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Epstein-Barr virus (EBV) expresses an immediate-earlyprotein, Rta, to activate the transcription of EBV lytic genes and the lytic cycle. This work identifies Ubc9 and PIAS1 as binding partners of Rta in a yeast two-hybrid screen. These bindings are verified by glutathione S-transferase pull-down assay, coimmunoprecipitation, and confocal microscopy. The interactions appear to cause Rta sumoylation, because not only can Rta be sumoylated in vitro but also sumoylated Rta can be detected in P3HR1 cells following lytic induction and in 293T cells after transfecting plasmids that express Rta and SUMO-1. Moreover, PIAS1 stimulates conjugation of SUMO-1 to Rta, thus acting as an E3 ligase. Furthermore, transfecting plasmids that express Ubc9, PIAS1, and SUMO-1 increases the capacity of Rta to transactivate the promoter that includes an Rta response element, indicating that the modification by SUMO-1 increases the transactivation activity of Rta. This study reveals that Rta is sumoylated at the Lys-19, Lys-213, and Lys-517 residues and that SUMO-1 conjugation at the Lys-19 residue is crucial for enhancing the transactivation activity of Rta. These results indicate that sumoylation of Rta may be important in EBV lytic activation.Small ubiquitin-like modifiers (SUMOs) 1 are a group of proteins that conjugate a wide range of proteins in the cell (1-3). In human cells, three types of SUMO, i.e. SUMO-1, SUMO-2, and SUMO-3, have been identified (4 -7). These SUMO molecules conjugate to their target proteins through an isopeptide bond formed between the C-terminal glycine residue of SUMO and a lysine residue in the substrate, frequently found at a conserved KXE motif; where represents a hydrophobic amino acid residue, including Leu, Ile, Val, or Phe (8,9). As is generally known, in a SUMO conjugation reaction, SUMO hydrolase first removes the four C-terminal amino acids of SUMO, exposing a glycine residue to facilitate SUMO conjugation. The SUMO molecule is then adenylated and covalently linked to a SUMO-activating E1 enzyme (10, 11). Subsequently, SUMO is transferred to the SUMO-conjugating E2 enzyme, Ubc9, which catalyzes the transfer of SUMO to its target proteins (12-15). The E3 ligase, which stimulates SUMO-1 conjugation to target proteins, has only recently been identified. Three proteins, including PIAS, RanBP2, and Pc2, are currently known to participate in the process of sumoylation (16 -20). Sumoylation may influence protein functions in many ways. An important function of SUMO is to stabilize its target proteins by acting as an antagonist to ubiquitin-mediated proteolysis (21). For instance, SUMO modification blocks ubiquitination and destruction of IB by the SCF(␤-TrCP) E3 ubiquitin ligase complex (21), thus stabilizing the ability of IB to inhibit NF-B. SUMO modification is also known to influence protein localization. For example, SUMO-1 modification not only targets promyelocytic leukemia protein (PML) to discrete subnuclear structures called PML nuclear bodies (22) but also is necessary for RanGAP1 binding t...

Mouse and human cDNA encoding AES (amino-terminal enhancer of split) and ESG (enhancer of split groucho) proteins with strong similarity to Drosophilu enhancer of split groucho protein were isolated and sequenced. Mouse AES-1 and AES-2 proteins, probably resulting from alternative splicing, contain 202 and 196 amino acids, respectively, while mouse ESG protein consists of 771 amino acids. The amino acid sequences of mouse and human AES proteins were found to exhibit approximately 50% identity to the amino-terminal region of Drosophilu groucho, mouse ESG and human transducin-like enhancer of split (TLE) proteins. Mouse AES transcripts of 1.5 kb and 1.2 kb were abundantly expressed in muscle, heart and brain. Human AES transcripts of 1.6 kb and 1.4 kb were predominantly present in muscle, heart and placenta. Mouse ESG (homolog of human TLE 3) transcripts of 3.3 kb and 4.0 kb were found only in testis, while human TLE 1 transcripts of 4.5 kb was more abundant in muscle and placenta compared to heart, brain, lung, liver, kidney and pancreas. Human AES, TLE 1 and TLE 3 genes were mapped to chromosomes 19, 9 and 15, respectively, using human and Chinese hamster hybrid cell lines.In the fruitfly Drosophila melanagaster, neurogenesis is under the control of several loci, whose products appear to determine the fate of neuroectodermal cells during the embryo development [l 1. The enhancer of split gene complex is one of these neurogenic loci and it can be divided into two functional groups: the m5, m7, m8 group and the m9/10 group. The m5, m7 and m8 transcripts encode proteins containing a helix-loop-helix motif characteristic of certain transcription factors. The m9/10 transcription unit was originally identified by a viable mutant called groucho which has specific head bristle duplications. Abbreviations. AES, mammalian protein exhibiting strong similarity to amino-terminal region of Drosophila enhancer of split groucho protein; cdc2K, cdc2-kinase site; CKII, casein kinase I1 site; ESG, mammalian homolog of enhancer of split groucho protein; EST, expressed sequence tag ; G-protein, guanine-nucleotidebinding regulatory protein ; TLE, transducin-like enhancer of split; NLS, nuclear-localization sequence; WD-40 repeat, the repeat of approximately 40 amino acids demarcated by Trp-Asp (WD).Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data banks and are available under accession numbers X73357, X73358, X73359, X73360 and X73361.terized several mouse and human cDNA encoding AES (amino-terminal enhancer of split) and ESG (enhancer of split groucho) proteins. While this investigation was in progress, a study describing human homologs (designated as TLE, transducin-like enhancer of split) of Drosophila groucho protein was published [4]. We report here the cDNA cloning, nucleotide and deduced amino-acid sequences, and tissue-specific expression of mouse and human AES and ESG (TLE) genes. We have also mapped the chromosomal locations of human AES and TLE genes using human and Ch...

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