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Overexpression of PREP-1 in F9 Teratocarcinoma Cells Leads to a Functionally Relevant Increase of PBX-2 by Preventing Its Degradation

2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês

10.1074/jbc.m304704200

ISSN

1083-351X

Autores

Elena Longobardi, Francesco Blasi,

Tópico(s)

Genomics and Chromatin Dynamics

Resumo

To bind DNA and to be retained in the nucleus, PBX proteins must form heterodimeric complexes with members of the MEINOX family. Therefore the balance between PBX and MEINOX must be an important regulatory feature. We show that overexpression of PREP-1 influences the level of PBX-2 protein maintaining the PREP-1-PBX balance. This effect has important functional consequences. F9 teratocarcinoma cells stably transfected with PREP-1 had an increased DNA binding activity to a PREP-PBX-responsive element. Because PREP-1 binds DNA efficiently only when dimerized to PBX, the increased DNA binding activity suggests that the level of PBX might also have increased. Indeed PREP-1-overexpressing cells had a higher level of PBX-2 and PBX-1b proteins. PBX-2 increase did not depend on increased mRNA level or a higher rate of translation but rather because of a protein stabilization process. Indeed, PBX-2 level drastically decreased after 3 h of cycloheximide treatment in control but not in PREP-1-overexpressing cells and the proteasome inhibitor MG132 prevented PBX-2 decay in control cells. Hence, dimerization with PREP-1 appears to decrease proteasomal degradation of PBX-2. Retinoic acid induces differentiation of F9 teratocarcinoma cells with a cascade synthesis of HOX proteins. In PREP-1-overexpressing cells, HOXb1 induction was more sustained (3 days versus 1 day) and the induced level of MEIS-1b, another TALE (three amino acid loop extension) protein involved in embryonal development, was higher. Thus an increase in PREP-1 leads to changes in the fate-determining HOXb1 and has therefore important functional consequences. To bind DNA and to be retained in the nucleus, PBX proteins must form heterodimeric complexes with members of the MEINOX family. Therefore the balance between PBX and MEINOX must be an important regulatory feature. We show that overexpression of PREP-1 influences the level of PBX-2 protein maintaining the PREP-1-PBX balance. This effect has important functional consequences. F9 teratocarcinoma cells stably transfected with PREP-1 had an increased DNA binding activity to a PREP-PBX-responsive element. Because PREP-1 binds DNA efficiently only when dimerized to PBX, the increased DNA binding activity suggests that the level of PBX might also have increased. Indeed PREP-1-overexpressing cells had a higher level of PBX-2 and PBX-1b proteins. PBX-2 increase did not depend on increased mRNA level or a higher rate of translation but rather because of a protein stabilization process. Indeed, PBX-2 level drastically decreased after 3 h of cycloheximide treatment in control but not in PREP-1-overexpressing cells and the proteasome inhibitor MG132 prevented PBX-2 decay in control cells. Hence, dimerization with PREP-1 appears to decrease proteasomal degradation of PBX-2. Retinoic acid induces differentiation of F9 teratocarcinoma cells with a cascade synthesis of HOX proteins. In PREP-1-overexpressing cells, HOXb1 induction was more sustained (3 days versus 1 day) and the induced level of MEIS-1b, another TALE (three amino acid loop extension) protein involved in embryonal development, was higher. Thus an increase in PREP-1 leads to changes in the fate-determining HOXb1 and has therefore important functional consequences. TALE (three amino acid loop extension) proteins, including a PBC and a MEINOX subfamily, are homeodomain transcription factors regulating the activity of various HOX proteins in segmentation, differentiation, and organogenesis (1Gehring W.J. Affolter M. Burglin T. Annu. Rev. Biochem. 1994; 63: 487-526Crossref PubMed Scopus (854) Google Scholar, 2Krumlauf R. Cell. 2001; 78: 191-201Abstract Full Text PDF Scopus (1734) Google Scholar, 3Moens C.B. Prince V.E. Dev. Dyn. 2002; 224: 1-17Crossref PubMed Scopus (158) Google Scholar, 4Burglin T. Nucleic Acids Res. 1997; 25: 4173-4180Crossref PubMed Scopus (491) Google Scholar). Proteins of the PBX subfamily (Extradenticle in Drosophila, lazarus in zebrafish, CEH20 in Caenorhabditis elegans, and PBX in mammals) interact with anterior HOX, increasing DNA target selectivity. In the presence of their target DNA, HOX proteins form molecular complexes with PBX through the homeodomains and nearby amino acid sequences (5Mann R.S. Chan S.-K. Trends Genet. 1996; 12: 258-262Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 6Chang C.P. Shen W.F. Rozenfeld S. Lawrence H.J. Largman C. Cleary M.L. Genes Dev. 1995; 9: 663-674Crossref PubMed Scopus (352) Google Scholar, 7Knoepfler P.S. Kamps M.P. Mol. Cell. Biol. 1995; 15: 5811-5819Crossref PubMed Scopus (144) Google Scholar, 8Phelan M.L. Rambaldi I. Featherstone M.S. Mol. Cell. Biol. 1995; 15: 3989-3997Crossref PubMed Scopus (202) Google Scholar, 9Shen W.-F. Rozenfeld S. Lawrence H.J. Largman C. J. Biol. Chem. 1997; 272: 8198-8206Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Proteins of the MEINOX subfamily (homothorax in Drosophila, CEH25 in C. elegans, and MEIS and PREP in vertebrates) (4Burglin T. Nucleic Acids Res. 1997; 25: 4173-4180Crossref PubMed Scopus (491) Google Scholar, 10Kurant E. Pai C.-Y Sharf R. Halachmi N. Sun Y.H. Salzberg A. Development. 1998; 125: 1037-1048Crossref PubMed Google Scholar, 11Steelman S. Moskow J.J. Muzynski K. North C. Druck T. Montgomery J.C. Huebner K. Daar I.O. Buchberg A.M. Genome Res. 1997; 7: 142-156Crossref PubMed Scopus (49) Google Scholar, 12Knoepfler P.S. Calvo K.R. Chen H. Antonarakis S.E. Kamps M.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14553-14558Crossref PubMed Scopus (168) Google Scholar, 13Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 14Berthelsen J. Zappavigna V. Ferretti E. Mavilio F. Blasi F. EMBO J. 1998; 17: 1434-1445Crossref PubMed Scopus (182) Google Scholar, 15Pai C.-Y. Kuo T.-S. Jaw T.J. Kurant E. Chen C.-T. Bessarab D.A. Salzberg A. Sun Y.H. Genes Dev. 1998; 12: 435-446Crossref PubMed Scopus (271) Google Scholar, 16Calvo K.R. Knoepfler P. McGrath S. Kamps M.P. Oncogene. 1999; 18: 8033-8043Crossref PubMed Scopus (38) Google Scholar) form specific DNA-independent dimeric complexes with PBX, expanding the specific target selectivity and transcriptional regulatory functions (10Kurant E. Pai C.-Y Sharf R. Halachmi N. Sun Y.H. Salzberg A. Development. 1998; 125: 1037-1048Crossref PubMed Google Scholar, 11Steelman S. Moskow J.J. Muzynski K. North C. Druck T. Montgomery J.C. Huebner K. Daar I.O. Buchberg A.M. Genome Res. 1997; 7: 142-156Crossref PubMed Scopus (49) Google Scholar, 12Knoepfler P.S. Calvo K.R. Chen H. Antonarakis S.E. Kamps M.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14553-14558Crossref PubMed Scopus (168) Google Scholar, 13Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 14Berthelsen J. Zappavigna V. Ferretti E. Mavilio F. Blasi F. EMBO J. 1998; 17: 1434-1445Crossref PubMed Scopus (182) Google Scholar, 15Pai C.-Y. Kuo T.-S. Jaw T.J. Kurant E. Chen C.-T. Bessarab D.A. Salzberg A. Sun Y.H. Genes Dev. 1998; 12: 435-446Crossref PubMed Scopus (271) Google Scholar, 16Calvo K.R. Knoepfler P. McGrath S. Kamps M.P. Oncogene. 1999; 18: 8033-8043Crossref PubMed Scopus (38) Google Scholar). Through the use of different interaction surfaces, PBX forms trimeric complexes with HOX and MEINOX proteins, further expanding DNA target selectivity of HOX (17Jacobs Y. Schnabel C.A. Cleary M.L. Mol. Cell. Biol. 1999; 19: 5134-5142Crossref PubMed Scopus (214) Google Scholar, 18Ryoo H.D. Marty T. Casares F. Affolter M. Mann R.S. Development. 1999; 126: 5137-5148Crossref PubMed Google Scholar, 19Ferretti E. Marshall H. Pspperl H. Maconochie M. KrumLauf R. Blasi F. Development. 2000; 127: 155-166Crossref PubMed Google Scholar). Vertebrates express multiple members of the PBC and MEINOX subfamilies, at least 6 PBX (four genes), 5 MEIS (three genes), and 2 PREP (two genes) (4Burglin T. Nucleic Acids Res. 1997; 25: 4173-4180Crossref PubMed Scopus (491) Google Scholar, 11Steelman S. Moskow J.J. Muzynski K. North C. Druck T. Montgomery J.C. Huebner K. Daar I.O. Buchberg A.M. Genome Res. 1997; 7: 142-156Crossref PubMed Scopus (49) Google Scholar, 20Nourse J. Mellentin J.D. Galili N. Wilkinson J. Stanbridge E. Smith S.D. Cleary M.L. Cell. 1990; 60: 535-545Abstract Full Text PDF PubMed Scopus (566) Google Scholar, 21Monica K. Galili N. Nourse J. Saltman D. Cleary M.L. Mol. Cell. Biol. 1993; 11: 6149-6157Crossref Scopus (260) Google Scholar, 22Moskow J.J. Bullrich F. Huebner K. Daar I.O. Buchberg A.M. Mol. Cell. Biol. 1995; 15: 5434-5443Crossref PubMed Scopus (285) Google Scholar, 23Chen H.M. Rossier C. Nakamura Y. Lynn A. Chakravarti A. Antonarakis S.E. Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar, 24Wagner K. Mincheva A. Korn B. Lichter P. Popperl H. Mech. Dev. 2001; 103: 127-131Crossref PubMed Scopus (61) Google Scholar, 25Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (150) Google Scholar, 26Fognani C. Kilstrup-Jensen C. Ferretti E. Zappavigna V. Blasi F. Nucleic Acids Res. 2002; 30: 2043-2051Crossref PubMed Scopus (40) Google Scholar). Thus a high number of possible MEINOX-PBX heterodimers may form but very little is known regarding the role of individual heterodimers. The formation of heterodimers between PBX and MEIS/PREP has multiple biological consequences that include the regulation of nuclear localization (26Fognani C. Kilstrup-Jensen C. Ferretti E. Zappavigna V. Blasi F. Nucleic Acids Res. 2002; 30: 2043-2051Crossref PubMed Scopus (40) Google Scholar, 27Abu-Shaar M. Ryoo H.D. Mann R.S. Genes Dev. 1999; 13: 935-945Crossref PubMed Scopus (192) Google Scholar, 28Berthelsen J. Kilstrup-Nielsen C. Blasi F. Mavilio F. Zappavigna V. Genes Dev. 1999; 13: 946-953Crossref PubMed Scopus (199) Google Scholar, 29Jaw T.J. You L.R. Knoepfler P.S. Yao L.-C. Pai C.Y. Tang C.-Y. Chang L.-P. Berthelsen J. Blasi F. Kamps M.P. Sun Y.H. Mech. Dev. 2000; 91: 279-291Crossref PubMed Scopus (76) Google Scholar, 30Kurant E. Eytan D. Salzberg A. Genetics. 2001; 157: 689-698Crossref PubMed Google Scholar, 31Waskiewicz A.J. Rikhof H.A. Hernandez R.E. Moens C.B. Development. 2001; 128: 4139-4151PubMed Google Scholar). Neither PREP-1 nor PREP-2 contains a nuclear localization signal and thus requires PBX heterodimerization to enter the nucleus (26Fognani C. Kilstrup-Jensen C. Ferretti E. Zappavigna V. Blasi F. Nucleic Acids Res. 2002; 30: 2043-2051Crossref PubMed Scopus (40) Google Scholar, 28Berthelsen J. Kilstrup-Nielsen C. Blasi F. Mavilio F. Zappavigna V. Genes Dev. 1999; 13: 946-953Crossref PubMed Scopus (199) Google Scholar). On the other hand, PREP-1 dimerization prevents nuclear export of PBX (28Berthelsen J. Kilstrup-Nielsen C. Blasi F. Mavilio F. Zappavigna V. Genes Dev. 1999; 13: 946-953Crossref PubMed Scopus (199) Google Scholar, 29Jaw T.J. You L.R. Knoepfler P.S. Yao L.-C. Pai C.Y. Tang C.-Y. Chang L.-P. Berthelsen J. Blasi F. Kamps M.P. Sun Y.H. Mech. Dev. 2000; 91: 279-291Crossref PubMed Scopus (76) Google Scholar). Thus the balance between PREP and PBX may be functionally important; therefore, its disruption may lead to important functional consequences. Because these proteins are expressed both in the embryo and in the adult, such consequences may be felt not only in embryogenesis but also in the adult life. In man, Prep1 maps at chromosome 21q22.3 (23Chen H.M. Rossier C. Nakamura Y. Lynn A. Chakravarti A. Antonarakis S.E. Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar, 32Berthelsen J. Viggiano L. Schulz H. Ferretti E. Consalez G.G. Rocchi M. Blasi F. Genomics. 1998; 47: 323-324Crossref PubMed Scopus (24) Google Scholar) and hence it can be present in triple copy in chromosomes of Down's syndrome patients. Indeed, PREP-1 is overexpressed in Down's syndrome fetal brains along with at least one of its target genes (33Sanchez-Font A.F. Bosch-Comas A. Gonzales-Duarte R. Marfany G. Nucleic Acids Res. 2003; 31: 2769-2777Crossref PubMed Scopus (32) Google Scholar). Because PREP-1 binds DNA only as a heterodimer with PBX proteins, one would predict that increased PREP-1 could not affect the expression of other genes unless PBX proteins also were increased. Data in Drosophila and zebrafish show that homothorax and MEIS proteins stabilize PBX (30Kurant E. Eytan D. Salzberg A. Genetics. 2001; 157: 689-698Crossref PubMed Google Scholar, 31Waskiewicz A.J. Rikhof H.A. Hernandez R.E. Moens C.B. Development. 2001; 128: 4139-4151PubMed Google Scholar). If PREP-1 had the same effect, its increase would cause a generalized variation of the gene expression pattern in various tissues. Here we have overexpressed PREP-1 in cultured F9 teratocarcinoma cells. We find that the increase of PREP-1 results in increase of PBX-2 and PBX-1b proteins, that the effect on PBX-2 is post-transcriptional, and that PREP-1 prolongs its half-life, preventing its rapid degradation. The functional significance of this finding is underlined by the change in the kinetics of HOXb1 synthesis during retinoic acid-induced differentiation of F9 teratocarcinoma cells. Transfections—F9 (mouse embryonal carcinoma) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Cells were transfected with either pBOSPREP-1 (25Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (150) Google Scholar) plus pcDNA3neo (Invitrogen) plasmids or with pcDNA3neo alone by electroporation (single electric pulse of 53 ms at 500 microfarads and 230 V). Cells were plated in 10-cm dishes, and stable clones were obtained by selection with 380 μg/ml neomycin. To induce differentiation, trans-retinoic acid (RA) 1The abbreviations used are: RA, retinoic acid; CHX, cycloheximide; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase. from Sigma was added to a final concentration of 10 nm. The cells were collected after 0-7 days of incubation, and nuclear extracts were prepared as described below. Cell Extracts—Nuclear and cytoplasmic extracts were obtained as described previously (34Berthelsen J. Vandekerkhove J. Blasi F. J. Biol. Chem. 1996; 271: 3822-3830Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). One million cells were washed twice with cold phosphate-buffered saline, collected with 300 μl of cold buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride) into an Eppendorf tube, left 10 min on ice, and lysed by the addition of Triton X-100 to a final concentration of 0.3%. The nuclei were collected by centrifugation. The supernatant was removed to a new tube, and 0.11 volume of buffer B was added (0.3 m HEPES, pH 7.9, 1.4 m KCl, 30 mm MgCl2), incubated for 30 min at 4 °C, and centrifuged. The resulting supernatant was denoted as cytoplasmic extract. Nuclear extracts were prepared by resuspending the pelleted nuclei in 60 μl of buffer C (20 mm HEPES, pH 7.9, 25% glycerol (v/v), 0.42 m NaCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride) for 30 min on ice. The extract was cleared by centrifugation. Total extracts were obtained as follows. Cells were washed twice with cold phosphate-buffered saline and lysed with radioimmune precipitation buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). The extracts were cleared by centrifugation. Cycloheximide (CHX) and MG132 treatments—F9 clones were starved for 30 min in fetal bovine serum-free medium. After a 30-min incubation in complete medium, the medium was supplemented with 10 μg/ml CHX or 5 μm MG132 and lysed 3 h later with radioimmune precipitation buffer as described above. In other experiments, a time course was carried out by incubating cells for 0, 30, or 60 min with 5 μm MG132 at 37 °C. In all of the cases, at the end of incubation cells were lysed and the total or nuclear extracts were prepared as described above. Immunoblotting—Total (or nuclear and cytoplasmic) protein extracts were resolved by 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore) in a semi-dry blotting apparatus. The membranes were incubated with anti-PREP-1 (1:10000), anti-PBX-1,2,3 (1:4000), anti-PBX-1 (1:1000), anti-PBX-2 (1:2000), anti-HOXb1 (1:500), anti-MEIS-1 (1:5000), and anti-α-actin (1:1000) antibodies. The polyclonal rabbit antibodies against PREP-1 have been described previously (25Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (150) Google Scholar). Antibodies against PBX and actin proteins were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PBX-1,2,3 recognizes a common C-terminal peptide present in all of the 50-kDa splice variants. Antibodies against PBX-1 and PBX-2 are reactive for the N terminus of the proteins. Antibodies against HOXb1 were from Babco (Richmond, CA), and antibodies against MEIS-1 were a kind gift of Dr. A. M. Buchberg. Blots were developed with SuperSignal Westpico chemiluminescence substrate or with the more sensitive SuperSignal Westdura extended duration substrate (Pierce). Electrophoretic Mobility Shift Assays (EMSAs)—EMSAs were done essentially as described previously (34Berthelsen J. Vandekerkhove J. Blasi F. J. Biol. Chem. 1996; 271: 3822-3830Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 1 μg of nuclear extract was incubated with 2 μg of poly(dI·dC) in 9 μl of H2K150 (25 mm HEPES, pH 7.9, 20% glycerol (v/v), 150 mm KCl, 1 mm MgCl2, 0.2 mm EDTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride) on ice for 10 min. 25,000 cpm of 32P-labeled oligonucleotide was added alone or together with 100-fold concentrated unlabeled oligonucleotide or with specific antibodies and left at room temperature for 30 min. The reactions were analyzed by 5% PAGE in 0.5× Tris borate-EDTA. The oligonucleotide probe used for the EMSA (binding sites underlined) was B2PP2, 5′-GGAGCTGTCAGGGGGCTAAGATTGATCGCCTCA-3′. B2PP2 derived from the HOXb2 r4 enhancer contains both a PREP-MEIS (CTGTCA) and a PBX-HOX site (AGATTGATCG) (underlined) (19Ferretti E. Marshall H. Pspperl H. Maconochie M. KrumLauf R. Blasi F. Development. 2000; 127: 155-166Crossref PubMed Google Scholar). Northern Blotting and RT-PCR—Total RNA was extracted according to standard methods. For Northern blot analysis, 10-20 μg of total RNA were electrophoresed through a 1% agarose, 2.2 m formaldehyde gel and transferred to a Hybond NH+ membrane with 20× SSC. The filter was then washed in 5× SSC at room temperature for 5 min and dried, and the RNA was fixed to the membrane by UV. The filter was prehybridized in formamide prehybridization/hybridization solution (5× SSC, 5× Denhardt solution, 50% (w/v) formamide, 1% SDS, 100 μg/ml of sheared salmon sperm DNA) for 2 h at 60 °C and hybridized in the same buffer containing the radiolabeled probe. The filter was washed at room temperature twice in 2× SSC, 0.1% SDS; twice in 0.2× SSC, 0.1% SDS; and then once in 0.2× SSC, 0.1%SDS at 42 °C. The human PREP-1 and mouse PBX-2 cDNAs, used as probe, were labeled with [α-32P]dCTP by the random primer method. RT-PCRs were carried out following the Invitrogen Superscript Choice System protocol using oligo(dT) for first strand cDNA synthesis. Amplification of specific amplicons was obtained with the following primers pairs: PBX-2 primers (493-bp product): PBX-2F, 5′-GCCACAGCCGCACCAGCTCT-3′, and PBX-2R, 5′-GGACACCCCACTCTCCCTG-3′; PREP-1 primers (183-bp product): PREP-1FRT, 5′-AAGATCTCAGCATCTTGC-3′, and PREP-1RRT, 5′-TGTTGACTTGGAGTAGTGTC-3′; and β-actin primers (685-bp product): Act1, 5′-GGCATCCTGACCCTGAAGT-3′, and Act2, 5′-CGGATGTCAACGTCACACTT-3′. Amplification involved a first denaturation step at 97 °C for 2 min followed by 25 cycles (32 cycles in the case of PREP-1), each consisting of a denaturation step at 95 °C for 30 s, an annealing step at 63 °C (for PBX-2 and actin) or 55 °C (for PREP-1) for 30 s, and an extension step at 72 °C for 1 min. To end the reaction, an extension step of 10 min at 72 °C was used. Overexpression of PREP-1 in Mouse F9 Cells Results in PBX-2 Increase—Five F9 mouse teratocarcinoma cell clones, each transfected with either pcDNA3-neo vector (controls, C1-C5) or with both pcDNA3-neo and pBOS-PREP-1 (P1-P5), have been isolated. Immunoblotting analysis performed on nuclear extracts from different clones of control (C1-C4) and PREP-1-transfected F9 cells (clones P1-P4) shows an increase in PREP-1 (Fig. 1A). To test whether overexpression of PREP-1 affected PBX protein levels, we employed different PBX antibodies. Using an antibody (PBX-1,2,3) that does not distinguish between PBX-1a, PBX-2, and PBX-3a, we found a clear increase in the intensity of the PBX band in P1-P4 clones. To identify the form of PBX increased in PREP-1-overexpressing cells, we used isoform-specific antibodies. As shown in Fig. 1A, the PBX-2 and PBX-1b bands were clearly increased in the different clones. PBX-3 antibodies gave an ambiguous result (data not shown). The reactivity toward anti-α-actin antibodies was comparable in all of the lanes. The same result was obtained with another clone, P5 (data not shown). We then tested the subcellular localization of PREP-1 and PBX proteins by analyzing nuclear and cytoplasmic extracts by immunoblotting. In untransfected F9 cells (and in all of the control clones, data not shown), PREP-1 was present exclusively in the nuclear extract (Fig. 1B). The cytoplasm showed two weaker bands of different sizes that may represent cross-reacting components. In overexpressing P2 and P5 clones, PREP1 was found increased in the nucleus but sometimes also in the cytoplasm. The presence of PREP-1 in the cytoplasm correlated with its level of expression (compare clones P5 and P2). We have then analyzed by immunoblotting the same nuclear and cytoplasmic extracts using PBX-1,2,3 antibodies. As shown in Fig. 1B, the PBX band was present only in the nuclear extracts of PREP-1-overexpressing cells. The increase in PBX was higher in the extract of clone P2 than in clone P5, in agreement with the higher expression of PREP-1 in clone P2. The absence of PBX bands in the immunoblots of control cells in Fig. 1B is probably because of a lower efficiency of the anti-PBX antibodies with respect to anti-PREP-1 antibodies and shorter time of development of the film. As a control, we tested the level of another nuclear protein, HMG1. As shown in Fig. 1B, HMG1 level was found unaltered in both control and PREP-1-overexpressing cells and was present uniquely in the nuclear extracts. In conclusion, the data show a net increase of some PBX proteins in PREP-1-overexpressing clones and their localization in the nucleus. As PBX-2 was the PBX isoform mainly affected by PREP-1 overexpression (see Fig. 1A), we quantitated the level of PBX-2 by immunoblotting with anti-PBX-2 antibodies using diluted nuclear extracts of the P2 (PREP-1 overexpressing) clone and compared it with that of the undiluted extract from control clone C3. The data show that PBX-2 was increased ∼4-fold in the nuclei of PREP-1-overexpressing clone (Fig. 1C). The increase in PREP-1 and PBX proteins should be reflected in an increase of DNA binding activity to the specific DNA sequence recognized by the PBX-PREP-1 heterodimer. To characterize the DNA binding activity of control and transfected F9 cells, we used oligonucleotide b2PP2 from the HOXB2 gene (19Ferretti E. Marshall H. Pspperl H. Maconochie M. KrumLauf R. Blasi F. Development. 2000; 127: 155-166Crossref PubMed Google Scholar) that carries both the PBX-HOX and the PREP-MEIS sites (see "Experimental Procedures") and that binds with high affinity all of the PREP-1-PBX dimers (19Ferretti E. Marshall H. Pspperl H. Maconochie M. KrumLauf R. Blasi F. Development. 2000; 127: 155-166Crossref PubMed Google Scholar). Fig. 2 shows an EMSA comparing the binding activity of a nuclear extract of control clone C3 with that of the overexpressing clone P2. C3 displayed one very weak electrophoretically retarded band (band A), the migration of which is compatible with a complex of PREP-1 with one of the long forms of PBX (25Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (150) Google Scholar). In the overexpressing clone P2, two bands were observed (bands A and B) and the intensity of A was increased over control extract. The migration of the weaker faster migrating band (band B) is consistent with a PREP-1 complex with one of the short forms of PBX (see below). The binding was specific in both C3 and P2 extracts as it was eliminated by incubation with a 100-fold excess of unlabeled oligonucleotide. To identify the binding components, we used specific antibodies. Anti-PREP-1 antibodies totally inhibited binding in all of the cases, showing the presence of PREP-1 in all of the binding complexes. The A band of control C3 extracts was inhibited by both PBX-1,2,3 and anti-PBX-2-specific antibodies but not inhibited by anti-PBX-3 antibodies, suggesting that the DNA-binding forms were mostly PREP-1-PBX-1a and PREP-1-PBX-2 heterodimers. In PREP-1-overexpressing P2 cells, the slower migrating A band was strongly inhibited (in addition to PREP-1 antibodies) by anti-PBX-1,2,3 and anti-PBX-2 and very weakly by anti-PBX-1 and anti-PBX-3 antibodies. Therefore, it represents mostly a PREP-1·PBX-2 complex. On the other hand, the B band in P2 extracts was inhibited only by PBX-1 and PREP-1 antibodies, suggesting a PREP-1-PBX-1b dimer. In conclusion, the data show in PREP-1-overexpressing cells an increase in DNA binding activity and a shift in heterodimer composition from a mostly PREP-1-PBX-1 heterodimer in control cells to mostly PREP-1-PBX-2 plus PREP-1-PBX-1b heterodimers in overexpressing cells. These results are in agreement with the immunoblotting data, which show an increase of PBX-2 and PBX-1b. PBX-2 mRNA Is Not Increased in PREP-1-overexpressing Cells and Is Not More Efficiently Translated—We went back to PREP-1-overexpressing cells to test whether the increase of PBX-2 was dependent on an increase of its mRNA. As shown in Fig. 3A, Northern blot analysis of the total RNA showed an abundant PREP-1 mRNA signal in the overexpressing P2 clone under conditions in which no signal was visible with RNA extracted from control clone C3. On the other hand, the signal obtained with a PBX-2 probe was very weak and was found not to be different in the two types of clones (Fig. 3B). We also used RT-PCR to check the levels of the two mRNAs. When we used PBX-2-specific primers, a band of the expected size (Fig. 3C, arrow on the right) was visible with total RNA from both control (C2, C3, and C4) and PREP-1-overexpressing clones P1, P2, and P3. The band had the same intensity in all of the clones, suggesting that the clones had comparable levels of transcript. As a control, we also amplified the endogenous actin mRNA (Fig. 3C, arrow, left side). Amplicons derived from endogenous β-actin mRNA also showed no difference in intensity. The amplification assay for PBX-2 mRNA was in the linear range. A control experiment showed a linear relationship between the intensity of the PCR-amplified band and the amount of input PBX-2 cDNA (Fig. 3C). Thus an increase in PBX-2 mRNA would have been detected if present. RT-PCR with PREP-1-specific primers showed an increase of PREP-1 mRNA in overexpressing P1, P2, and P3 clones when compared with control C2, C3, and C4 clones as expected (Fig. 3D). Therefore, we conclude that the increased level of PBX-2 in PREP-1-overexpressing clones does not result from an increase of PBX-2 mRNA, suggesting a post-transcriptional mechanism for the elevation of PBX-2. We also tested whether the difference in PBX-2 production could be attributed to a differential translation of PBX-2 mRNA in PREP-1-overexpressing cells. We pulsed cells with [35S]methionine for 0-3 h, extracted the proteins at different times, and immunoprecipitated them with specific anti-PBX-2 antibodies. SDS-PAGE and autoradiography analysis showed a time-dependent increase of the intensity of the PBX-2 band but showed no difference between control and overexpressing clones (data not shown). Therefore, we conclude that the effect of PREP-1 overexpression is neither at the level of transcription or stability of message nor at the level of its translation. PBX-2 Has a Short Half-life, Which Is Increased in PREP-1 Overexpressing Cells and by Proteasome Inhibitors—We tested for degradation of PBX-2 in one control (C3) clone by exposing cells for 3 h to the protein synthesis inhibitor (CHX) and testing total protein extracts by immunoblotting. CHX was used at 10 μg/ml, a concentration at which it inhibited [35S]methionine incorporation by 92% (data not shown). As shown in Fig. 4A, the level of both PREP-1 and PBX-2 in total extracts of C3 control clone decreased substantially after CHX addition. However, the decrease of PBX-2 was much lower in the extracts of PREP-1-overexpressing cells (clone P2). This result has been reproduced several times. Immunoblotting with anti-actin antibody is also shown. To examine the mechanism of degradation of PBX-2, we employed MG132 (5 μm), an inhibitor of the proteasome. As shown in Fig. 4B, in control C3 cells the level of PBX-2 increased in the extract after the addition of MG132 for 3 h. We also compared the effect of MG132 on the level of PBX-2 in nuclear and cytoplasmic extracts of C3 control cells. The data show (Fig. 4C) that in the presence of MG132 there is a time-dependent accumulation of PBX-2 and PREP-1 in the cytoplasm. We conclude that the level of PBX-2 is controlled by degradation, most probably in the proteasome because it is inhibited by MG132. It seems reasonable to conclude that when PREP-1 is overexpresse

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