p21 Disrupts the Interaction between cdk2 and the E2F-p130 Complex

Abstract

In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G 1 phase is associated with a cyclin-dependent binding of the cyclindependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L-cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex. We have also analyzed the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32؇C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculate that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.The transcription factors of the E2F family play a central role in cell proliferation. Several growth-associated genes, including c-myc, n-myc, B-myb, cdc2, dhfr, and the thymidine synthetase, thymidine kinase, and DNA polymerase ␣ genes, contain E2F-binding sites in their regulatory regions (6,12, 29,33,50,55,56,66,70). Some of these genes have been shown to depend on E2F for their cell cycle-regulated expression (6,12,34,38,43,49,53,64). The activity of E2F is inhibited by proteins of the retinoblastoma family, such as the retinoblastoma tumor suppressor protein (pRB), p107, and p130. Inhibition of E2F activity coincides with a suppression of cell growth by these retinoblastoma family proteins (2,3,4,5,7,8,10,11,14,23,31,32,44,45,57,59,63,67,68,71,73,75).The tumor suppression function of pRB also appears to correlate with its ability to repress E2F-dependent transcription. The naturally occurring loss-of-function mutants of pRB are unable to regulate E2F (2,31,32,57). Recent studies indicated that pRB, p107, and p130 suppress cell growth by biochemically distinct pathways involving different members of the E2F family. pRB regulates E2F1, E2F2, and E2F3, whereas p107 and p130 regulate E2F4 and E2F5 (17, 20, 27, 28, 39, 42, 46, 59, 61, 67, 73). The E2F-regulatory function and growth suppression function of pRB are inactivated by cyclincyclin-dependent kinase (cdk)-dependent phosphorylation of pRB (8,9,13,16,36). p107 and p130 also associate with cyclin-cdk2 complexes (7,11,14,19,24,45,47,48,63,73). Recent experiments indicated that phosphorylation of p107 by a cyclin-cdk complex could regulate its interaction with E2F (74). p107 has been shown to interact with cyclin A-cdk2 through sequences that are distinct fro...

Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes

“…P21 has been shown to compete with both p107 and p130 for interactions with cyclin A/ cdk2 and cyclin E/cdk2 (Zhu et al, 1995b;Shiyanov et al, 1996). The competition for cyclin/cdk interactions may extend to kinase substrates which do not form stable interactions with cyclin/cdk complexes if this activity requires the same binding site occupied by p130, p107, p21 cip1 and p27 kip1 .…”

Small contribution of G1 checkpoint control manipulation to modulation of p53-mediated apoptosis

“…Inhibition or repression of E2F activity by Rb and related proteins appears to be critical for p53-mediated G1 arrest, since expression of either the adenovirus E1A or human papillomavirus (HPV) E7 viral oncogenes, both of which disrupt E2F binding to Rb and related molecules, or overexpression of E2F-1 itself, can overcome IR-induced G1 arrest (Slebos et al, 1994;Demers et al, 1994;Lowe et al, 1993b;Degregori et al, 1995a,b). Consistent with this model, overexpression of p21 signi®cantly inhibits E2F transcriptional activity, presumably by preventing phosphorylation of Rb and related molecules by G1 cyclin-cdk's (Dimri et al, 1996;Shiyanov et al, 1996).…”

Loss of anti-mitotic effects of Bcl-2 with retention of anti-apoptotic activity during tumor progression in a mouse model

“…Both Bcl-2 and p21 are reported to promote formation of E2F-p130 complexes, a feature of nonproliferating cells (Lind et al, 1999;Shiyanov et al, 1996). Release of E2F promotes progression through the cell cycle.…”

“…Release of E2F promotes progression through the cell cycle. p21 has been shown to suppress cell growth by enhancing formation of E2F-p130 complexes through disruption of the interaction between the cyclin-dependent kinase Cdk2 and the E2F-p130 complex (Shiyanov et al, 1996). p107 and p130 appear to both bind to Cdk2 and inhibit its activity (Coats et al, 1999;Castano et al, 1998;De Luca et al, 1997).…”