Identification of the E2A Gene Products as Regulatory Targets of the G1 Cyclin-dependent Kinases
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m008371200
ISSN1083-351X
Autores Tópico(s)Ubiquitin and proteasome pathways
ResumoThe E2A gene products, E12 and E47, are multifunctional transcription factors that as homodimers regulate B cell development, growth, and survival. In this report, the E2A gene products are shown to be targets for regulation by the G1 cyclin-dependent kinases. Two novel G1 cyclin-dependent kinase sites are identified on the N-terminal domain of E12/E47. One site displays homology to a preferential D-type cyclin-dependent kinase site (serine 780) on the retinoblastoma susceptibility gene product (pRB) and, consistent with this homology, is more efficiently phosphorylated by cyclin D1-CDK4 than by the other cyclin-dependent kinases (CDK) that were tested. The second kinase site is phosphorylated by both cyclin D1-CDK4 and cyclin A/E-CDK2 complexes. Mutation studies indicated that phosphorylation of the cyclin D1-CDK4 site, or more potently, of both the cyclin D1-CDK4 and cyclin A/E-CDK2 sites, negatively regulates the growth suppressor function associated with the N-terminal domain of E12/E47. Transient expression studies showed that ectopic expression of cyclin D1 or E negatively regulates sequence-specific activation of gene transcription by E12/E47. Analysis of site mutants, however, indicated that inhibition of E12/E47 transcriptional activity did not require the N-terminal G1cyclin-dependent kinase sites. Together, the results suggest that the growth suppressor and transcriptional activator functions of E12/E47 are targets for regulation by G1cyclin-dependent kinases but that the mechanisms of regulation for each function are distinct. The E2A gene products, E12 and E47, are multifunctional transcription factors that as homodimers regulate B cell development, growth, and survival. In this report, the E2A gene products are shown to be targets for regulation by the G1 cyclin-dependent kinases. Two novel G1 cyclin-dependent kinase sites are identified on the N-terminal domain of E12/E47. One site displays homology to a preferential D-type cyclin-dependent kinase site (serine 780) on the retinoblastoma susceptibility gene product (pRB) and, consistent with this homology, is more efficiently phosphorylated by cyclin D1-CDK4 than by the other cyclin-dependent kinases (CDK) that were tested. The second kinase site is phosphorylated by both cyclin D1-CDK4 and cyclin A/E-CDK2 complexes. Mutation studies indicated that phosphorylation of the cyclin D1-CDK4 site, or more potently, of both the cyclin D1-CDK4 and cyclin A/E-CDK2 sites, negatively regulates the growth suppressor function associated with the N-terminal domain of E12/E47. Transient expression studies showed that ectopic expression of cyclin D1 or E negatively regulates sequence-specific activation of gene transcription by E12/E47. Analysis of site mutants, however, indicated that inhibition of E12/E47 transcriptional activity did not require the N-terminal G1cyclin-dependent kinase sites. Together, the results suggest that the growth suppressor and transcriptional activator functions of E12/E47 are targets for regulation by G1cyclin-dependent kinases but that the mechanisms of regulation for each function are distinct. basic helix-loop-helix retinoblastoma susceptibility gene product retinoblastoma cyclin-dependent kinases fetal bovine serum Dulbecco's minimum essential medium polyacrylamide gel electrophoresis polymerase chain reaction chloramphenicol acetyltransferase phosphate-buffered saline fluorescence-activated cell sorter cytomegalovirus The products of the e2a gene are basic helix-loop-helix (bHLH)1DNA-binding proteins that control gene expression as homodimers in B cells and as heterodimers in other tissues (1Murre C. Bain G. van Dijk M.A. Engel I. Furnari B.A. Massari M.E. Matthews J.R. Quong M.W. Rivera R.R. Stuiver M.H. Biochim. Biophys. Acta. 1994; 1218: 129-135Crossref PubMed Scopus (405) Google Scholar, 2Reya T. Grosschedl R. Curr. Opin. Immunol. 1998; 10: 158-165Crossref PubMed Scopus (60) Google Scholar). Expression of thee2a gene produces two splice products, E12 and E47 (3Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1837) Google Scholar, 4Sun X.H. Baltimore D. Cell. 1991; 64: 459-470Abstract Full Text PDF PubMed Scopus (316) Google Scholar). One of these two proteins, E47, was originally identified by its ability to bind specific sites (E boxes) in the enhancer regions of the immunoglobulin genes (3Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1837) Google Scholar, 5Henthorn P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (335) Google Scholar, 6Schlissel M. Voronova A. Baltimore D. Genes Dev. 1991; 5: 1367-1376Crossref PubMed Scopus (195) Google Scholar). The E2A gene products later were found to be part of a larger, widely expressed family of bHLH regulators (the E proteins) that function primarily as heterodimers with tissue-specific bHLH proteins (1Murre C. Bain G. van Dijk M.A. Engel I. Furnari B.A. Massari M.E. Matthews J.R. Quong M.W. Rivera R.R. Stuiver M.H. Biochim. Biophys. Acta. 1994; 1218: 129-135Crossref PubMed Scopus (405) Google Scholar). Functional heterodimers between the E proteins and other tissue-specific bHLH regulators have been observed in differentiating neuronal, myogenic, and pancreatic cells (7Lassar A.B. Buskin J.N. Lockshon D. Davis R.L. Apone S. Hauschka S.D. Weintraub H. Cell. 1989; 58: 823-831Abstract Full Text PDF PubMed Scopus (567) Google Scholar, 8Cordle S.R. Henderson E. Masuoka H. Weil P.A. Stein R. Mol. Cell. Biol. 1991; 11: 1734-1738Crossref PubMed Google Scholar, 9Lassar A.B. Davis R.L. Wright W.E. Kadesch T. Murre C. Voronova A. Baltimore D. Weintraub H. Cell. 1991; 66: 305-315Abstract Full Text PDF PubMed Scopus (684) Google Scholar, 10Murre C. Voronova A. Baltimore D. Mol. Cell. Biol. 1991; 11: 1156-1160Crossref PubMed Scopus (132) Google Scholar, 11Johnson J.E. Birren S.J. Saito T. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3596-3600Crossref PubMed Scopus (133) Google Scholar). The E2A gene products function as homodimers in B cells (10Murre C. Voronova A. Baltimore D. Mol. Cell. Biol. 1991; 11: 1156-1160Crossref PubMed Scopus (132) Google Scholar, 12Bain G. Gruenwald S. Murre C. Mol. Cell. Biol. 1993; 13: 3522-3529Crossref PubMed Scopus (138) Google Scholar, 13Jacobs Y. Vierra C. Nelson C. Mol. Cell. Biol. 1993; 13: 7321-7333Crossref PubMed Scopus (66) Google Scholar, 14Shen C.P. Kadesch T. Mol. Cell. Biol. 1995; 15: 4518-4524Crossref PubMed Scopus (160) Google Scholar), and mature B cells are absent from e2a gene null mutant mice (15Bain G. Maandag E.C. Izon D.J. Amsen D. Kruisbeek A.M. Weintraub B.C. Krop I. Schlissel M.S. Feeney A.J. van Roon M. van der Valk M. te Riele H. Berns A. Murre C. Cell. 1994; 79: 885-892Abstract Full Text PDF PubMed Scopus (643) Google Scholar, 16Zhuang Y. Soriano P. Weintraub H. Cell. 1994; 79: 875-884Abstract Full Text PDF PubMed Scopus (576) Google Scholar). On the other hand, development of other tissues, including muscle and nerves, appears normal in these mice. This result suggests that homodimers of the E2A gene products are essential for B cell development but that their role in forming active heterodimers with tissue-specific bHLH proteins may be played by other members of the E protein family (1Murre C. Bain G. van Dijk M.A. Engel I. Furnari B.A. Massari M.E. Matthews J.R. Quong M.W. Rivera R.R. Stuiver M.H. Biochim. Biophys. Acta. 1994; 1218: 129-135Crossref PubMed Scopus (405) Google Scholar, 3Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1837) Google Scholar, 5Henthorn P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (335) Google Scholar, 17Hu J.S. Olson E.N. Kingston R.E. Mol. Cell. Biol. 1992; 12: 1031-1042Crossref PubMed Scopus (251) Google Scholar).Homodimers of E2A gene products bind and activate transcription from a minimal reporter construct containing four tandem repeats of μE5, μE2, and μE3 sites ([5,2,3]x4-TATA-CAT). In the absence of ectopically expressed E2A gene products, expression of this reporter is restricted to B cells (18Ruezinsky D. Beckmann H. Kadesch T. Genes Dev. 1991; 5: 29-37Crossref PubMed Scopus (65) Google Scholar). The reporter is activated in NIH3T3 cells when ITF-1 (E47/E2-5) is expressed ectopically (18Ruezinsky D. Beckmann H. Kadesch T. Genes Dev. 1991; 5: 29-37Crossref PubMed Scopus (65) Google Scholar). The function of E2A gene products as transcriptional activators is critical for the completion of immunoglobulin gene rearrangement and normal B cell development. In e2a gene null mice, B cell development is severed between the pre-B and pro-B cell stages, and rearrangement of the immunoglobulin genes is interrupted (15Bain G. Maandag E.C. Izon D.J. Amsen D. Kruisbeek A.M. Weintraub B.C. Krop I. Schlissel M.S. Feeney A.J. van Roon M. van der Valk M. te Riele H. Berns A. Murre C. Cell. 1994; 79: 885-892Abstract Full Text PDF PubMed Scopus (643) Google Scholar, 16Zhuang Y. Soriano P. Weintraub H. Cell. 1994; 79: 875-884Abstract Full Text PDF PubMed Scopus (576) Google Scholar). The importance of the E2A gene products for initiation of immunoglobulin gene rearrangement is underscored by experiments showing that ectopic expression in T cells induces immunoglobulin gene rearrangement (6Schlissel M. Voronova A. Baltimore D. Genes Dev. 1991; 5: 1367-1376Crossref PubMed Scopus (195) Google Scholar). Since transcriptionally active E2A gene products are found in B cells during most stages of their development, it is likely that they also perform critical functions in cells that are more mature than pro-B cells, although the nature and importance of these functions has not been well explored.The DNA-binding and dimer assembly domain (the bHLH) of E12/E47 maps to the extreme C-terminal region of the proteins, leaving a large N-terminal domain. The transcriptional activation domains of the E2A gene products have been mapped to this N-terminal domain and are conserved among members of the E protein family (19Quong M.W. Massari M.E. Zwart R. Murre C. Mol. Cell. Biol. 1993; 13: 792-800Crossref PubMed Scopus (130) Google Scholar, 20Massari M.E. Jennings P.A. Murre C. Mol. Cell. Biol. 1996; 16: 121-129Crossref PubMed Scopus (86) Google Scholar). One activation domain has been structurally characterized as a loop followed by an α-amphipathic helix and is referred to as a loop-helix motif (19Quong M.W. Massari M.E. Zwart R. Murre C. Mol. Cell. Biol. 1993; 13: 792-800Crossref PubMed Scopus (130) Google Scholar). Within an acidic stretch of residues immediately N-terminal to the bHLH are two sites that are phosphorylated by casein kinase II and protein kinase A (21Sloan S.R. Shen C.P. McCarrick-Walmsley R. Kadesch T. Mol. Cell. Biol. 1996; 16: 6900-6908Crossref PubMed Scopus (82) Google Scholar). In electrophoretic mobility shift studies, the DNA binding ability of E47 homodimers was blocked by phosphorylation of these residues, whereas the DNA binding ability of MyoD/E47 heterodimers was unaffected (21Sloan S.R. Shen C.P. McCarrick-Walmsley R. Kadesch T. Mol. Cell. Biol. 1996; 16: 6900-6908Crossref PubMed Scopus (82) Google Scholar). Consistent with this, phosphorylation of E47 at these sites was observed only in non-B cells (21Sloan S.R. Shen C.P. McCarrick-Walmsley R. Kadesch T. Mol. Cell. Biol. 1996; 16: 6900-6908Crossref PubMed Scopus (82) Google Scholar), suggesting a unique mechanism for restricting the transcriptional activity of E12/E47 homodimers to B cells.Recent studies have indicated that the E2A gene products activate transcription of the cyclin-dependent kinase inhibitor p21CIP/WAF/SD11 (22Prabnu S. Ignatiova A. Park S. Sun X.-H. Mol. Cell. Biol. 1997; 27: 5888-5896Crossref Scopus (285) Google Scholar), suggesting a direct mechanism through which E12/E47 can regulate cell growth. In addition, other functions for E12/E47 in the regulation of cell growth that are not directly associated with the activation of gene transcription have been described. These functions have been structurally mapped to the N-terminal segment of E12/E47 and include regulation of apoptosis (23Inaba T. Inukai T. Yoshihara T. 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Recent studies have shown thate2a gene null mice develop T cell lymphomas and that ectopic expression of E12/E47 in these lymphoma cells induces their death (27Engel I. Murre C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 996-1001Crossref PubMed Scopus (88) Google Scholar). A role for E47 in growth suppression was first observed in NIH3T3 cells (29Peverali F.A. Ramqvist T. Saffrich R. Pepperkok R. Barone M.V. Philipson L. EMBO J. 1994; 13: 4291-4301Crossref PubMed Scopus (185) Google Scholar). In colony-forming assays, deletion mutants of E47 that contained the N-terminal region suppressed growth of NIH3T3 fibroblasts as well as wild-type E47 (29Peverali F.A. Ramqvist T. Saffrich R. Pepperkok R. Barone M.V. Philipson L. EMBO J. 1994; 13: 4291-4301Crossref PubMed Scopus (185) Google Scholar). The growth suppressor activity of E47 contrasts that of MyoD (30Crescenzi M. Fleming T.P. Lassar A.B. Weintraub H. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8442-8446Crossref PubMed Scopus (174) Google Scholar, 31Sorrentino V. Pepperkok R. Davis R.L. Ansorge W. Philipson L. Nature. 1990; 345: 813-815Crossref PubMed Scopus (177) Google Scholar) and other bHLH proteins, as E47 does not require the bHLH domain for this function. Growth suppression by E47 is induced during entry into or progression through the G1 phase of the cell cycle (29Peverali F.A. Ramqvist T. Saffrich R. Pepperkok R. Barone M.V. Philipson L. EMBO J. 1994; 13: 4291-4301Crossref PubMed Scopus (185) Google Scholar), suggesting that the G1cyclin-dependent kinases (CDKs) may negatively regulate this function under appropriate growth conditions.In this report, we identify the E2A gene products as novel targets for regulation by G1 cyclin-dependent kinases. Two novel G1 cyclin-dependent kinase sites are identified on the N-terminal domain of E12/E47, and evidence is presented that phosphorylation of these sites negatively regulates the growth suppressor activity of E47. One of the sites displays a strong preference for phosphorylation by cyclin D1-dependent kinase, a characteristic previously associated only with certain kinase sites of another class of growth suppressors, the retinoblastoma susceptibility gene (RB) family. In addition, ectopic expression of cyclin D1 or E is shown to regulate negatively site-specific transcriptional activation by E12/E47 but through a mechanism that does not require the N-terminal cyclin-dependent kinase sites. Together, the results suggest that the products of the e2agene are targets through which the G1cyclin-dependent kinases may regulate the proliferation and gene expression of certain cell types.EXPERIMENTAL PROCEDURESCell CultureC3H10T1/2 mouse embryonic fibroblasts (given by H. Weintraub, Fred Hutchinson Cancer Research Center) and CV-1 monkey kidney fibroblasts (provided by P. Palese, Mt. Sinai School of Medicine) were grown in Dulbecco's minimum essential medium (DMEM, Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS, HyClone). NIH3T3 mouse fibroblasts (given by S. Aaronson, Mt. Sinai School of Medicine) were maintained in DMEM supplemented with 10% newborn calf serum (Life Technologies, Inc.). Mantle lymphoma cell line MO2058 (given by T. Meeker, University of Kentucky) was grown in suspension in DMEM supplemented with 15% FBS. Sf9 insect cells (PharMingen) were maintained as monolayers in supplemented Grace's Medium (Life Technologies, Inc.) supplemented with 10% FBS.Eukaryotic Expression PlasmidsTranscriptional activities of E47 and site mutants were measured with an E protein-specific reporter, [μE5 + μE2 + μE3]x4-TATA-CAT (18Ruezinsky D. Beckmann H. Kadesch T. Genes Dev. 1991; 5: 29-37Crossref PubMed Scopus (65) Google Scholar) generously provided by T. Kadesch (University of Pennsylvania). Parallel transient expression assays were performed with a control reporter, pRSV-CAT (32Gorman C.M. Merlino G.T. Willingham M.C. Pastan I. Howard B.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6777-6781Crossref PubMed Scopus (877) Google Scholar) generously provided by E. Johnson (Mt. Sinai School of Medicine). The cDNAs coding for human cyclin A (33Pines J. Hunter T. Nature. 1990; 346: 760-763Crossref PubMed Scopus (528) Google Scholar), cyclin E (34Koff A. Cross F. Fisher A. Schumacher J. Leguellec K. Philippe M. Roberts J.M. Cell. 1991; 66: 1217-1228Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 35Lew D.J. Dulic V. Reed S.I. Cell. 1991; 66: 1197-1206Abstract Full Text PDF PubMed Scopus (661) Google Scholar), and cyclin D1 (36Motokura T. Bloom T. Kim H.G. Jüppner H. Ruderman J.V. Kronenberg H.M. Arnold A. Nature. 1991; 350: 512-515Crossref PubMed Scopus (1153) Google Scholar) were generously provided as pRc/CMV (Invitrogen) expression constructs by P. Hinds and R. Weinberg (Whitehead Institute). The vector, pECE (37Ellis L. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (694) Google Scholar), and pECE expression construct coding for the human E47·(ITF-1/E2-5 (5Henthorn P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (335) Google Scholar)) cDNA with a C-terminal SV40 tag were given by B. Wold (California Institute of Technology). E47 deletion mutants were generated that code residues 1–402 (with additional amino acids at the C terminus, GRQRDQAGGEGGRGEHVSG; pECE-E47Not) and residues 1–45 and 415–559 (pECE-E47bHLH). The E47 site mutants E47-S48A, E47-S154A, and E47-S48A/S154A were prepared as described below and were ligated into the BglII and XbaI sites of pECE. The β-galactosidase expression construct pCMV-β (Invitrogen) was used in some transient expression assays as an internal control.Baculovirus Expression ConstructsThe cDNAs of cyclin A, cyclin E, and cyclin D1 were blunt-inserted into the transfer vectors, pVL1392 or pVL1393 (PharMingen). The cDNA for mouse CDK4 (38Matsushime H. Ewen M.E. Strom D.K. Kato J.Y. Hanks S.K. Roussel M.F. Sherr C.J. Cell. 1992; 71: 323-334Abstract Full Text PDF PubMed Scopus (777) Google Scholar) was given by C. Sherr (St. Jude Children's Research Hospital) and subcloned into theEcoRI site of the transfer vector, pAcGHLT-A (PharMingen).Bacterial Expression ConstructsThe cDNA for human p21 (39El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7888) Google Scholar, 40Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (707) Google Scholar, 41Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5216) Google Scholar, 42Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3156) Google Scholar, 43Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1309) Google Scholar) was given by D. Beach (Cold Spring Harbor Laboratory) and inserted into the NcoI andEcoRI sites of pTAT-HA (44Ezhevsky S.A. Nagahara H. Vocero-Akbani A.M. Gius D.R. Wei M.C. Dowdy S.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10699-10704Crossref PubMed Scopus (247) Google Scholar), generously provided by S. Dowdy (Washington University School of Medicine). The His6-tagged expression constructs were prepared by inserting PCR fragments containing coding sequence into theBamHI and HindIII sites of pQE30 (Qiagen). Wild-type and mutant E47 His6-tagged expression constructs were prepared by using sequences coding for residues 9–559. Sequence coding for residues 1–912 of mouse CDK4 was used to prepare the His6-tagged CDK4 expression construct.Site-directed MutagenesisA method was devised to generate site mutants based on two separate PCRs. The gene was divided into two fragments flanking the mutation site and extending to vector sequences at the 5′ and 3′ ends of the gene. To generate the 5′ fragment, a forward (sense) primer containing a vector restriction site 5′ to the gene (e.g. BglII) and a backward (antisense) primer starting with the mutant nucleotide and extending into the gene were used in one PCR. To generate the 3′ fragment, a forward (sense) primer starting 1 nucleotide down from the mutant and a backward (antisense) primer starting from a vector restriction site 3′ to gene (e.g. XbaI) were used in a second PCR. A protocol modified from that described by Liang and Pardee (45Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4687) Google Scholar) was used to perform the PCRs. Reactions were carried out by a Techne Progene thermal cycler programmed to perform 35 cycles of a three-stage process and a final elongation step. The 1st stage was set at 95 °C for 40 s, 2nd stage set at 59 °C for 2 min, and 3rd stage set at 72 °C for 1 min. Reactions were extracted with phenol/chloroform to remove proteins and dNTPs. The 5′ and 3′ PCR fragments were digested byBglII and XbaI, respectively, whereas the vector was digested by both BglII and XbaI and then further digested with CIP (New England Biolabs). The two fragments and vector were combined and incubated with ligase, which resulted in a blunt-end ligation at the site of the mutation and sticky-end ligations at the BglII and XbaI sites. Vent DNA polymerase (New England Biolabs), a thermophilic DNA polymerase that generates blunt ends instead of overhangs, was used for the PCRs. Competent XL-1 blue cells were transformed with the ligation reactions as described elsewhere (46Ausubel F.M. Bent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley — Sons, New York1998Google Scholar). Plasmid DNA was isolated from randomly selected clones with the Wizard SV Mini-prep DNA Isolation Kit (Promega) and digested with both BglII and XbaI to check for inserts. Positive clones were then sequenced to confirm mutations and correct junctions.DNA SequencingPlasmid DNA was denatured under alkaline conditions and used as template for sequencing by the Sanger method (Sequenase version 2.0 DNA Sequencing Kit supplied by Amersham Pharmacia Biotech/U. S. Biotechnology Corp.). Sequence reactions were then run on a 6% polyacrylamide/urea gel in TBE buffer (46Ausubel F.M. Bent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley — Sons, New York1998Google Scholar). The gel was fixed, dried under vacuum, and then exposed to x-ray film. Sequences were read directly from the autoradiographs.Transient Expression AssaysCells were transfected at 80% confluency with a standard calcium phosphate method (55Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and incubated for 18 h. After transfection, cells were grown in low mitogen medium (DMEM supplemented with 3% horse serum) for another 48 h. Cells were then harvested for CAT activity. CAT assays were performed as described elsewhere (47Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5288) Google Scholar). CAT activities were then quantified from TLC plates by PhosphorImager analysis. To control for nonspecific effects on [μE5 + μE2 + μE3]x4-TATA-CAT reporter activity, parallel experiments were performed with a constitutively active reporter, pRSV-CAT. Relative CAT activities were then determined by normalizing CAT values from [μE5 + μE2 + μE3]x4-TATA-CAT reporter with respective values obtained with the control reporter (pRSV-CAT).Generation of Recombinant ProteinsProteins were prepared by protocol modified from that described elsewhere (Qiagen His6 Protein Expression Manual). TB-1 competent cells expressing pREP4 were transformed with expression constructs by a standard protocol, and small scale protein preparations were performed to isolate high protein-expressing clones. Clones were grown overnight in LB medium containing both ampicillin and kanamycin and were then used for large scale protein preparations. For large scale protein preparations, bacteria were grown for 1 h in LB medium at 37 °C and then induced to express protein with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for 4–5 h. Cells were harvested, washed with ice-cold TES (10 mm Tris, pH 7.5, 1 mm EDTA, pH 8.0, 150 mm NaCl), and lysed in sonication buffer (25 mmHEPES, pH 8.0, 150 mm NaCl, and 8 m urea). Lysates were sonicated, clarified, and then loaded onto nickel-nitrilotriacetic acid-agarose (Qiagen) columns that were prewashed with wash buffer (25 mm HEPES, pH 8.0, 150 mm NaCl, 8 m urea). Columns were washed several times with wash buffer, and His6-tagged proteins were eluted with 150 mm imidazole in wash buffer. Proteins were then dialyzed twice against 10 mm HEPES, pH 8.0, 100 mm NaCl. Samples were aliquoted and flash-frozen.Preparation of Active Cyclin-CDK ComplexesThe four steps used in the preparation of active cyclin-CDK complexes included generation of baculoviruses, titration of baculoviruses, coinfection with cyclin and CDK baculoviruses, and purification of active cyclin-CDK complexes. The baculovirus expressing HA-tagged CDK2 (34Koff A. Cross F. Fisher A. Schumacher J. Leguellec K. Philippe M. Roberts J.M. Cell. 1991; 66: 1217-1228Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 48Elledge S.J. Spottswood M.R. EMBO J. 1991; 10: 2653-2659Crossref PubMed Scopus (196) Google Scholar, 49Ninomiya-Tsuji J. Nomoto S. Yasuda H. Reed S.I. Matsumoto K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9006-9010Crossref PubMed Scopus (154) Google Scholar, 50Tsai L.H. Harlow E. Meyerson M. Nature. 1991; 353: 174-177Crossref PubMed Scopus (328) Google Scholar) was generously provided by C. Prives (Columbia University). Baculoviruses expressing human MO15/CDK7 (51Darbon J.M. Devault A. Taviaux S. Fesquet D. Martinez A.M. Galas S. Cavadore J.C. Dorée M. Blanchard J.M. Oncogene. 1994; 9: 3127-3138PubMed Google Scholar, 52Fisher R.P. Morgan D.O. Cell. 1994; 78: 713-724Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 53Tassan J.P. Schultz S.J. Bartek J. Nigg E.A. J. Cell Biol. 1994; 127: 467-478Crossref PubMed Scopus (215) Google Scholar, 54Wu L. Yee A. Liu L. Carbonaro-Hall D. Venkatesan N. Tolo V.T. Hall F.L. Oncogene. 1994; 9: 2089-2096PubMed Google Scholar) and His6-tagged cyclin H (52Fisher R.P. Morgan D.O. Cell. 1994; 78: 713-724Abstract Full Text PDF PubMed Scopus (552) Google Scholar) were gifts from D. Morgan (University of California, San Francisco).Generation of BaculovirusesThe baculoviruses expressing cyclin A, cyclin E, cyclin D1, and CDK4 were generated with the Baculovirus Protein Expression Kit from PharMingen. Monolayers of Sf9 insect cells (2 × 106) passaged into T25 flasks were cotransfected with transfer vectors (driven by the polyhedrin promoter) that contain the gene of interest and BaculoGoldTM DNA (linearized viral DNA) by a calcium phosphate method for 4 h. The use of BaculoGoldTM DNA results in the generation of baculoviruses that express only the protein of interest, thus eliminating the need to select positive expressing clones. After transfection, cells were grown in fresh medium and incubated for 5 days. The medium was collected, and 1 ml was used to infect 2 × 107 Sf9 cells seeded on a 15-cm tissue culture plate. After 3 days of infection, the medium was collected, and the titer of the virus was initially measured by end point dilution assay. Multiple rounds of amplification were performed until viral stocks reached titers greater than or equal to 1 × 108 plaque-forming units/cell.Titration of BaculovirusesSince cells are infected with two viruses simultaneously, infection efficiencies may differ from one virus to another. Infection efficiencies of cyclin and CDK viruses were normalized by comparing relative protein expression levels at different virus concentrations. Sf9 cells were passaged into 24-well plates containing 2 × 105 cells/well. Amplified virus was added to the wells at dilutions values of 1:10, 1:20, 1:40, 1:100, 1:200, 1:400, and 1:1000. After 2 days of infection, cells were then harvested, and lysates were analyzed by SDS-PAGE (55Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Proteins were visualized by staining gels with Coomassie Blue R-250 solution (55Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).Coinfection with Cyclin and CDK VirusesThe dilution value where maximum protein expression levels we
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