. This effect results in a stimulation of the overall amount of transcript formation. These observations are similar to those proposed for transcriptional activation of c-Jun by phorbol ester-induced phosphorylation of the transactivation domain (31Franklin C.C. Sanchez V. Wagner F. Woodgett J.R. Kraft A.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7247-7251Crossref PubMed Scopus (96) Google Scholar). The importance of PKA-phosphorylated CREB-1 in vivo was described in a transgenic mouse model study by Struthers et al. (32Struthers R.S. Vale W.W. Arias C. Sawchenko P.E. Montminy M.R. Nature. 1991; 350: 622-624Crossref PubMed Scopus (257) Google Scholar), where expression of a phosphorylation-deficient mutant CREB impaired transcriptional activation of the growth hormone gene and produced growth abnormalities. The enhanced transactivation property of phosphorylated CREB also has been examined by nuclear microinjection in fibroblasts; the results showed that phosphorylated CREB, but not unphosphorylated CREB, stimulated CREB-dependent gene expression (33Alberts A.S. Arias J. Hagiwara M. Montminy M.R. Feramisco J.R. J. Biol. Chem. 1994; 269: 7623-7630Abstract Full Text PDF PubMed Google Scholar). The transcriptional activation of target genes by the Ser133-phosphorylated form of CREB has been proposed to be due to increased association with the phospho-CREB-binding protein (CBP) (34Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1758) Google Scholar). Further, in transient expression studies, overexpression of CBP can stimulate cAMP-responsive promoter activity in a CREB phosphorylation-dependent manner (35Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H. Brennan R. Roberts S. Green M. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar). CREB phosphorylation, however, has not always been found to mediate activation of CREB-dependent promoters. In several studies (33Alberts A.S. Arias J. Hagiwara M. Montminy M.R. Feramisco J.R. J. Biol. Chem. 1994; 269: 7623-7630Abstract Full Text PDF PubMed Google Scholar, 36Wadzinski B.E. Wheat W.H. Jaspers S. Peruski L.F. Lickteig R.L. Johnson G.L. Klemm D.J. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (281) Google Scholar, 37Sun P. Schoderbek W.E. Maurer R.A. Mol. Endocrinol. 1992; 6: 1858-1866PubMed Google Scholar) with in vitro transcription system, no difference in transactivation properties was found with phosphorylated and unphosphorylated CREB. In some reports, it was suggested that the failure to observe phosphorylated CREB-dependent activation of genes was due to rapid dephosphorylation of CREB by nuclear phosphatases (37Sun P. Schoderbek W.E. Maurer R.A. Mol. Endocrinol. 1992; 6: 1858-1866PubMed Google Scholar, 38Hagiwara M. Alberts A. Brindle P. Meinkoth J. Feramisco J. Deng T. Karin M. Shenolikar S. Montminy M. Cell. 1992; 70: 105-113Abstract Full Text PDF PubMed Scopus (404) Google Scholar). Recent studies of Brindle and Montminy (24Brindle P.K. Montminy M.R. Curr. Opin. Genet. Dev. 1992; 2: 199-204Crossref PubMed Scopus (256) Google Scholar) suggested that, in addition to Ser133-phosphorylated CREB, other proteins are required to mediate CREB·CBP complex formation and transcriptional activation of target genes. Data from our studies indicate that phosphorylation of CREB at Ser133 by MNNG treatment of cells, perhaps in association with CBP, correlates with β-pol gene activation in the HeLa cell system. Explanations for the differences between these results and those from some other studies are unknown at the moment but could be related with differences in promoter sequence context and as well as details of extract preparation.In summary, we have made use of in vitro transcription systems to understand a potential mechanism for up-regulation of the β-pol promoter after mammalian cells are treated with a monofunctional DNA-alkylating agent. Since this up-regulation appears to require DNA alkylation damage (39Srivastava D.K. Rawson T.Y. Showalter S.D. Wilson S.H. J. Biol. Chem. 1995; 270: 16402-16408Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), it should be interesting to study the linkage between DNA damage and the signal transduction system. INTRODUCTIONDNA polymerase β (β-pol) 1The abbreviations used are: β-polDNA polymerase βATFactivating transcription factorCREcAMP response elementCREBCRE-binding proteinMNNGN-methyl-N′-nitro-N-nitrosoguanidinePKAprotein kinase ANEHeLa nuclear extractNEdCREB-depleted HeLa nuclear extractRPcclosed preinitiation complexPAGEpolyacrylamide gel electrophoresisphospho-CREB-1protein kinase A-phosphorylated recombinant CREB-1CBPCREB-binding protein. is a eukaryotic cellular polymerase involved in "gap-filling" synthesis during base excision repair (for review, see Ref. 1Singhal R.K. Wilson S.H. J. Biol. Chem. 1993; 268: 15906-15911Abstract Full Text PDF PubMed Google Scholar). Accordingly, β-pol has been found to promote repair of a G:U base pair (2Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), a monofunctional DNA adduct (3Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (260) Google Scholar, 4Jenkins T.M. Saxena J.K. Kumar A. Wilson S.H. Ackerman E.J. Science. 1992; 258: 475-478Crossref PubMed Scopus (47) Google Scholar), and an abasic site in DNA (5Matsumoto Y. Bogenhagen D.F. Mol. Cell. Biol. 1989; 9: 3750-3757Crossref PubMed Scopus (52) Google Scholar). Embryonic mouse fibroblasts with a homozygous β-pol gene deletion are deficient in base excision repair activity and are hypersensitive to monofunctional DNA-alkylating agents (6Sobol R.W. Horton J.K. Kühn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar). The core promoters of the human, bovine, and rodent β-pol genes have been described (7Widen S.G. Kedar P. Wilson S.H. J. Biol. Chem. 1988; 263: 16992-16998Abstract Full Text PDF PubMed Google Scholar, 8Yamaguchi M. Hayashi Y. Matsuoka S. Takahashi T. Matsukage A. Eur. J. Biochem. 1994; 221: 227-237Crossref PubMed Scopus (34) Google Scholar, 9Chen K.-H. Wood T. He F. Narayan S. Wilson S.H. Gene (Amst.). 1995; 164: 323-327Crossref PubMed Scopus (14) Google Scholar). These cloned β-pol promoters lack typical TATA and CCAAT elements, are G+C-rich, and have distinct binding elements for Sp1 and activating transcription factor (ATF)/cAMP response element-binding protein, referred to as CREB throughout. The CRE site in the β-pol promoters is required for full promoter activity (7Widen S.G. Kedar P. Wilson S.H. J. Biol. Chem. 1988; 263: 16992-16998Abstract Full Text PDF PubMed Google Scholar), and a purified CREB from bovine testis binds specifically to the conserved CRE site of the β-pol promoters and stimulates promoter activity in vitro (7Widen S.G. Kedar P. Wilson S.H. J. Biol. Chem. 1988; 263: 16992-16998Abstract Full Text PDF PubMed Google Scholar, 10Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar). The human and bovine β-pol promoters also are known to have a functional binding element for the YY1 family of initiation site binding proteins (11He F. Narayan S. Wilson S.H. Biochemistry. 1996; 35: 1775-1782Crossref PubMed Scopus (12) Google Scholar).β-Pol gene expression is induced after exposure of cells to the DNA-alkylating agent MNNG. This induction required transcription (12Fornace A.J. Zmudzka B. Hollander C. Wilson S.H. Mol. Cell. Biol. 1989; 9: 851-853Crossref PubMed Scopus (147) Google Scholar), and use of a transfected β-pol core promoter fusion gene revealed transcriptional up-regulation of the β-pol promoter after MNNG treatment. This response is mediated through the CRE of the β-pol promoter (13Kedar P.S. Widen S.G. Englander E.W. Fornace A.J. Wilson S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3729-3733Crossref PubMed Scopus (43) Google Scholar), and up-regulation of promoter activity is dependent upon the protein kinase A (PKA) signal transduction pathway (14Englander E.W. Wilson S.H. DNA Cell Biol. 1992; 11: 61-69Crossref PubMed Scopus (13) Google Scholar, 15Englander E.W. Wilson S.H. Nucleic Acids Res. 1992; 20: 5527-5531Crossref PubMed Scopus (18) Google Scholar).Using an in vitro transcription assay system, we found that the rate of transcript formation from a chimeric human β-pol promoter was much higher with nuclear extract (NE) from MNNG-treated HeLa cells than with extract from control HeLa cells. Further results indicated that this up-regulation was dependent upon CREB. The role of CREB from normal and MNNG-treated cells on the transcription initiation process has been described; CREB from MNNG-treated cells supports recruitment of more RPc than CREB from control cells (16Narayan S. Beard W.A. Wilson S.H. Biochemistry. 1995; 34: 73-80Crossref PubMed Scopus (35) Google Scholar). The present investigation was conducted to isolate RPc assembled from control and MNNG-treated HeLa cells and to characterize the CREB family member(s) present and its possible modification as a function of cellular MNNG treatment.
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