Figure 4. HPV18 E1^E4 association with active CDK-cyclin complexes is dependent on RXL motifs.
(A) The presence of cyclin A, B1 and E proteins in co-precipitates formed in a HeLa cell lysate using GST-fusion proteins of wild-type (WT) and mutant HPV18 E1^E4 proteins (immunoblot). Two forms of cyclin B1, indicated with arrows, are detected in the cell lysates, the slower migrating form being a phosphorylated form of the cyclin. Two forms of cyclin E (identified by arrowheads) were recognised by the antibody, the upper represents the mature form of cyclin E, ~ 47 kDa. Ponceau stain identifies loading of all of the GST-E4 proteins, with GST being in excess. (B) Phosphorylation of histone H1 protein by GST and GST-HPV18 E1^E4 co-precipitates prepared from HeLa cell lysates in the presence and absence of the CDK-inhibitor roscovitine (autoradiograph). The level of GST and histone H1 proteins is visualised by a Coomassie stain. (C) Phosphorylation of histone H1 by GST, wild-type (WT) and mutant GST-18E4 coprecipitates prepared from HeLa cell lysates. The Coomassie-stained gel shows loadings of the fusion proteins.