Figure 3.

An RXL motif in E1 is required for association with and phosphorylation by cyclin E/Cdk2. (a) Domains and positions of candidate Cdk phosphorylation sites in HPV E1 are shown. (b) Mutations in the RXL motif of E1 abolish association with cyclin E in vitro. Equal quantities (0.8 μg) of EE–E1 (lanes 1–5), EE–E1^ARA (lanes 6–10), and EE–E1^ΔCdk (lanes 11–15) were used for binding with increasing quantities of insect-cell lysates with or without cyclin E/Cdk2 and bound proteins detected by immunoblotting. To control for levels of input proteins, immunoblots of reaction mixtures prior to bead washing were performed (Top). (c) EE–E1 (lanes 1–4), EE–E1^ARA (lanes 5–8), and EE–E1^ΔCdk (lanes 9–12) immobilized on anti-EE beads were incubated with purified cyclin E/Cdk2 (1, 10, and 100 nM, respectively) and [γ-³²P]ATP (see Materials and Methods). Products were separated by using SDS/PAGE and transferred to nitrocellulose before immunoblotting (Top) and autoradiography (Bottom). (d) E1^ARA and E1^ΔCdk interact with E2. Anti-EE immune complexes of insect-cell lysates derived from the indicated infections were separated by using SDS/PAGE and visualized with Coomassie blue. (e) Immobilized EE–E1 or EE–E1/E2 complexes were assembled with cyclin E/Cdk2 in vitro and the excess kinase removed before assay in the presence (lanes 5–8) or absence (lanes 1–4) of histone H1. As a control, serial dilutions of cyclin E/Cdk2 (1–1,000 ng) were used in histone H1 kinase assays (lanes 9–12).