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• W2072010065 abstract "In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control. Using a sensitive photoaffinity labeling assay that measured [α-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that the [α-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [α-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells. Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth. In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control. Using a sensitive photoaffinity labeling assay that measured [α-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that the [α-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [α-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells. Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth. Growth factor binding to cell-surface receptors can initiate signals that are propagated through the cell by a cascade of protein-protein interactions, ultimately to impact upon specific cellular functions and regulate cell growth. The activities of signaling molecules must be tightly regulated to maintain the integrity of cellular communication, as loss of regulation in these processes can give rise to defects in cell growth and metabolism that may lead to human disease. Given the importance of signaling processes in cell growth, a great deal of effort has gone into the elucidation of proteins participating in signaling pathways that start at the level of receptor activation and culminate in the stimulation of a nuclear activity. Multiple cascades have now been identified that result in the activation of different nuclear mitogen-activated protein kinases, including the extracellular receptor-activated kinases and the stress-responsive c-Jun N-terminal kinase/stress-activated protein kinase and p38 (1Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar, 2Hunter T. Cell. 1997; 88: 333-346Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). Extracellular receptor-activated kinase activation is the outcome of mitogen-stimulated Ras signaling, whereas c-Jun N-terminal kinase/stress-activated protein kinase and p38 activities are often stimulated by pathways involving the Cdc42 and Rac GTP-binding proteins (2Hunter T. Cell. 1997; 88: 333-346Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 3Coso O.A. Chiariell M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar, 4Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar, 5Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 6Bagrodia S. Derigard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). Although these different signaling pathways were originally thought to be independently regulated, later work showed that cross-talk between the individual mitogen-activated protein kinase pathways exists. A common functional outcome of the activation of these signaling pathways is a translocation of the activated mitogen-activated protein kinase to the nucleus and subsequent activation of specific transcription factors and gene expression (2Hunter T. Cell. 1997; 88: 333-346Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 3Coso O.A. Chiariell M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar, 4Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar, 5Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 6Bagrodia S. Derigard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). How other nuclear functions might be influenced in response to extracellular stimulation is less clear. However, it is attractive to envision how critical nuclear activities such as RNA metabolism and export, nuclear protein import, and cell cycle control might be subject to regulation as downstream targets of extracellular stimuli. With this in mind, we set out to identify novel nuclear activities that were growth factor-responsive. Using a photoaffinity labeling approach, we identified the nuclear cap-binding complex (CBC) 1The abbreviations used are: CBC, cap-binding complex; NGF, nerve growth factor; EGF, epidermal growth factor; TBS, Tris-buffered saline; DTT, dithiothreitol; AMP-PNP, adenosine 5′-(β,γ-iminotriphosphate); GMP-PNP, guanosine 5′-(β,γ-iminotriphosphate); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HA, hemagglutinin; eIF, eukaryotic initiation factor; snRNA, small nucleotide RNA. as such an activity based on the enhanced ability of its ∼20-kDa subunit (CBP20) to undergo a photocatalyzed incorporation of [α-32P]GTP in response to extracellular stimulation. The CBP20 protein and its 80-kDa binding partner, CBP80, constitute a functional CBC (7Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 8Kataoka N. Ohno M. Kangawa K. Tokoro Y. Shimura Y. Nucleic Acids Res. 1994; 22: 3861-3865Crossref PubMed Scopus (39) Google Scholar, 9Izaurralde E. Lewis J. Gamberi C. Jarmolowski A. McGuigan C. Mattaj I.W. Nature. 1995; 376: 709-712Crossref PubMed Scopus (288) Google Scholar, 10Kataoka N. Ohno M. Moda I. Shimura Y. Nucleic Acids Res. 1995; 23: 3638-3641Crossref PubMed Scopus (40) Google Scholar). This nuclear complex binds cotranscriptionally to the monomethylated guanosine cap structure (m7G) of RNA polymerase II-transcribed RNAs (7Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 11Izaurralde E. Stepinski J. Darzynkiewicz E. Mattaj I.W. J. Cell Biol. 1992; 118: 1287-1295Crossref PubMed Scopus (125) Google Scholar, 12Visa N. Izaurralde E. Ferreira J. Daneholt B. Mattaj I.W. J. Cell Biol. 1996; 133: 5-14Crossref PubMed Scopus (198) Google Scholar) and has been reported to play a role in diverse aspects of RNA metabolism: it increases the splicing efficiency of cap proximal introns (7Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 13Ohno M. Sakamoto H. Simura Y. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5187-5191Crossref PubMed Scopus (97) Google Scholar, 14Lewis J.D. Izaurralde E. Jarmolowski A. McGuigan C. Mattaj I.W. Genes Dev. 1996; 10: 1683-1698Crossref PubMed Scopus (188) Google Scholar, 15Lewis J.D. Goerlich D. Mattaj I.W. Nucleic Acids Res. 1996; 24: 3332-3336Crossref PubMed Scopus (91) Google Scholar), positively affects the efficiency of 3′-end processing (16Flaherty S. Fortes P. Izaurralde E. Mattaj I.W. Gilmartin G.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11893-11898Crossref PubMed Scopus (174) Google Scholar), and is required for the efficient transport of U snRNAs (9Izaurralde E. Lewis J. Gamberi C. Jarmolowski A. McGuigan C. Mattaj I.W. Nature. 1995; 376: 709-712Crossref PubMed Scopus (288) Google Scholar). We demonstrate that the incorporation of [α-32P]GTP by CBP20 reflects the activation of the CBC and is correlated with its ability to bind capped RNA. A variety of growth factors and other cellular stimuli can activate the CBC under conditions that can give rise to a stimulation of the splicing of precursor mRNAs in an in vitro assay system. The implications of CBP20 functioning as a novel end point in signal transduction highlight the importance of RNA metabolism in regulated cell growth. Rat pheochromocytoma (PC12) cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 10% horse serum, and antibiotic/antimycotic solution (Sigma). All other cell types, including HeLa, BHK21, and COS-7 cells, were maintained in Dulbecco's modified Eagle's medium with the addition of 10% fetal bovine serum and antibiotic/antimycotic solution. Prior to growth factor treatment, cells were switched to serum-free medium for 40 h. Growth factors (NGF (Life Technologies, Inc.), heregulin β1 (residues 177–244; a generous gift from Dr. Mark Sliwkowski, Genentech), and EGF (Calbiochem) or 25% fetal bovine serum) were then added to the serum-free medium in the concentrations and for the times indicated under “Results” at 37 °C. Following treatment, the growth factor-containing medium was removed, and the cells were washed twice with Tris-buffered saline (TBS; 25 mm Tris-Cl, pH 7.4, 140 mm NaCl, and 1.0 mm EDTA) and then lysed (see below). Cell cycle blocks were performed in HeLa cells. A G0 block was achieved by switching to serum-free medium for 22–24 h. For G1/S phase arrest, 2.5 mm thymidine was added to the growth medium for 22–24 h. 80 ng/ml nocodazole was added to the growth medium for 22–24 h to achieve arrest in M phase. After treatment, cells were collected, washed twice with TBS, and lysed. To challenge cells with UV irradiation, the medium was removed from serum-starved cells, and the cells were then exposed to UV light for 2 min. Following exposure, cells were replenished with serum-free medium and allowed to recover at 37 °C for the times indicated below. Tissue culture cells were washed twice on the plate with TBS and then lysed in a buffer containing Hanks' solution (20 mm Hepes, pH 7.4, 5 mm KCl, 137 mm NaCl, 4 mmNaHCO3, 5.5 mmd-glucose, and 10 μm EDTA), 0.3% (v/v) Nonidet P-40, 1 mmsodium orthovanadate, 1 mm DTT, 1 mmphenylmethanesulfonyl fluoride, and 10 μg/ml each leupeptin and aprotinin. The lysate was then centrifuged at 800 rpm for 15 min at 4 °C. The supernatant was microcentrifuged for 10 min at 4 °C, and then the resulting supernatant was saved as the cytoplasmic fraction. The nuclear pellet was washed twice with an equal volume of Hanks' solution with 0.2% (v/v) Triton X-100 and centrifuged at 800 rpm for 15 min at 4 °C. The resulting pellet was treated as the purified nuclear fraction. The nuclei were then lysed in a buffer containing 50 mm Tris, pH 7.4, 1% (v/v) Triton X-100, 400 mm KCl, 1 mm sodium orthovanadate, 1 mm DTT, and protease inhibitors as described above. The samples were incubated on ice for 30 min and microcentrifuged for 10 min at 4 °C, and the supernatant was used as the whole nuclear fraction. For nuclear fractionation, nuclei were isolated from tissue culture cells, and nuclear membranes and nuclear soluble fractions were then prepared as described by Davis and Blobel (17Davis L.I. Blobel G. Cell. 1986; 45: 699-709Abstract Full Text PDF PubMed Scopus (450) Google Scholar) with some modification. The whole nuclear fraction was resuspended in 50 mm Tris-HCl, pH 7.4, 10% (w/v) sucrose, 1 mmsodium orthovanadate, 1 mm DTT, 1 mmMgCl2, and protease inhibitors. DNase-I (5 mg/ml) and RNase A (1 mg/ml) were added, and the nuclei were then incubated for 15 min at 37 °C. Following the incubation with DNase-I, the nuclei were underlaid with 30% sucrose and then subjected to centrifugation in a swinging bucket rotor for 10 min at 20,000 × g to generate a soluble nuclear fraction and a nuclear membrane fraction. Photoaffinity labeling of cellular proteins with [α-32P]GTP was performed as described previously (18Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar). In brief, the UV cross-linking reaction was carried out in a buffer containing 50 mm Hepes, pH 7.4, 2 mm EGTA, 1 mm DTT, 20% (v/v) glycerol, 100 mm NaCl, and 500 μm AMP-PNP. Samples (20 μl) prepared from the cell fractionation procedures, described above, were incubated for 10 min at room temperature with an equal volume of cross-linking buffer containing [α-32P]GTP (2–3 μCi/sample, 3000 Ci/mmol; NEN Life Science Products ) in a 96-well, non-tissue culture-treated plate. The samples were then placed in an ice bath and irradiated with UV light (254 nm) for 15 min. After irradiation, samples were mixed with 5× Laemmli buffer and boiled. SDS-PAGE was performed using 15% acrylamide gels. The gels were then typically silver-stained and dried, and autoradiography was performed (typically overnight) using Kodak X-Omat XAR-5 film at −80 °C. To perform competition experiments, competing nucleotides (m7GpppG and GpppG (New England Biolabs Inc.) and m7GTP and GTP (Sigma)) were added to the sample prior to the addition of the [α-32P]GTP-containing cross-linking buffer. This buffer did not contain AMP-PNP. The samples were then subjected to UV cross-linking as described above. Bovine retinas were obtained frozen from J. A. & W. L. Lawson Co. (Lincoln, NE). The retinas (typically 200/batch) were thawed in a buffer containing 50 mm Tris, pH 8.0, 25 mm KCl, 5 mmMgCl2, and protease inhibitors as described for cell lysate preparations and then homogenized with a motor-driven Dounce homogenizer. The homogenate was centrifuged at 2500 rpm in a swinging bucket rotor to yield a crude nuclear pellet. The nuclei were purified from this crude preparation using the method described by Blobel and Potter (19Blobel G. Potter V.R. Science. 1966; 154: 1662-1665Crossref PubMed Scopus (986) Google Scholar), and the soluble nuclear contents were then extracted as described above. The 18-kDa activity was precipitated using 40–75% ammonium sulfate, resuspended in 3–5 ml of Buffer A (50 mmTris, pH 8.0, 100 mm NaCl, 10 mm MgCl2, 1 mm EDTA, 1 mm DTT, 20 mm KCl), and loaded onto a fast protein liquid chromatography Superdex-200 Highload 16/60 column as described above. The purification of this activity was monitored by both silver staining and UV cross-linking to [α-32P]GTP. The fractions eluted from the Superdex-200 column were assayed for [α-32P]GTP incorporation into the 18-kDa protein, and six peak fractions (eluting with molecular masses of ∼100–150 kDa) were pooled in a final volume of 12 ml and loaded directly onto a fast protein liquid chromatography ion-exchange Mono Q 5/5 column (Amersham Pharmacia Biotech) equilibrated in Buffer A minus KCl. Bound proteins were eluted from the Mono Q 5/5 column with a 28-ml linear gradient of 100–500 mm NaCl. [α-32P]GTP-incorporating activity eluted from the Mono Q 5/5 column with ∼300 mm NaCl in a volume of 5 ml. Peak activity as assayed by [α-32P]GTP incorporation was eluted from the Mono Q column and applied directly to a Bio-Gel HPHT hydroxylapatite column (Bio-Rad) equilibrated in 10 mmpotassium phosphate, pH 6.8, 2.5 mm MgCl2, 0.01 mm CaCl2, and 1 mm DTT. Bound proteins were then eluted, first by stepping the potassium phosphate to 100 mm and then by a 20-ml linear gradient of 100–300 mm potassium phosphate. Peak activity as assayed by the light-catalyzed incorporation of [α-32P]GTP was found to elute with ∼250 mm phosphate. CBP20 was cloned by polymerase chain reaction from HeLa cell cDNA (a generous gift from Dr. Wannian Yang, Cornell University). 5′- and 3′-primers were designed using the published sequence for Homo sapiensCBP20 (GenBankTM accession P52298), and theCBP20 gene was then amplified from the HeLa cell cDNA using 40 polymerase chain reaction cycles (1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C). The 470-base product was inserted into a cloning vector (pCR2.1) using a TA cloning kit (Invitrogen) and then subcloned into the mammalian expression vector pcDNA3 (Invitrogen) and into the Escherichia coli expression vector pGEX-2TK. E. coli cells transformed with the pGEX-2TK-CBP20 vector were grown in a 1-liter culture, and expression of glutathioneS-transferase (GST)-CBP20 protein was induced for 3 h using isopropyl-β-d-thiogalactopyranoside. Following induction, the cells were pelleted by centrifugation (5000 rpm for 10 min in a JA-10 rotor). The harvested cells were resuspended in 15 ml of 50 mm Tris-HCl, pH 8.0, 50 mm EDTA, 1 mm DTT, and protease inhibitors (as described above) and then lysed using 15 mg of lysozyme, followed by the addition of 200 mm MgCl2 and 1 mg of DNase-I. Following centrifugation (100,00 × g for 30 min at 4 °C), the supernatant was incubated with glutathione-agarose beads for 1 h at 4 °C to bind the GST-CBP20 protein. Glutathione-agarose-bound CBP20 was washed with 50 mm Tris-HCl, pH 8.0, 0.5% (v/v) Triton X-100, 200 mm KCl, and 1 mm DTT and then stored in a buffer containing 50 mm Tris-HCl, pH 8.0, 1 mm EDTA, 1 mm DTT, 10 μm GTP, and protease inhibitors. GST-CBP20 was eluted from the glutathione-agarose beads using 10 mm glutathione, pH 8.0, and the GST moiety was cleaved from CBP20 by the addition of 500 units of thrombin for 30 min at room temperature. Using the LipofectAMINE protocol (Life Technologies, Inc.), a hemagglutinin-tagged form of CBP20 (HA-CBP20) was transiently transfected into BHK21 cells according to the manufacturer's directions. Following a 5-h incubation with serum-free medium containing the lipid-DNA complex, the medium was removed and replaced with medium containing 10% fetal bovine serum. Cells were allowed to grow in the presence of serum for ∼20 h and were then switched to serum-free medium for 40 h prior to stimulation with serum. A polyclonal antibody generated against recombinant CBP80 (αCBP80) was prepared as described previously (7Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar). Cytosolic and nuclear lysates were prepared as described above. Prior to immunoprecipitation, the cytosolic lysate was adjusted to 100 mm NaCl, and the nuclear lysate was diluted 3-fold with 50 mm Tris-HCl, pH 8.0, 1 mm DTT, and 1 mm sodium orthovanadate. The lysates were then allowed to incubate at 4 °C for 1 h, with or without the addition of 5 μl of 12CA5 monoclonal antibody or αCBP80 polyclonal antibody. Following the first incubation, 40 μl of protein A-Sepharose beads were added to each sample, and the samples were incubated for another hour at 4 °C. The samples were then centrifuged, and the immunoprecipitated pellets were washed four times with 50 mm Tris-HCl, pH 8.0, 133 mm KCl, 0.33% Triton X-100, 1 mm DTT, and 1 mm sodium orthovanadate. The resulting immunoprecipitated pellets were resuspended in 20 μl of UV cross-linking buffer and were incubated with [α-32P]GTP and UV cross-linked as described above. For Western blot analysis, proteins were transferred to polyvinylidene difluoride membranes following SDS-PAGE. The polyvinylidene difluoride membranes were blocked with 2.5% (w/v) bovine serum albumin in TBS plus 0.1% Tween 20 for 1 h at room temperature. After blocking, the membranes were incubated with either 12CA5 or αCBP80 antibody for 1 h at room temperature, washed with several changes of TBS and 0.1% Tween 20, and incubated for 30 min at room temperature with sheep anti-rabbit or sheep anti-mouse horseradish peroxidase-conjugated antibody (Amersham Pharmacia Biotech) as appropriate. Immunolabeling was detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) according to the manufacturer's instructions. UV cross-linking was done essentially as described by Rozen and Sonenberg (20Rozen F. Sonenberg N. Nucleic Acids Res. 1987; 15: 6489-6500Crossref PubMed Scopus (29) Google Scholar), except that the RNA probe was transcribed from BamHI-cleaved pBluescript II KS with T3 RNA polymerase (Promega). Splicing extracts were prepared from HeLa cells (serum-starved for 40 h prior to stimulation with 100 nm heregulin for 24 h) as described by Lee and Green (21Lee K.A. Green M.R. Methods Enzymol. 1990; 181: 20-30Crossref PubMed Scopus (63) Google Scholar). pBSAd1 precursor linearized bySau3AI was transcribed using T3 RNA polymerase in the presence of m7GpppG dinucleotide cap. Splicing reactions were then carried out as described by Izaurralde et al. (7Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar). In brief, 60 μg of splicing extract were preincubated for 15 min at 30 °C with 1 mm MgCl2. 5 mmcreatine phosphate, 1.5 mm ATP, 2.5 × 104cpm of labeled precursor mRNA, and an additional 1 mmMgCl2 were then added in a final volume of 20 μl, and the reactions were incubated for 2 h at 30 °C. Splice products were visualized by separation on a 10% denaturing polyacrylamide gel, followed by autoradiography. The overall goal of these studies was to identify nuclear activities that could represent novel downstream targets in receptor-coupled signaling pathways. One of the assays we used to identify such activities was the photocatalyzed incorporation of [α-32P]GTP into nuclear proteins. The rationale for this approach was that it would provide a very sensitive assay for identifying guanine nucleotide-binding activities in the nucleus, in a manner analogous to the use of phosphorylation assays to identify growth factor-sensitive phosphosubstrates. Using this assay, we identified an 18-kDa protein that strongly incorporated [α-32P]GTP in serum-treated but not serum-starved cells (see below). We found this activity to be exclusively nuclear and present in every cell line we examined, including HeLa, PC12, COS-7, and BHK21 cells, as well as in various mammary epithelial cells. A similar activity was also observed in the yeast Saccharomyces cerevisiae. A purification scheme was developed using bovine retinal nuclei, which were a particularly rich source of this 18-kDa nuclear activity. A series of three chromatography steps resolved the activity, as assayed by [α-32P]GTP incorporation, from the majority of contaminating low molecular mass proteins (see “Experimental Procedures”). These steps also resolved an 80-kDa protein (designated p80), detected by silver staining, which co-purified with the 18-kDa activity. This putative protein complex was reminiscent of the nuclear CBC, as the CBC comprises an 18-kDa nuclear protein, CBP20 (forcap-binding protein20), stably complexed with an 80-kDa protein, designated CBP80. The formation of the CBP20-CBP80 heterodimer enables the CBC to bind a guanine derivative, the 7-methylguanosine cap structure (m7GpppN), on RNAs transcribed by RNA polymerase II (7Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 8Kataoka N. Ohno M. Kangawa K. Tokoro Y. Shimura Y. Nucleic Acids Res. 1994; 22: 3861-3865Crossref PubMed Scopus (39) Google Scholar, 9Izaurralde E. Lewis J. Gamberi C. Jarmolowski A. McGuigan C. Mattaj I.W. Nature. 1995; 376: 709-712Crossref PubMed Scopus (288) Google Scholar, 10Kataoka N. Ohno M. Moda I. Shimura Y. Nucleic Acids Res. 1995; 23: 3638-3641Crossref PubMed Scopus (40) Google Scholar). The similarities between the 18-kDa nuclear activity and CBP20 (both in complex formation and substrate binding) led us to investigate whether the CBC was a nuclear target for extracellular signals. First, we assayed directly the ability of recombinant E. coli-expressed CBP20 to incorporate [α-32P]GTP. Fig. 1 A shows GST-CBP20, thrombin-cleaved CBP20, and the complexed CBC proteins (His-tagged CBP20 (9Izaurralde E. Lewis J. Gamberi C. Jarmolowski A. McGuigan C. Mattaj I.W. Nature. 1995; 376: 709-712Crossref PubMed Scopus (288) Google Scholar) and CBP80 (7Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar)) as visualized by staining with Coomassie Blue. Fig. 1 B shows that the recombinant CBP20 proteins were all capable of incorporating [α-32P]GTP in a photoaffinity labeling assay. This activity was greatly enhanced by the presence of CBP80 (see lane 5), consistent with previous studies that have demonstrated that complex formation between CBP20 and CBP80 is necessary for capped RNA binding. The GST control did not show any cross-linking to [α-32P]GTP. We next examined whether the ability of CBP20 to incorporate [α-32P]GTP could be regulated in response to serum. BHK21 cells were transiently transfected with a HA-tagged CBP20 construct. Following 40 h of serum starvation, the cells were stimulated with 25% fetal bovine serum for 1.5 h, and HA-CBP20 was immunoprecipitated from cytosolic and nuclear lysates prepared from either serum-starved or stimulated cells. The immunoprecipitates were then assayed for the photocatalyzed incorporation of [α-32P]GTP into CBP20. HA-CBP20 was present in both the cytosolic and nuclear fractions (Fig.2 B), and CBP80 co-immunoprecipitated with nuclear localized HA-CBP20 equally well under conditions of either serum starvation or stimulation (Fig.2 A). The large percentage of HA-CBP20 localized to the cytosol is presumably the result of its overexpression. Nuclear CBP20 demonstrated a marked serum-dependent incorporation of [α-32P]GTP (Fig. 2 C). Given that the m7GpppN RNA cap structure is a known substrate for the CBC, the stimulated incorporation of [α-32P]GTP into CBP20 may reflect an enhanced ability of the CBC to bind the cap structure on RNA. To address this issue, we first examined the relative binding affinities of the CBC for different cap analogs by testing their ability to inhibit the incorporation of [α-32P]GTP into CBP20. PC12 cell nuclear lysates were immunoprecipitated with antibodies generated against CBP80 (i.e. the binding partner of CBP20) (7Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar), and the immunoprecipitates were then assayed for photocatalyzed incorporation of [α-32P]GTP into CBP20 in the absence and presence of RNA cap analogs or GTP. CBP20 proteins that co-immunoprecipitated with CBP80 could be efficiently labeled with [α-32P]GTP. This activity was strongly inhibited by the addition of low concentrations of cap analogs to the [α-32P]GTP cross-linking assay and yielded the following binding specificity: m7GpppG > m7GTP > GpppG > GTP (Fig. 3 A). Indeed, the m7GpppG analog competed with [α-32P]GTP for binding to CBP20 ∼1000 times more effectively than GTP, suggesting that the CBC most likely binds RNA, rather than GTP, in cells. We further examined whether the CBC shows a regulated binding to capped RNAs using a PC12 cell line that stably expresses HA-tagged CBP20. Following starvation, these cells were stimulated with NGF. HA-CBP20 was then immunoprecipitated from the cytoplasmic and nuclear lysates and assayed for the incorporation of either [α-32P]GTP (Fig. 3 B, upper panel) or m7G32pppN-capped RNA (lower panel). Both substrates were incorporated into nuclear HA-CBP20 strictly in a growth factor-dependent manner. Thus, these findings indicate that the growth factor-stimulated incorporation of [α-32P]GTP into CBP20 accurately reflects the activation of the CBC, such that it is induced to bind m7GpppN-capped RNAs. We took further advantage of the high" @default.
• W2072010065 created "2016-06-24" @default.
• W2072010065 creator A5008056151 @default.
• W2072010065 creator A5011351945 @default.
• W2072010065 creator A5073747429 @default.
• W2072010065 creator A5079119381 @default.
• W2072010065 creator A5080948421 @default.
• W2072010065 creator A5087460044 @default.
• W2072010065 date "1999-02-01" @default.
• W2072010065 modified "2023-09-28" @default.
• W2072010065 title "The Nuclear Cap-binding Complex Is a Novel Target of Growth Factor Receptor-coupled Signal Transduction" @default.
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