1992)

1992). wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, PLX-4720 efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. TNFRSF4 In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK?)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor. cell line that bears a mutation in the ubiquitin-activating enzyme gene. Several lines of evidence show that degradation of p27 can be recapitulated in an in vitro system (Pagano et al. 1995; Brandeis and Hunt 1996; Millard et al. 1997) and that it requires both ubiquitination and the PLX-4720 activity of the proteasome (Pagano et al. 1995; Loda et al. 1997). First, extracts from proliferating cells degrade p27 faster than extracts from quiescent cells; second, incubation of p27 with proteasome-depleted extracts results in a block of its degradation and upon readdition of purified proteasome particles, p27 degradation is restored completely; third, ATP depletion prevents the appearance of p27 polyubiquitinated species and inhibits its proteolysis; fourth, addition of ATP–S, an ATP analog that can be hydrolyzed by the E1 ubiquitin-activating enzyme but not by the proteasome, leads to a substantial decrease in p27 proteolysis and to the accumulation of p27 polyubiquitinated species. Phosphorylation plays a major role in ubiquitin-mediated proteolysis. In cDNA was transcribed and translated in vitro in the presence of [35S] methionine and subjected to a ubiquitination reaction with a HeLa extract, as described in the Materials and Methods. Samples shown in lanes were incubated with hexokinase and deoxyglucose to deplete extract from ATP (ATP depl.). Then, AMPCPNP (lane was incubated with staurosporine (Stauro.). Samples in lanes and were incubated with the indicated concentrations of flavopiridol (Flav.), used as described in Materials and Methods. Samples in the last three lanes were depleted with control beads (lane and and were incubated in the presence of staurosporine (Stauro.) added to the ubiquitination reaction either before (lane is from G1-enriched cells (75% in G1, 6% in S, and 19% in G2/M); sample in lane is from proliferating cells (58% in G1, 21% in S, and 21% in G2/M); and sample in lane is from G1-depleted cells (4% in G1, 35% PLX-4720 in S, and 61% in G2/M). Lanes represent immunoprecipitations with a mouse anti-p27 monoclonal antibody. (and are from G1-depleted cells; sample in lane is from G1-enriched cells; and sample in lane is from proliferating cells. (Lane lacked okadaic acid (?OA); samples in lanes and were incubated in the presence of the indicated concentrations of flavopiridol (Flav.). The reaction products were analyzed by SDS-PAGE and autoradiography. The bracket (1and are from two separate experiments. The phosphorylation of p27 by different amounts of cyclin/Cdk complexes was performed as described in Materials and Methods. Phosphorylated p27 was detected by autoradiography (32P p27). The binding of p27 to different cyclin/Cdks was assayed by incubating in vitro-translated p27 with His-tagged cyclin/Cdk complexes and then purifying them with nickel-agarose. Bound p27 was then detected by immunoblot with an anti-p27 monoclonal antibody (Cdk-Bound p27).The stimulation of p27 ubiquitination by different amount of cyclin/Cdk complexes was assayed by incubating in vitro-translated p27 with these complexes and extracts from G1 HeLa cells. The reaction products were analyzed by SDS-PAGE and autoradiography. The bracket (for 10 min. The supernatant (S-10) was divided into smaller samples and frozen at ?80C. This method of extract preparation based on the use of a cell nitrogen-disruption bomb extract preserves the activity of in vitro ubiquitinate p27 better than the method described previously (Pagano et al. 1995) (data not shown). In vitro ubiquitination assay In vitro-translated 35S-labeled p27 (1 l) was incubated at PLX-4720 30C for different times (0C75 min) in 10 l of.