[CANCER RESEARCH 61, 1347–1353, February 15, 2001] Advances in Brief Phosphorylation- and SKP1-independent in Vitro Ubiquitination of E2F1 by Multiple ROC-Cullin Ligases1 Tomohiko Ohta2 and Yue Xiong3 Lineberger Comprehensive Cancer Center [T. O., Y. X.], Department of Biochemistry and Biophysics [Y. X.], and Program in Molecular Biology and Biotechnology [Y. X.], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295 Abstract Previously, we and others identified a RING finger protein known as ROC1 (13, 14), also called Rbx1 for RING-box protein (15, 16) or Ubiquitin-dependent proteolysis plays a critical role in the control of Hrt1 (17), as an essential subunit of CUL1/CDC53 ubiquitin ligases in many cellular processes and is mediated by a cascade of enzymes involving catalyzing F box-dependent ubiquitination of phosphorylated I␬B␣, ubiquitin activating (E1), conjugating (E2), and ligating (E3) activities. Cullin 1/CDC53 functions as an E3 ligase by interacting with RING finger G1 cyclin Cln2, and CDK inhibitor Sic1. Deficiency of yeast ROC1/ protein ROC1 and recruiting phosphorylated substrate. We report here Rbx1/Hrt1 can be functionally rescued by mammalian ROC1 and its that E2F1 transcription factor can be ubiquitinated in vitro and in vivo by homologue ROC2 but not yeast APC11, which shares a high degree of multiple ROC-cullin ligases. In vitro, E2F1 can be ubiquitinated by E2/ sequence similarity with ROC1 (13, 15, 17), demonstrating an evo- Ubc5 but not by E2/CDC34, is dependent on catalytically active ROC1, lutionary conservation and functional specificity for the ROC gene and is protected by the Rb protein. In contrast to substrates of the family (18–20). ROC1 and ROC2 commonly interact with all cullins SKP1-Cullin 1-F box (SCF) complexes, in vitro ubiquitination of E2F1 by (13), suggesting the existence of a potentially large number of het- CUL1-ROC1 ligase does not require E2F1 phosphorylation, is not stim- erodimeric ROC-cullin complexes. In vivo, there exits a large number ulated by overexpression of F box protein SKP2, and is not affected by of RING finger proteins. Several RING finger proteins with diverse immunodepletion of SKP1 or mutations in CUL1 disrupting SKP1 bind- structure and function, including oncoprotein MDM2 (21), tyrosine ing. These results suggest a novel, SKP1-independent mechanism for targeting E2F1 ubiquitination. kinase negative regulator c-Cbl (22), and several poorly uncharacter- ized RING finger proteins (23), were linked to ubiquitination, sug- Introduction gesting a broad and general function of RING fingers in activating E3 ligase activity. Surprisingly, APC11 alone, in the absence of cullin- Through a cascade of enzymes involving ubiquitin activating (E1), like APC2, can interact with UBC4 and is sufficient to promote E1- conjugating (E2), and ligating (E3) activities, the ubiquitin-protea- and E2-dependent multiubiquitin chain formation (24, 25). These some pathway catalyzes the formation of polyubiquitin chains onto findings are consistent with an idea that cullins function as scaffold substrate proteins via isopeptide bonds. Polyubiquitinated substrates proteins to bring together the RING-E2 ligase and substrates, as are then rapidly delivered to and degraded by the 26S proteasome opposed to participation in the catalysis directly. (1–4). Although E1 and E2 both represent structurally related proteins The E2F family of transcription factors controls the expression of and are relatively well characterized biochemically, the E3 ubiquitin several genes involved in the G1-to-S transition and in DNA replica- ligases was conceptually defined to contain two distinct activities: a tion. Ectopic overexpression of the prototypic member, E2F1, can ubiquitin ligase activity that catalyzes isopeptide bond formation; and induce quiescent cells to enter S-phase, followed by apoptosis, and a substrate-targeting activity. One of the best characterized E3 activ- can elicit neoplastic transformation in immortalized rodent cells (26, ities is the SCF4 complex in which SKP1 protein simultaneously binds 27). Conversely, mice deficient for E2F1 exhibited impaired apopto- to and thereby brings together CDC53/cullin 1 and an F box protein sis and increased tumor incidence (28, 29). These results indicate the that in turn binds to a phosphorylated substrate protein (5–8). CUL1/ importance of proper control of the intracellular concentration of CDC53 represents an evolutionarily conserved multigene family that E2F1, which is tightly regulated during the cell cycle by both tran- includes three genes in budding yeast, seven in Caenorhabditis el- scriptional activation and ubiquitin-dependent degradation (26, 27). egans, and at least six in mammalian cells (9, 10). A subunit of the Although transcriptional activation of the E2F1 promoter by one or mitotic APC E3 complex, APC2, was found to contain limited se- more E2F species during G0 exit has been identified as the mechanism quence similarity to cullins (11, 12), reinforcing the notion that cullins largely responsible for the accumulation of E2F1 mRNA in late G1, function in proteolysis. the mechanism underlying ubiquitin-mediated E2F1 degradation dur- ing late S is unclear. The only regulatory signal that has been clearly Received 9/18/00; accepted 12/28/00. linked to the control of E2F1 stability is the binding with, and The costs of publication of this article were defrayed in part by the payment of page protection from, degradation by the retinoblastoma protein, both in charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vitro (30–32) and in vivo (33). In this report, we demonstrate that 1 This study was supported by Research Project Grant RPG-00-048 from the American multiple ROC-cullin ligases can specifically catalyze E2F1 ubiquiti- Cancer Society (to Y. X.). T. O. is supported in part by the Department of Surgery, St. Marianna University School of Medicine, Japan. Y. X. is the recipient of a Career nation in vitro through a SKP1- and substrate phosphorylation-inde- Development Award from the Breast Cancer Research Program of the United States Army pendent manner. Medical Research and Materiel Command and is a Pew Scholar in Biomedical Science. 2 Present address: Department of Surgery, St. Marianna University School of Medi- cine, Kawasaki 216, Japan. Materials and Methods 3 To whom requests for reprints should be addressed, at Lineberger Comprehensive Cancer Center, CB 7295, The University of North Carolina at Chapel Hill, Chapel Hill, Plasmids and Purification of Recombinant Proteins. Full-length mam- NC 27599-7295. Phone: (919) 962-2142; Fax: (919) 966-8799; E-mail: yxiong@ malian cullin, ROC1, ROC2, APC11, APC2, SKP1, and SKP2 expression email.unc.edu. 4 plasmids were described by Ohta et al. (13) and Michel and Xiong (34). The The abbreviations used are: SCF, SKP1-Cullin 1-F box; APC, anaphase-promoting ␤ complex; ROC, regulator of cullins; CDK, cyclin-dependent kinase; IP, immunoprecipi- -TrCP clone was a gift from Dr. Yinon Ben-Neriah (The Hebrew University- tation; LLnL, N-acetyl-leucinyl-leucinyl-norleucinal; HA, hemagglutinin antigen. Hadassah Medical School, Jerusalem, Israel). E2 Ubc5c was amplified from a 1347 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2001 American Association for Cancer Research. IN VITRO UBIQUITINATION OF E2F1 Fig. 1. In vitro ubiquitination of E2F1 by ROC1-CUL1. A, puri- fied E2F1 was phosphorylated and 32P-labeled with CDK2-cyclin A and incubated with E1, E2, ubiquitin, and ROC1 immunocomplexes derived from untransfected or transfected 293T cells as indicated. B, in vitro E2F1 ubiquitination was performed as in A, except that unphosphorylated E2F1 was used as a substrate and E2F1 ubiquiti- nation was examined by anti-E2F1 immunoblotting. Association of CUL1, SKP1, and myc-SKP2 with HA-ROC1 was confirmed by immunoblotting the HA-ROC1 immunoprecipitate with antibodies to HA, CUL1, SKP1, and myc, respectively (bottom panel). C, purified recombinant E2F1 was incubated with HA-ROC1/CUL1 immuno- complexes for various lengths of time, and E2F1 ubiquitination was examined by anti-E2F1 immunoblotting. D, 293T cells were cotrans- fected with cullin 1 and either vector DNA control, wild type, or two ROC1 mutants. ROC1-CUL1 complex formation was examined by coupled IP-Western blot (bottom panel), and ubiquitination of E2F1(unphosphorylated) was examined by anti-E2F1 immunoblot- ting. HeLa cDNA library by PCR and inserted into a T7 bacterial expression vector with either 1 ml of anti-HA supernatant, anti-myc supernatant, or SKP1 sera fused in-frame with a hexahistidine tag. A GST-Rb379–928-expressing plasmid for 1 h. Approximately 1.5 mg of cell lysate from HA-ROC1/CUL1-trans- was a gift from Dr. Jiri Lukas (Danish Cancer Society, Copenhagen, Den- fected cells were incubated with 30 ␮l of each antibody-coated beads for three mark), and an HA-Ub-expressing plasmid was a gift from Dr. Dirk Bohmann time periods (3 h, 3 h, and overnight) and once with 30 ␮l of uncoated protein (EMBL, Heidelberg, Germany). Purified rabbit E1 (Exeter, United Kingdom) A beads for1htoremove residual antibody. Ten ␮l of the lysate after and ubiquitin (Sigma) were purchased commercially. Ubc5c, E2F1, and p21 depletion were subjected to direct Western, whereas 200 ␮l of the lysate were proteins were expressed in bacteria using the pET-3E-6xHis vector with used for immunoprecipitation (with anti-HA antibody) and