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Protein Kinase A-anchoring Protein AKAP95 Interacts with MCM2, a Regulator of DNA Replication

2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês

10.1074/jbc.m300765200

ISSN

1083-351X

Autores

Turid Eide, Kristin A. Taskén, Cathrine R. Carlson, Gareth Williams, Tore Jahnsen, Kjetil Taskén, Philippe Collas,

Tópico(s)

Microtubule and mitosis dynamics

Resumo

Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1–195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1–195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2. Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1–195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1–195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2. Eukaryotic DNA replication involves at least 20 different proteins and is a process largely conserved from yeast to human (1Lei M. Tye B.K. J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar). A key player in the regulation of DNA replication is the hexameric minichromosome maintenance (MCM) 1The abbreviations used are: MCM, minichromosome maintenance; pre-RC, pre-replication complex; PKA, protein kinase A; PKI, PKA inhibitor; ORC, origin of recognition complex; mAb, monoclonal antibody; GST, glutathione S-transferase; Pipes, 1,4-piperazinediethane-sulfonic acid; RII, regulatory subunit of PKA-type II.1The abbreviations used are: MCM, minichromosome maintenance; pre-RC, pre-replication complex; PKA, protein kinase A; PKI, PKA inhibitor; ORC, origin of recognition complex; mAb, monoclonal antibody; GST, glutathione S-transferase; Pipes, 1,4-piperazinediethane-sulfonic acid; RII, regulatory subunit of PKA-type II. complex. MCMs are proteins identified initially for their role in plasmid maintenance and cell cycle progression (2Maine G.T. Sinha P. Tye B.K. Genetics. 1984; 106: 365-385Crossref PubMed Google Scholar). Six of the MCM proteins, including MCM2, are highly conserved and form a complex that plays a direct role in both initiation of replication and DNA chain elongation (1Lei M. Tye B.K. J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, 3Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar). The pre-replication complex (pre-RC) assembles during late M and G1 phases by sequential loading of Cdc6, Cdt1, and MCM proteins onto the origin of recognition complex (ORC) at specific chromosomal sites (1Lei M. Tye B.K. J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, 3Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar, 4Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1385) Google Scholar). Once the MCM complex is assembled, S-phase cyclin-dependent kinase activity and the Cdc7-Dbf4 protein kinase (an MCM2-7 kinase) trigger firing of DNA synthesis (5Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (244) Google Scholar). After origin firing, the pre-RC is dissociated, and it is not reassembled until the next cell cycle. A proportion of MCM proteins are phosphorylated and believed to be released from chromatin, although a DNA unwinding or helicase function of MCM proteins has been shown during elongation (6Labib K. Diffley J.F. Curr. Opin. Genet. Dev. 2001; 11: 64-70Crossref PubMed Scopus (125) Google Scholar). Moreover, depletion of MCMs after initiation of DNA replication irreversibly blocks progression of replication forks in Saccharomyces cerevisiae, indicating that MCM proteins are essential for elongation (7Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (515) Google Scholar). We and others have cloned and characterized a 95-kDa protein kinase A (PKA, or cAMP-dependent kinase)-anchoring protein, AKAP95 (8Coghlan V.M. Langeberg L.K. Fernandez A. Lamb N.J. Scott J.D. J. Biol. Chem. 1994; 269: 7658-7665Abstract Full Text PDF PubMed Google Scholar, 9Eide T. Coghlan V. Orstavik S. Holsve C. Solberg R. Skålhegg B.S. Lamb N.J. Langeberg L. Fernandez A. Scott J.D. Jahnsen T. Tasken K. Exp. Cell Res. 1998; 238: 305-316Crossref PubMed Scopus (89) Google Scholar). AKAP95 is a nuclear AKAP that in interphase primarily co-fractionates with the nuclear matrix, a nonionic detergent-, nuclease-, and salt-resistant structure, whereas a minor fraction associates with chromatin (8Coghlan V.M. Langeberg L.K. Fernandez A. Lamb N.J. Scott J.D. J. Biol. Chem. 1994; 269: 7658-7665Abstract Full Text PDF PubMed Google Scholar, 10Collas P. Le Guellec K. Tasken K. J. Cell Biol. 1999; 147: 1167-1180Crossref PubMed Scopus (108) Google Scholar). At mitosis, and upon nuclear envelope breakdown in vitro, AKAP95 dissociates from the matrix and binds chromatin (10Collas P. Le Guellec K. Tasken K. J. Cell Biol. 1999; 147: 1167-1180Crossref PubMed Scopus (108) Google Scholar, 11Steen R.L. Cubizolles F. Le Guellec K. Collas P. J. Cell Biol. 2000; 149: 531-536Crossref PubMed Scopus (68) Google Scholar), onto which it has been proposed to recruit the condensin complex and thereby play a role in chromosome condensation (11Steen R.L. Cubizolles F. Le Guellec K. Collas P. J. Cell Biol. 2000; 149: 531-536Crossref PubMed Scopus (68) Google Scholar, 12Eide T. Carlson C. Tasken K.A. Hirano T. Tasken K. Collas P. EMBO Rep. 2002; 3: 426-432Crossref PubMed Scopus (44) Google Scholar). Mapping of chromatin condensation and condensin targeting functions of AKAP95 has shown that these processes are independent of PKA binding but require both the ZF1 and ZF2 zinc finger motives in the C-terminal half of AKAP95 (12Eide T. Carlson C. Tasken K.A. Hirano T. Tasken K. Collas P. EMBO Rep. 2002; 3: 426-432Crossref PubMed Scopus (44) Google Scholar). A yeast two-hybrid screen has identified p68 RNA helicase as a binding partner for AKAP95, suggesting a putative scaffolding role of AKAP95 in the assembly of transcription complexes (13Akileswaran L. Taraska J.W. Sayer J.A. Gettemy J.M. Coghlan V.M. J. Biol. Chem. 2001; 276: 17448-17454Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The c-Myc-binding protein, AMY-1, also binds AKAP95 near the PKA-RII-binding domain (14Furusawa M. Taira T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2002; 277: 50885-50892Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). AMY-1 can form a ternary complex with AKAP95 and RII that prevents targeting of the PKA catalytic subunit (14Furusawa M. Taira T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2002; 277: 50885-50892Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Any other putative function of AKAP95 in the interphase nucleus has remained unexplored. DNA replication has been connected to the nuclear matrix, based on indications that many DNA polymerases are immobilized on an insoluble intranuclear network in so-called replication factories (15Iborra F.J. Cook P.R. Curr. Opin. Cell Biol. 2002; 14: 780-785Crossref PubMed Scopus (19) Google Scholar, 16Cook P.R. Science. 1999; 284: 1790-1795Crossref PubMed Scopus (627) Google Scholar). Here, we present evidence for a direct interaction between AKAP95 and MCM2. Disruption of this interaction abolishes initiation and elongation phases of replication in vitro. Removing AKAP95 from nuclei also partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication. Our results suggest a role of AKAP95 as an MCM2 anchor and an involvement of the AKAP95-MCM2 association in DNA synthesis. Antibodies and Peptides—Anti-AKAP95 polyclonal antibodies were from Upstate Biotechnology (10Collas P. Le Guellec K. Tasken K. J. Cell Biol. 1999; 147: 1167-1180Crossref PubMed Scopus (108) Google Scholar). Monoclonal antibodies (mAbs) against AKAP95 and MCM2 (Bm28) were from Transduction Laboratories. Anti-GST, anti-Orc2, anti-Cdc6, and anti-MCM2 polyclonal antibodies were from Santa Cruz Biotechnology. mAbs against Gal4AD or Gal4BD domains were from Clontech. Antibodies against lamina-associated polypeptide 2β and B-type lamins were from Dr. J.-C. Courvalin (17Buendia B. Courvalin J.-C. Exp. Cell Res. 1997; 230: 133-144Crossref PubMed Scopus (77) Google Scholar). Anti-HA95 antibodies were as described previously (18Orstavik S. Eide T. Collas P. Han I.O. Tasken K. Kieff E. Jahnsen T. Skålhegg B.S. Biol. Cell. 2000; 92: 27-37Crossref PubMed Scopus (23) Google Scholar). Full-length AKAP95 was amplified by PCR using AKAP95 cDNA (9Eide T. Coghlan V. Orstavik S. Holsve C. Solberg R. Skålhegg B.S. Lamb N.J. Langeberg L. Fernandez A. Scott J.D. Jahnsen T. Tasken K. Exp. Cell Res. 1998; 238: 305-316Crossref PubMed Scopus (89) Google Scholar) as template to generate restriction sites for subcloning into pGEX-KG, expression from which yielded a GST-AKAP95-(1–692) fusion protein (9Eide T. Coghlan V. Orstavik S. Holsve C. Solberg R. Skålhegg B.S. Lamb N.J. Langeberg L. Fernandez A. Scott J.D. Jahnsen T. Tasken K. Exp. Cell Res. 1998; 238: 305-316Crossref PubMed Scopus (89) Google Scholar). This expression vector was used as template to generate deletion constructs and for site-directed mutagenesis (QuikChange; Stratagene) (12Eide T. Carlson C. Tasken K.A. Hirano T. Tasken K. Collas P. EMBO Rep. 2002; 3: 426-432Crossref PubMed Scopus (44) Google Scholar). GST-AKAP95 peptides produced were AKAP95-(1–692), AKAP95-(1–195), AKAP95-(387–692), AKAP95-(387–602), AKAP95-(387–569), AKAP95-(387–524), and AKAP95-(387–450) and peptides with a mutation in zinc fingers ZF1 and ZF2 or in the PKA-binding domain as described in “Results” (12Eide T. Carlson C. Tasken K.A. Hirano T. Tasken K. Collas P. EMBO Rep. 2002; 3: 426-432Crossref PubMed Scopus (44) Google Scholar). MCM2-(398–543) was ligated into pGEX5X-3 (Amersham Biosciences) to produce a GST-MCM2-(398–543) protein. All constructs were sequenced. Expression and purification of GST-tagged proteins were done as described previously (19Solberg R. Tasken K. Wen W. Coghlan V.M. Meinkoth J.L. Scott J.D. Jahnsen T. Taylor S.S. Exp. Cell Res. 1994; 214: 595-605Crossref PubMed Scopus (36) Google Scholar). Protein concentrations were determined by the Bradford method. Cells—HeLa cells were grown in Eagle's minimal essential medium/10% fetal calf serum (Life Technologies, Inc.). Cells were synchronized in M phase with 1 μm nocodazole for 18 h (10Collas P. Le Guellec K. Tasken K. J. Cell Biol. 1999; 147: 1167-1180Crossref PubMed Scopus (108) Google Scholar). To allow cell cycle reentry, mitotic cells were harvested and replated at 2.5 × 106 cells/162-cm2 flask. G1- and S-phase cells were harvested 2 and 12 h, respectively, after release from mitotic arrest (20Martins S. Eikvar S. Furukawa K. Collas P. J. Cell Biol. 2003; 160: 177-188Crossref PubMed Scopus (57) Google Scholar). Nuclei and Chromatin—Intact nuclei were isolated from unsynchronized and G1- or S-phase HeLa cells, as indicated, by Dounce homogenization (21Martins S.B. Eide T. Steen R.L. Jahnsen T. Skålhegg B.S. Collas P. J. Cell Sci. 2000; 113: 3703-3713Crossref PubMed Google Scholar) and used for immunoprecipitation and replication assays, respectively. Concentration of nuclei was determined using an automated cell counter (Coulter). Purified S-phase nuclei were depleted of AKAP95 by loading anti-NuMA antibodies and subsequent exposure to a mitotic HeLa cell extract as described previously (11Steen R.L. Cubizolles F. Le Guellec K. Collas P. J. Cell Biol. 2000; 149: 531-536Crossref PubMed Scopus (68) Google Scholar). To reconstitute nuclear architecture, AKAP95-depleted chromatin masses were recovered from the extract by sedimentation through 1 m sucrose and incubated for 1.5 h in a nuclear reassembly extract containing cytosol, membrane vesicles, ATP-regenerating system, and GTP to promote nuclear envelope formation (20Martins S. Eikvar S. Furukawa K. Collas P. J. Cell Biol. 2003; 160: 177-188Crossref PubMed Scopus (57) Google Scholar). Reconstituted nuclei were sedimented at 1,000 × g through 1 m sucrose for analysis. To prepare chromatin, confluent interphase HeLa cells were harvested and suspended in hypotonic buffer (10 mm Tris, pH7.6, 10 mm NaCl, 3 mm MgCl2, 0.5 mm phenylmethylsulfonyl fluoride, and a protease inhibitor mixture) containing 0.5% Nonidet P-40. Nuclei were sedimented, resuspended in CSK buffer (10 mm Pipes, pH 6.8, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and a protease inhibitor mixture) containing 1% Triton X-100, and extracted at 4 °C for 15 min. The extract was centrifuged at 2,000 × g for 5 min. The pellet was digested in CSK buffer containing 0.5% Triton X-100 and 5 units/μl micrococcal nuclease at 37 °C for 15 min. The lysate was sedimented at 10,000 × g, and the supernatant constituted the chromatin fraction. Yeast Two-hybrid Screening—As bait, a full-length AKAP95 was amplified by PCR (9Eide T. Coghlan V. Orstavik S. Holsve C. Solberg R. Skålhegg B.S. Lamb N.J. Langeberg L. Fernandez A. Scott J.D. Jahnsen T. Tasken K. Exp. Cell Res. 1998; 238: 305-316Crossref PubMed Scopus (89) Google Scholar) and subcloned into yeast vector pAS2-1 (Clontech) to produce a Gal4BD-AKAP95 fusion protein. The construct was transformed into yeast strain PJ69-2A containing Ade and His reporters. Yeasts expressing the bait were mated to yeasts containing a human HeLa Matchmaker cDNA pre-transformed library (5 × 106 independent clones in pGADGH, strain Y187; Clontech) to screen for interacting proteins. Growth on medium lacking Trp, His, Ade, and Leu (high stringency conditions) and β-galactosidase assays were used to isolate cDNAs encoding AKAP95-interacting candidates. 3-Amino-1,2,4-triazole (20 mm), a competitive inhibitor of the yeast His protein, was used to suppress background growth. Positive clones were isolated according to the YPH user manual (Clontech). To confirm protein-protein interactions, bait and prey were re-transformed into strains Y187 and PJ69-2A, respectively, and mated. Diploids were selected onto synthetic drop out minus Trp and Leu (double drop out) plates before re-streaking onto synthetic drop out minus Leu, Trp, Ade, and His (quadruple drop out) to select for interaction. Diploids were tested for β-galactosidase activity by colony-lift filter assay. Expression and Isolation of Proteins from Yeast—Yeasts were harvested in log phase. Cells were resuspended in radioimmune precipitation assay buffer (150 mm NaCl, 1% Triton X-100, 0.1% SDS, and 50 mm Tris, pH 8.0), boiled, and lysed by vortexing with glass beads for 10 min at 4 °C. Proteins were boiled in SDS-loading buffer and analyzed by SDS-PAGE. In Vitro Transcription and Translation of MCM2 and AKAP95—The T7 promoter was incorporated by PCR upstream of MCM2 or AKAP95 using the Clontech Matchmaker Co-Immunoprecipitation kit. In vitro transcription and translation were performed using a TNT T7 Coupled Reticulocyte Lysate System (Promega) and [35S]Met labeling (Amersham Biosciences). Proteins were analyzed by SDS-PAGE. Immunological Procedures—Immunoblotting analysis was performed as described previously (21Martins S.B. Eide T. Steen R.L. Jahnsen T. Skålhegg B.S. Collas P. J. Cell Sci. 2000; 113: 3703-3713Crossref PubMed Google Scholar) using antibodies against AKAP95 (polyclonal antibodies), MCM2 (monoclonal and polyclonal antibodies), Cdc6, and Orc2. Immunofluorescence analysis of purified nuclei was done as described previously (21Martins S.B. Eide T. Steen R.L. Jahnsen T. Skålhegg B.S. Collas P. J. Cell Sci. 2000; 113: 3703-3713Crossref PubMed Google Scholar). For immunoprecipitations, HeLa cells (2 × 107) were sonicated and extracted in radioimmune precipitation assay buffer, and the lysate was centrifuged at 13,000 × g. The supernatant was pre-cleared with protein A/G-agarose beads, and AKAP95 was immunoprecipitated using anti-AKAP95 mAbs (0.25 μg/μl). Immune precipitates were eluted in SDS-loading buffer. Alternatively, AKAP95 was immunoprecipitated from HeLa nuclear extracts in immunoprecipitation buffer (10 mm Hepes, pH 7.5, 10 mm KCl, 2 mm EDTA, 1% Triton X-100, 1 mm dithiothreitol, and protease inhibitors) using polyclonal antibodies (21Martins S.B. Eide T. Steen R.L. Jahnsen T. Skålhegg B.S. Collas P. J. Cell Sci. 2000; 113: 3703-3713Crossref PubMed Google Scholar). These AKAP95 immune precipitates were used in dissociation experiments. GST Precipitation—In vitro-translated [35S]Met-labeled MCM2 or AKAP95 peptides were incubated separately with 200 nm GST-AKAP95 or GST-MCM2-(398–543) peptides in GST binding buffer (20 mm Tris-HCl, pH 7.4, 300 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, and 0.1% Triton X-100) for 30 min. Alternatively, pre-cleared radioimmune precipitation assay extracts from HeLa cells of HeLa nuclei (2 × 107) were incubated overnight with the indicated GST-AKAP95 peptide (400 nm) bound to glutathione-agarose beads. GST-proteins were precipitated, washed in GST binding buffer, and subjected to SDS-PAGE and autoradiography or immunoblotting. Loading of Nuclei with GST-AKAP95 Peptides—Peptide loading into isolated G1- or S-phase HeLa nuclei was performed as described previously (10Collas P. Le Guellec K. Tasken K. J. Cell Biol. 1999; 147: 1167-1180Crossref PubMed Scopus (108) Google Scholar). Briefly, nuclei were mildly permeabilized with lysolecithin, excess lysolecithin was quenched with 3% bovine serum albumin for 5 min, and nuclei were incubated for 1 h with 100 μm of the indicated GST-AKAP95 peptides or GST alone. Nuclei were washed through sucrose and held on ice until use. Lysolecithin-treated nuclei supported import and DNA replication in vitro (21Martins S.B. Eide T. Steen R.L. Jahnsen T. Skålhegg B.S. Collas P. J. Cell Sci. 2000; 113: 3703-3713Crossref PubMed Google Scholar) (see “Results”). DNA Replication Assay—Replication extracts were prepared from S-phase HeLa cells collected 15 h after release from nocodazole-induced mitotic arrest. Cells were lysed by Dounce homogenization (20Martins S. Eikvar S. Furukawa K. Collas P. J. Cell Biol. 2003; 160: 177-188Crossref PubMed Scopus (57) Google Scholar) and briefly sonicated on ice to lyse nuclei and release soluble nuclear components. The lysate was sedimented at 15,000 × g for 15 min and then at 200,000 × g for 2 h at 4 °C. Protein concentration of the extract was ∼18 mg/ml. Replication was assayed by incorporation of [α-32P]dCTP (20Martins S. Eikvar S. Furukawa K. Collas P. J. Cell Biol. 2003; 160: 177-188Crossref PubMed Scopus (57) Google Scholar, 22Stoeber K. Mills A.D. Kubota Y. Krude T. Romanowski P. Marheineke K. Laskey R.A. Williams G.H. EMBO J. 1998; 17: 7219-7229Crossref PubMed Scopus (129) Google Scholar, 23Krude T. Jackman M. Pines J. Laskey R.A. Cell. 1997; 88: 109-119Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Purified G1- or S-phase nuclei, loaded with GST-AKAP95 peptides or GST alone as control, were incubated for 3 h at 5,000 nuclei/μl in 40 μl of replication extract containing an ATP-regenerating system (1.2 μl), 100 μm GTP (0.4 μl), buffered deoxynucleotide triphosphates (40 mm Hepes, pH 7.8, 7 mm MgCl2, and 0.1 mm each of dATP, dGTP, dTTP, and dCTP; 2 μl), and [α-32P]dCTP (3,000 Ci/mmol; Nycomed-Amersham) (20Martins S. Eikvar S. Furukawa K. Collas P. J. Cell Biol. 2003; 160: 177-188Crossref PubMed Scopus (57) Google Scholar). At the end of incubation, samples were mixed with 1 volume of 20 mm Tris (pH 7.5) and digested for 2 h at 37 °C with 1 mg/ml proteinase K. Samples were mixed by pipetting, and aliquots were electrophoresed through 0.8% agarose. Gel loading was assessed by ethidium bromide staining. Signals were detected by autoradiography. Isolation of MCM2 as an AKAP95-binding Protein by Two-hybrid Screening—A full-length AKAP95 clone was fused to the C terminus of Gal4-DNA-BD (16 kDa), expressed in yeast and detected as a ∼120-kDa product (Fig. 1A). Screening of a pre-transformed HeLa cell library (5 × 106 clones) yielded 290 candidate clones after quadruple drop out and β-galactosidase selection, of which 7% were lacZ+, His+, and Ade+ in the absence of bait and eliminated. One hundred and twenty positive clones were sequenced. Whereas most clones were excluded as being false positives or artifacts because the open reading frame was out of frame or in the wrong orientation with respect to the fused Gal4 activation domain, nine clones appeared to be putative interaction partners. Of these, one represented the AKAP95-binding protein AMY-1 (14Furusawa M. Taira T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2002; 277: 50885-50892Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and seven are currently subject to further investigation. One clone encoded residues 398–543 of MCM2, a component of the pre-RC, with a mobility of 30 kDa when fused to the 12-kDa Gal4AD domain (Fig. 1B). This clone was lacZ+, His+, and Ade+ only in presence of AKAP95, but not with Gal4-DNA-BD alone (Fig. 1C, left panel). AMY-1, detected in the screen, was used as positive control, and pGADGH was used as negative control (Fig. 1C). Interaction of MCM2 and AMY-1 with AKAP95 was confirmed by β-galactosidase assay (Fig. 1C). GST Precipitation of 35S-AKAP95 and MCM2 and Mapping of Interaction Domain—To verify the interaction between AKAP95 and MCM2, in vitro-translated [35S]Met-labeled AKAP95 was incubated with GST-MCM2 or GST alone, and GST-protein complexes were precipitated. 35S-AKAP95 co-precipitated with GST-MCM2, but not with GST alone (Fig. 2A). Furthermore, a GST-AKAP95-(1–692) fusion protein, but not GST alone, co-precipitated endogenous MCM2 from a HeLa cell extract, as shown by anti-MCM2 immunoblot analysis (Fig. 2B). This indicates that AKAP95 interacts directly with MCM2. We next mapped the MCM2 interaction domain of AKAP95. In vitro-translated [35S]Met-labeled MCM2 was incubated with GST-AKAP95 deletion peptides. GST precipitations show that AKAP95-(1–692) and AKAP95-(1–195) interacted with MCM2, whereas interaction was nearly abolished with AKAP95-(387–692) (Fig. 2C). This result was confirmed in a precipitation of GST-AKAP95 peptides from HeLa nuclear extracts. GST-AKAP95-(1–195) or GST-AKAP95-(1–692) co-precipitated endogenous MCM2, whereas GST-AKAP95-(397–692) (or shorter deletions thereof; data not shown) was ineffective (Fig. 2D). Notably, neither AKAP95 peptide co-precipitated Orc2 or Cdc6, two other components of the pre-RC (Fig. 2D), arguing toward specificity of the association between AKAP95-(1–195) and MCM2. Collectively, the data argue that the first 195 residues of AKAP95 are involved in the interaction with MCM2. AKAP95 Peptides Dissociate MCM2 from AKAP95 Immune Complexes—AKAP95 and MCM2 co-immunoprecipitated from HeLa cell extracts (Fig. 3A). Fractionation of purified HeLa nuclei further showed that AKAP95 co-fractionated with both nuclear matrix and chromatin, whereas MCM2 co-fractionated primarily with chromatin (Fig. 3B). AKAP95 and MCM2 co-immunoprecipitated from the chromatin fraction (data not shown), suggesting that both proteins reside in a same complex associated with chromatin. Whether GST-AKAP95 peptides would disrupt the interaction between AKAP95 and MCM2 was determined. Anti-AKAP95 immune precipitates from HeLa nuclei were incubated for 1 h with 100 μm GST-AKAP95 fragments, immune precipitates were sedimented, and dissociation of MCM2 from AKAP95 was monitored by immunoblotting. AKAP95-(1–195) dissociated most MCM2 from the AKAP95 immune precipitates, whereas AKAP95-(387–692), shorter C-terminal AKAP95 fragments (data not shown), or GST alone was ineffective (Fig. 3C). This result corroborates the interaction of AKAP95 with MCM2 via residues 1–195 and raises the possibility of disrupting this interaction in nuclei for functional studies. Disruption of the AKAP95-MCM2 Interaction in G 1 Inhibits Initiation and Elongation Phases of DNA Replication—To explore a putative role of the AKAP95-MCM2 interaction on DNA replication, dissociation of MCM2 from AKAP95 was elicited intranuclearly, and the effect of this disruption on replication in a cell-free system was investigated. GST-AKAP95 peptides or, as control, GST alone was introduced into nuclei isolated from G1-phase HeLa cells after gentle permeabilization of the nuclei with lysolecithin. Control and peptide-loaded nuclei were incubated for 3 h in a nuclear and cytosolic extract from S-phase HeLa cells containing [α-32P]dCTP, deoxynucleotide triphosphates, GTP, and an ATP-regenerating system to promote replication. In the absence of peptide or with GST alone, nuclei synthesized DNA, and replication was inhibited by 50 μm aphidicolin in the extract (Fig. 4A, lanes 1 and 2). AKAP95-(1–692) and AKAP95-(1–195) abolished replication, whereas AKAP95-(387–692), AKAP95-(387–602), AKAP95-(387–569), and AKAP95-(387–524) only partially, but significantly, affected replication (Fig. 4A, lanes 3–8). In contrast, AKAP95-(387–450) did not affect replication (Fig. 4A, lane 9). Disruption of AKAP95 zinc finger ZF1 in AKAP95-(387–692) by mutation of two of the zinc-chelating cysteines (C392S and C395S) or similarly mutating ZF1 in AKAP95-(387–450) did not alter the effect of these peptides on replication (Fig. 4A, ZF1*, lanes 11 and 13). In contrast, mutation of the C-terminal zinc finger ZF2 (C481S and C484S) in AKAP95-(387–692) restored a control level of DNA synthesis (Fig. 4A, ZF2*, lane 12), supporting an effect of a region containing ZF2 on replication. Disruption of the PKA-binding domain of AKAP95 by an I582P mutation did not alter the effect of AKAP95-(387–692) on replication efficiency (Fig. 4A, PKA*, lane 14). To provide evidence that 32P labeling in G1 nuclei was due to initiation of replication, we showed that replication was inhibited with 10 μm olomoucine, an inhibitor of the cyclin A-cyclin-dependent kinase 2 complex required for S phase entry (22Stoeber K. Mills A.D. Kubota Y. Krude T. Romanowski P. Marheineke K. Laskey R.A. Williams G.H. EMBO J. 1998; 17: 7219-7229Crossref PubMed Scopus (129) Google Scholar) (Fig. 4B). We also excluded the possibility that the DNA synthesis signal in G1 nuclei represented an elongation phase in already replicating nuclei because G1 nuclei incubated in a G0-phase extract did not replicate (data not shown). Furthermore, we showed that the localization of proteins of the inner nuclear membrane (lamina-associated polypeptide lamina-associated polypeptide 2β), nuclear lamina (B-type lamins), and endogenous AKAP95 was not altered in G1 nuclei containing GST-AKAP95 peptides (Fig. 4C). Lastly, G1 nuclei containing GST-AKAP95 peptides were capable of importing the replication factor Cdc6 in vitro (Fig. 4C). The results indicate that AKAP95 peptides containing the MCM2-binding domain abolish DNA replication in G1 nuclei in vitro without affecting global nuclear architecture or import. Partial inhibition of replication also occurs with AKAP95-(387–524) but not AKAP95-(387–450). This suggests that two regions of AKAP95 are implicated in replication, namely, amino acids 1–195 and a critical domain between residues 450 and 524 that includes ZF2. Whether the elongation phase of DNA replication was also affected by AKAP95 peptides was determined. Nuclei isolated from S-phase HeLa cells were loaded with GST-AKAP95 peptides and incubated in S-phase extract under conditions promoting replication. AKAP95-(1–195) largely inhibited DNA synthesis (Fig. 4D); however, in contrast to G1 nuclei, replication in S-phase nuclei was not affected by any other AKAP95 fragment including those bearing mutations in ZF1, ZF2, or the PKA-binding domain (data not shown). Note that some DNA synthesis occurred in S-phase nuclei incubated in extract from G0 cells, reflecting the replicating state of these nuclei (data not shown). We concluded that, as with initiation of DNA replication, elongation is inhibited by AKAP95-(1–195). However, C-terminal AKAP95 peptides have no effect on the elongation phase of DNA replication. PKI Abolishes Initiation of Replication in Vitro—To address the involvement of PKA in DNA replication, the effect of the AKAP-RII disruptor, Ht31 (24Carr D.W. Stofko-Hahn R.E. Fraser I.D.

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