Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-23
2002-04-09
Nolan, Patrick J. (Department: 1664)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S183000, C435S320100, C536S023500
Reexamination Certificate
active
06368809
ABSTRACT:
The present invention relates to the interaction of transcription factor E2F with ubiquitin, and to assays for modulators of this interaction.
E2F transcription factors control the expression of at least three groups of genes that are involved in cell cycle regulation. First, E2F sites have been found in the promoter of the immediate early gene c-myc (Hiebert et al., 1989; Oswald et al., 1994). In addition, E2F contributes to the regulation of several genes whose expression is activated in the G1 phase of the cell cycle, including cyclin E, E2F-1 and p107 (Degregori et al., 1995; Johnson et al., 1994; Neuman et al., 1994; Zhu et al., 1995). Finally, E2F contributes to the cell cycle-regulated expression of a number of genes that are required during S phase, such as cyclin A, dihydrofolate reductase, DNA polymerase a and thymidine kinase (reviewed by Farnham et al., 1993). E2F transcription factors are heterodimers that contain one of five related E2F polypeptides and one of two DP polypeptides (reviewed by Beijersbergen and Bernards, 1996).
The activity of the various E2F transcription factors is regulated at three different levels. First, the abundance of E2F is regulated at the level of transcription. For example, E2F-4 is the most prominent E2F species in quiescent cells, whereas E2F-1 is absent from quiescent cells and is transcriptionally induced in late G1 after serum stimulation (Johnson et al., 1994; Sardet et al., 1995). Second, transactivation by E2Fs is negatively regulated by complex formation with one of three members of the retinoblastoma pocket protein family, pRb, p107 and p130 (reviewed by Beijersbergen and Bernards, 1996). E2F-1, 2, and 3 interact preferentially with pRb; E2F-4 with p107 and p130 (Beijersbergen et al., 1994; Ginsberg et al., 1994; Vairo et al., 1995); E2F-5 with p130 only (Hijmans et al., 1995). These E2F-pocket protein complexes are likely to perform different functions during the cell cycle as the timing of their appearance differs. Most quiescent cells have one major E2F complex that consists of E2F-4 in complex with p130 (Chittenden et al., 1993; Cobrinik et al., 1993; Vairo et al., 1995). Exponentially growing cells contain significant amounts of free E2F and E2F-p107 complexes (Beijersbergen et al., 1995; Cobrinik et al., 1993; Lees et al., 1992; Shirodkar et al., 1992). E2F-pocket protein complexes are regulated by phosphorylation of the pocket proteins by G1 cyclin/cyclin-dependent kinase (cdk) complexes. pRb can be phosphorylated by cyclin D/cdk4, cyclin E/cdk2 and cyclin A/cdk2 kinase complexes (Dowdy et al., 1993; Ewen et al., 1993; Hinds et al., 1992). In contrast, p107 is only efficiently phosphorylated by cyclin D/cdk4 (Beijersbergen et al., 1995). In addition, several viral oncoproteins, including adenovirus E1A, can disrupt E2F-pocket protein complexes through high affinity binding to the pocket proteins (Whyte et al., 1988). A third level of regulation of E2F activity concerns the regulation of DNA binding activity. Krek et al. (1994) have shown that E2F-1 can interact directly with cyclin A, which results in phosphorylation of DP-1 in S phase, causing down-regulation of E2F DNA binding activity. Down-regulation of E2F in S phase appears to be important in cellular homeostasis, as over-expression of E2F, or mutants of E2F that resist cyclin A down-regulation, can cause apoptosis and transformation (Beijersbergen et al., 1994; Johnson et al., 1994; Krek et al., 1995; Qin et al., 1994; Singh et al., 1994; Wu and Levine, 1994).
E2F DNA binding sites in promoters can act both as positive and negative regulatory elements, depending on the promoter context (Lam and Watson, 1993). The action of E2F sites as negative regulatory elements is most readily explained by the finding that pocket proteins can mediate active transcriptional Thus, E2F/pocket protein complexes found in quiescent cells may contribute to maintaining quiescence through active transcriptional silencing of growth factor-activated genes.
Many of the proteins that contribute to regulation of the cell cycle appear and disappear rapidly, often in a cell cycle-regulated manner. Degradation of unstable proteins frequently involves the ubiquitin-proteasome pathway (Hilt and Wolf, 1996; Hochstrasser, 1995; Jentsch, 1992; Jentsch and Schlenker, 1995; Rubin and Finley, 1995). This system acts by covalent attachment of multiple ubiquitin polypeptides to the substrate. Ubiquitin is a highly conserved 76 amino acid protein found in eukaryotic cells. Ubiquitination requires the action of three different enzymes. A ubiquitin-activating enzyme (E1), which binds ubiquitin and transfers it to an ubiquitin conjugating enzyme (UBC or E2) (Haas and Rose, 1982; Pickart and Rose, 1985), which in turn may need the assistance of a ubiquitin ligase (E3) to attach the ubiquitin residue covalently to the substrate at a lysine residue. Each ubiquitin covalently attached to a lysine residue of the substrate protein is further ubiquitinated at a lysine residue in the ubiquitin sequence itself. Multi-ubiquitination acts as a sorting signal which targets substrates for rapid degradation by the proteasome, a complex of proteolytic enzymes (Chau et al., 1989).
An important link between the ubiquitin-proteasome machinery and cell cycle regulation came from the finding that CDC34, a yeast gene required for the G1 to S transition, was identical to yeast UBC3, a ubiquitin conjugating enzyme (Goebl et al., 1994). Other substrates of Cdc34 include yeast G1 cyclins and the cyclin/cdk inhibitor p40
sic1
(Deshaies et al., 1995; Schwob et al., 1994; Yaglom et al., 1995). In mammalian cells, the p27 cyclin/cdk inhibitor was recently shown to be a degraded in a cell cycle dependent fashion by the ubiquitin-proteasome pathway (Pagano et al., 1995). In budding yeast, the S-phase cyclin Clb5 and the mitotic cyclin Clb2 are ubiquitinated through UBC9 (Seufert et al., 1995) and Xenopus mitotic cyclins have also been shown to be degraded through ubiquitination (Glotzer et al., 1991).
We report here that E2F transcription factors are unstable due to destruction by the ubiquitin-proteasome pathway and that their degradation is highly regulated.
Accordingly, the present invention provides an assay method for an inhibitor of transcription factor E2F ubiquitin-mediated degradation which method comprises:
a) bringing a polypeptide which contains a domain which renders E2F a substrate for ubiquitination into contact with a candidate inhibitor; and
b) determining whether or not the candidate inhibitor is capable of reducing ubiquintination of said polypeptide.
The polypeptide may be an E2F protein or fragment thereof which is capable of binding to a pocket protein or the polypeptide may comprise an indicator polypeptide such as LacZ.
Preferably, the domain which renders E2F a substrate for ubiquitination comprises the 63 amino acid C-terminal region of E2F-l, or the 138 amino acid C-terminal domain of E2F-4, or portion thereof such as the 112 C-terminal amino acids.
The assay will normally be conducted in a format where the polypeptide is expressed in a host cell from a recombinant expression vector, such as an expression vector where the polypeptide is operably linked to a CMV promoter.
In one embodiment, a DP-1 polypeptide is co-expressed in the host cell.
Assays of the invention may be conducted in the presence of a proteasome inhibitor.
In the assay of the invention the determining of whether or not the candidate inhibitor is capable of reducing ubiquintination of said polypeptide may be performed by providing ubiquitin and determining the amount of said ubiquitin which has been bound to said polypeptide. This may be achieved by ubiquitin or contains an expression vector capable of expressing ubiquitin. The ubiquitin may be tagged with an epitope capable of binding to a monoclonal antibody, such as an HA epitope.
In another aspect, the invention also provides a vector which comprises a nucleotide sequence encoding a truncated E2F polypeptide wherein said truncated polypeptide does not contain a C-terminal domain capable
Nixon & Vanderhye P.C.
Nolan Patrick J.
Prolifix Limited
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