[CANCER RESEARCH 63, 1430 –1437, March 15, 2003] E1A Deregulates the Centrosome Cycle in a Ran GTPase-dependent Manner 1 Antonio De Luca, 2 Rosamaria Mangiacasale, 2 Anna Severino, 2 Lorenzo Malquori, Alfonso Baldi, Antonella Palena, Anna Maria Mileo, Patrizia Lavia, 3 and Marco G. Paggi 3 Department for the Development of Therapeutic Programs, Laboratory “C,” Regina Elena Cancer Institute, CRS, Rome [A. D. L., A. S., A. B., A. M. M., M. G. P.], and Section of Genetics, Institute of Molecular Biology and Pathology, National Research Council, Rome [R. M., L. M., A. P., P. L.], Italy ABSTRACT By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization. Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines. The induction of supernumerary centrosomes requires func- tional RCC1, the nucleotide exchange factor for Ran and, hence, a func- tional Ran network. The E1A portion responsible for the interaction with Ran is the extreme NH 2 -terminal region (amino acids 1–36), which is also required for the induction of centrosome amplification. In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide ex- change on Ran, whereas an E1A mutant, deleted from the extreme NH 2 - terminal region, does not. In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoproteins functionally related to E1A. These findings suggest a common pathway of these oncoproteins in eliciting virus-induced genomic instability. INTRODUCTION Adenovirus E1A are potent oncoproteins engineered to heavily reprogram gene expression and, ultimately, the fate of host cells (1). The 289R and 243R forms of E1A are the most abundant translated products and are synthesized immediately after infection (2). Multi- ple, sometimes antithetical, functions have been attributed to these proteins, including as diverse effects as induction of cellular prolif- eration and transformation, inhibition of differentiation (3, 4), induc- tion of apoptosis (5–7), and tumor suppression (8 –11). Viral onco- proteins disrupt the coordination of cell cycle events mainly by modifying the activity of endogenous cellular factors. The identifica- tion of such factors, therefore, is of fundamental relevance to thor- oughly understand cell cycle regulatory networks and can help to devise novel targets that may be sensitive to anticancer therapies. To expand our current knowledge of these multifaceted viral tools and their molecular targets in host cells, we have focused on the ENT 4 region (amino acids 1–36), common to both 289R E1A and 243R E1A oncoproteins from human adenovirus (2). Recently, we identified RACK1, a receptor for activated C kinase, as an E1A antagonizing factor, that physically interacts with the ENT region of E1A (12). By means of the yeast two-hybrid system, we have now identified a novel physical interaction between adenovirus 2 E1A and the Ran GTPase. The Ran GTPase is the central element in a regulatory network that includes a GTPase-activating protein (RanGAP1), which catalyzes GTP hydrolysis on Ran producing RanGDP, and a guanine exchange factor termed RCC1, which catalyzes nucleotide exchange on Ran; both of these activities are modulated by RanBP1, which increases GTP hydrolysis by RanGAP1 and inhibits the exchange activity of RCC1. Activity of the network requires a full nucleotide exchange- and-hydrolysis cycle on Ran (13–15). Ran plays a primary regulatory role in nucleocytoplasmic transport and cell cycle progression (re- viewed in Refs. 14 and 16). Growing evidence also indicates a regulatory role of Ran in mitotic spindle organization (reviewed in Refs. 17 and 18). Ran network components specifically associate with mitotic structures in mammalian cells (19 –21). In addition, the induc- tion of imbalance among components by overexpressing the RanBP1 protein yields abnormal mitoses, often with monopolar or multipolar spindles (19). Spindle poles are organized by centrosomes, the major microtubule-organizing centers in eukaryotic cells. To ensure bipo- larity in the mitotic spindle, centrosomes duplicate only once per cell cycle during S phase and separate in late G 2 to give rise to two spindle poles. Errors in the centrosome duplication cycle give rise to multi- polar spindles and, therefore, are regarded as a major cause of genomic instability (reviewed in Refs. 22–25). Centrosome amplifi- cation occurs in many tumors and, in particular, in human carcinomas expressing the HPV16-derived E7 transforming protein (26, 27). Here we have sought to dissect the functional significance of the interaction between E1A and Ran. We have found that E1A, like E7, deregulates the centrosome duplication cycle. This effect depends on both the ability of E1A to interact with Ran and on the functional integrity of the Ran network. MATERIALS AND METHODS Yeast Two-Hybrid Selection. The NH 2 -terminal region (nt 1–108, corre- sponding to amino acids 1–36) of the adenovirus 2 E1A 243R gene product was cloned into the EcoRI-BamHI sites of vector pGBKT7 (BD Clontech, Palo Alto, CA) in frame with the GAL4 binding domain. The yeast strain AH109 (28), carrying UAS-His3, UAS-LacZ, and UAS-ADE2 reporter genes, was cotransformed with the pGBKT7-E1A1-36 bait and with a human HeLa cDNA library (BD Clontech) fused to the GAL4 activation domain in the pACT2 vector (BD Clontech). Transformation was carried out using the lithium acetate method (29). Cells were plated on minimal synthetic defined medium (BD Clontech) supplemented with the required bases and amino acids, and lacking tryptophan (Trp), leucine (Leu), histidine (His), and adenine (Ade). Plates were incubated for 7 days at 30°C, then His + Ade + transformants were isolated. The His + Ade + colonies, replica-plated on synthetic defined Leu-Trp-His-Ade medium and LacZ + , were identified by a filter-lifting assay for -galactosidase activity. Plasmid DNA was prepared from candidate clones and transformed into Escherichia coli XL1-blue cells (Stratagene, La Jolla, CA). Recovered library-derived plasmids were analyzed by DNA sequencing. Cell Lines. BHK21 baby hamster kidney epithelial cells, and its derivative tsBN2 cell line, carrying a temperature-sensitive RCC1 allele (S256F; Ref. 30), were kindly provided by T. Nishimoto (University of Fukuoka, Fukuoka, Japan) and routinely maintained at the permissive temperature of 32°C or shifted to 39°C, as indicated in the text. NIH/3T3 murine embryo fibroblasts, L929 murine epithelial cells, PtK kangoroo-rat epithelial cells, and 293 human Received 8/1/02; accepted 1/16/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the National Research Council (Consiglio Nazionale delle Ricerche) and by grants from Associazione Italiana Ricerca sul Cancro (to P. L. and M. G. P.), Ministero della Sanita ` (to M. G. P.), and Federazione Italiana Ricerca Cancro (FIRC; to A. D. L.). A. S. is a FIRC research fellow. 2 A. D. L., R. M., and A. S. contributed equally to this work. 3 To whom requests for reprints should be addressed, at CNR, Institute of Molecular Biology and Pathology, Section of Genetics, c/o Department of Genetics and Molecular Biology, University “La Sapienza,” Via degli Apuli, 4, 00185 Rome, Italy. Phone: 39-06-4991-7536; Fax: 39-06-445-7529; E-mail: patrizia.lavia@uniroma1.it; or at Regina Elena Cancer Institute, CRS, Via delle Messi d’Oro, 156, 00158 Rome, Italy. Phone: 39-06-5266-2550; Fax: 39-06-5266-2572; E-mail: paggi@temple.edu. 4 The abbreviations used are: ENT, extreme NH 2 terminus/terminal; RanBP1, Ran- binding protein 1; GST, glutathione S-transferase; BrdUrd, bromodeoxyuridine; GFP, green fluorescent protein; IF, immunofluorescence; FADD, Fas-associating protein with death domain; T Ag, T antigen. 1430 Research. on August 17, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from