Fig. 1. hHR6A CDK consensus phosphorylation sites and aligned sequences of hHR6A homologues. The CDK consensus phosphorylation sites on hHR6A are boxed. The sequence representing identical amino acids of hHR6A and its homologues, including human RAD6B (hHR6B), Drosophila melanogaster, Neurospora crassa, S.cerevisiae and Schizosaccharomyces pombe Rad6, is in bold and italicized.

Fig. 2. hHR6A is a CDK substrate in vitro and is phosphorylated during the G₂/M cell cycle phase in vivo. (A) Purified His₆-hHR6A (top panel) or GST–pRb^773–928 (bottom panel) was incubated in the presence of [γ-³²P]ATP, in either the absence (Control) or presence of purified cyclin D1–CDK4, cyclin E–CDK2, cyclin A–CDK2, cyclin A–CDK1 or cyclin B–CDK1, resolved by SDS–PAGE and visualized by autoradiography. (B) CHO cells transfected with either pCMV-Tag2 vector (Control) or pCMV-Tag2-hHR6A (hHR6A) were labelled with [³²P]phosphate and incubated with either dimethylsulfoxide (left and middle lanes) or 50 µM roscovitine (right lane) during the labelling period. Following labelling, hHR6A was immunoprecipitated, separated by SDS–PAGE and visualized by autoradiography. (C) CHO cells transfected with pCMV-Tag2-hHR6A (hHR6A) were synchronized by blocking in the G₂/M cell cycle phase with nocodazole, then initiated to re-enter the cell cycle by nocodazole removal, harvested at various time points for DNA analysis by flow cytometry, and the proportion of cells in G₁, S and G₂M phases determined. (D) CHO cells transfected with either pCMV-Tag2 vector (Control) or pCMV-Tag2-hHR6A (hHR6A) were synchronized by blocking in the G₂/M cell cycle phase with nocodazole (time = 0), initiated to re-enter the cell cycle by nocodazole removal and then lysed at various time points (as indicated). The cells were pulse-labelled with [³²P]phosphate for 3 h prior to lysis. Following lysis, hHR6A was immunoprecipitated, separated by SDS–PAGE and visualized by autoradiography (upper panel) or western blotting with an anti-FLAG antibody (lower panel). Phosphorylated hHR6A and the co-immunoprecipitating phosphoprotein (asterisk) are indicated with arrows.

Fig. 3. hHR6A is phosphorylated on Ser120 in vivo and on the same site by cyclin A–CDK2 in vitro. (A) Wild-type hHR6A was transfected into CHO cells. Following [³²P]phosphate labelling, hHR6A was immuno precipitated and phosphoamino acid analysis performed (upper panel, In vivo). Recombinant purified wild-type hHR6A was phosphorylated in vitro with cyclin A–CDK2 and subjected to phosphoamino acid analysis (lower panel, In vitro). The positions of phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr) are indicated with arrows. (B) Wild-type, S97A or S120A hHR6A was transfected into CHO cells. Following [³²P]phosphate labelling, hHR6A was immunoprecipitated and subjected to tryptic phosphopeptide mapping (upper panels, In vivo). Recombinant purified wild-type, S120A or T3A hHR6A was phosphorylated with cyclin A–CDK2 in vitro and subjected to tryptic phosphopeptide mapping (lower panels, In vitro). The major phosphopeptides are indicated with arrows. The origin is labelled ‘o’.

Fig. 4. Ser120 is critically important for ubiquitin-conjugating activity, and phosphorylation of this site increases hHR6A activity in vitro. (A) hHR6A was either unphosphorylated (lanes 1, 3, 4 and 6) or phosphorylated with cyclin A–CDK2 (lane 2). Following phosphorylation, cyclin A–CDK2 was immunodepleted and the ubiquitylation reaction performed. Lanes 3–6 are control lanes lacking histone H2A, GST–ubiquitin, hHR6A and E1, respectively (upper panel). hHR6A was either unphosphorylated or phosphorylated with cyclin A–CDK2 (as indicated) and the ubiquitylation reaction performed for either 20, 40 or 60 min (lower panel). Ubiquitylated histone H2A (H2A-Ub) and auto-ubiquitylated hHR6A (hHR6A-Ub) are indicated with arrows. (B) The ubiquitylation activity of wild-type, S120A, S120T, S120D and S120E hHR6As towards histone H2A. Ubiquitylated histone H2A (H2A-Ub) is indicated with an arrow. (C) Wild-type or S120E hHR6A was either unphosphorylated or phosphorylated with cyclin A–CDK2 (A/CDK2). Following phosphorylation, cyclin A–CDK2 was immunodepleted and the ubiquitylation reaction performed (30 min for wild-type hHR6A and 60 min for S120E hHR6A). Ubiquitylated histone H2A is indicated with an arrow.

Fig. 5. Increased ubiquitylation of histone H2B during the G₂/M cell cycle phase. CHO cells were synchronized by blocking in the G₂/M cell cycle phase with nocodazole, then initiated to re-enter the cell cycle by nocodazole removal, harvested at various time points (as indicated) and histones prepared. Histones were separated by SDS–PAGE and then western blotted with an anti-histone H2B (top panel) or anti-ubiquitin antibody (lower panel).

Fig. 6. Complementation of the S.cerevisiae rad6Δ growth defect with wild–type and mutant alleles of hHR6A. The S.cerevisiae rad6Δ strain was transformed with either empty plasmid or plasmid expressing either wild-type, S120A, S120T, S120D or S120E hHR6A. (A) The expression of wild-type and mutant hHR6As in S.cerevisiae rad6Δ. Lysates were prepared from either wild-type S.cerevisiae (RAD6) or the S.cerevisiae rad6Δ transformants grown at either 30 (top panel) or 37°C (lower panel). Equal amounts of protein were separated by SDS–PAGE, transferred to nitrocellulose and immunoblotted using an anti-hHR6A polyclonal antibody. (B) Wild-type S. cerevisiae (RAD6) or S.cerevisiae rad6Δ transformants were grown at either 30 or 37°C. Ten-fold serial dilutions (from left to right) of exponential phase cultures were spotted on plates and incubated at the indicated temperatures.

Fig. 7. Aligned sequences of hHR6A and other UBCs encompassing the region of hHR6A Ser120 from several species, including human (Homo sapiens), rabbit (Oryctolagus cuniculus), wheat (Triticum aestivum) and yeast (S.cerevisiae). Protein sequences were obtained from the Swiss-Prot database. Ser120 of hHR6A and the proline residue immediately C-terminal to it, and the equivalent aligned residues in the other UBCs are in bold and italicized.