Ubiquitin-dependent degradation of p27 requires Thr-187, Nedd8-conjugating activity, and proteasome activity. Ubiquitination reactions were performed by using in vitro-translated [³⁵S]His₆-p27 (lanes 1–5) or mutant (T187A) (lanes 6–10) in the presence or absence of S100 extract, proteasome inhibitor MG273, wild-type Nce1, or Nce1 C111S as indicated.

Reconstitution of p27 ubiquitination activity. (A) SCF^Skp2 and Cul1/Skp1 complexes were prepared from Sf9 cells coexpressing His₆-tagged Cul1, His₆-tagged Skp1, and Skp2 (lane 1), or His₆-tagged Cul1 and His₆-tagged Skp1. Proteins were purified with Ni-NTA resin and analyzed by SDS/PAGE and immunostaining with antibodies against Cul1, Skp1, and Skp2. (B) Ubiquitination reactions containing [³²P]-GST-p27, 30 μM ubiquitin, 100 nM E1 (ubiquitin activating enzyme), 2 μM ubiquitin aldehyde, 4 μM MG273, 1 μM okadaic acid, and 2 mM ATP were carried out by using in the absence (lanes 1 and 2) or presence of SCF^Skp2 (lanes 3–6) or Cul1/Skp1 (lane 7). Reaction mixtures were further supplemented with Nedd8 pathway components (0.29 μM Nedd8, 1 μM Nce1, and 100 nM Nae1; lanes 2, 4, 6, and 7) and 1 μM Cdc34 (lanes 2 and 5–7).

In vitro ubiquitination of p27^Kip1. (A) HeLa S100 contains a ubiquitin ligase activity specific for p27 phosphorylated on Thr-187. Reaction mixtures contained S100 extract as indicated and [³⁵S]His₆-p27 wild type (lanes 1 and 2) or mutant (T187A) (lanes 3 and 4). After incubation for 1 h at 30°C, [³⁵S]His₆-p27 and its polyubiquitinated forms were resolved on a 12% Tris-glycine SDS-gel, and analyzed by phosphorimaging. (B) Both FI and FII are required for robust ubiquination of in vitro-translated p27. [³⁵S]His₆-p27 was incubated with FI and/or FII as indicated, and reactions were analyzed as in A. Note that FII alone supported a basal level of ubiquitination (lane 2), which was likely caused by reticulocyte lysate in the reaction. (C) Ubiquitination of bacterially expressed p27 is FI and FII dependent. Reactions contained S100 extract, FI, and/or FII as indicated. GST-p27 and its polyubiquitinated forms were resolved on an 8% Tricine SDS-gel and immunoblotted by using anti-p27 antibodies. Note that robust p27 conjugate formation was detected only in reactions containing S100 or both FI and FII (lanes 2 and 5).

Nedd8 is required for p27 ubiquitination. (A) Ubiquitination of p27 requires a FI component. In vitro-translated [³⁵S]His₆-p27 was incubated in the absence (lane 1) or presence of FII (lanes 2–6). FII containing reactions were further supplemented by the addition of buffer alone (lane 2), a low molecular weight gel filtration fraction of FI (lane 3), or 0.29 μM Nedd8 (lane 4). Substitution of ubiquitin with Ub-K48R results in the formation of discrete low molecular weight p27-ubiquitin conjugates in the presence (lane 5) but not the absence of recombinant Nedd8 (lane 6). (B) Ubiquitination of p27 by FII requires both Nedd8 and the Nedd8-conjugating enzyme, Nce1. GST-p27 was incubated with FII alone (lane 1) or in the presence of 1 μM Nce1 and/or 0.29 μM recombinant Nedd8 as indicated (lanes 2–4). (C) Prior phosphorylation is required for p27 ubiquitination. Reactions were performed by using S100 extract as a source of ubiquitinating enzymes, and either in vitro-translated [³⁵S]His₆-p27 or bacterially expressed GST-p27 as substrates. Ubquitination of [³⁵S]His₆-p27 (lanes 1–3) was carried out in the absence (lane 1) or presence of 2 mM ATP (lane 2) or 2 mM AMP-PNP (lane 3). In lanes 4–6, GST-p27 (F62/64A) was prephosphorylated with cyclin E/CDK2 using [γ-³²P]ATP and repurified. Reactions containing [³²P]-GST p27 were conducted in the absence (lane 4) or presence of 2 mM ATP (lane 5) or 2 mM AMP-PNP (lane 6). (D) Ubiquitination of prephosphorylated [³²P]-GST-p27 by FII requires both Nedd8 and Nce1 (lanes 1–3).

The Nce1 C111S mutant exerts a dominant negative effect on p27 ubiquitination. (A) Ubiquitination of in vitro-translated [³⁵S]His₆-p27 was carried out with FII, 0.29 μM Nedd8 (lanes 2–9), and Nce1 mutant C111S in increasing concentrations: 0.08, 0.2, 0.3, 0.5, 1, 2, and 4 μM (lanes 3–9). (B) Wild-type Nce1 reverses inhibition by Nce1 C111S. Ubiquitination of in vitro-translated [³⁵S]His₆-p27 was carried out by using FII, 0.29 μM Nedd8 (lanes 2–9), 4 μM Nce1 C111S (lanes 3–9), and increasing wild-type Nce1: 0.02, 0.1, 0.2, 0.5, 1, and 2 μM (lanes 4–9, respectively). (C) Continuous Nce1 activity is required to sustain the p27 ubiquitination reaction. Ubiquitination of in vitro-translated [³⁵S]His₆-p27 was carried out by using S100 extract. Two reactions were incubated in the absence (lane 1) or presence (lane 2) of 4 μM Nce1 C111S for 60 min. The reaction in lane 3 was immediately mixed with Ni-NTA beads and kept on ice until completion of the experiment. The remaining reactions were incubated at 30°C for 15 min and either terminated by the addition of Ni-NTA and chilling on ice (lane 4), or mixed with Nce1 C111S (final concentration 4 μM, lanes 8–10), or mock buffer (lanes 5–7). Reactions were further incubated 30°C for an additional 15, 30, or 45 min before termination. (D) Polyubiquitinated [³⁵S]p27 reaction products (shown with brackets in C) were quantified by phosphorimage analysis. The arrow indicates the addition of Nce1 C111S (▴) or mock buffer (○).