Volume 16, Issue 1, Pages 47-58 (October 2004) An Rb-Skp2-p27 Pathway Mediates Acute Cell Cycle Inhibition by Rb and Is Retained in a Partial-Penetrance Rb Mutant  Peng Ji, Hong Jiang, Katya Rekhtman, Joanna Bloom, Marina Ichetovkin, Michele Pagano, Liang Zhu  Molecular Cell  Volume 16, Issue 1, Pages 47-58 (October 2004) DOI: 10.1016/j.molcel.2004.09.029

Figure 1 Reduction in Protein Levels of E2F Target Genes Follows a Slower Kinetics Than G1 Cell Cycle Arrest after Timed Reexpression of Rb in Saos-2 Cells Actively growing cells were induced (Tet−), and aliquots of cells were harvested at the indicated times for various analyses. (A) Rb expression determined with Western blot. (B) Cell cycle profiles of Saos-2 cells determined by FACS. Striped areas represent S phase cells. (C) Expression of cellular E2F target genes examined by Northern and Western blots. Molecular Cell 2004 16, 47-58DOI: (10.1016/j.molcel.2004.09.029)

Figure 2 Kinetics of Inhibition of Cdk Kinase Activity and Accumulation of p27 Protein Parallels with G1 Cell Cycle Arrest Samples of the same time course experiment used in Figure 1 were tested for cyclin E/Cdk2 and cyclin A/Cdk2 kinase activity toward histone H1 (A), for protein association with Cdk2 and p27 by immunoprecipitation-Western blot as indicated (B), and for protein levels of p27, p21, and p16 in total extracts by Western blot (C). Molecular Cell 2004 16, 47-58DOI: (10.1016/j.molcel.2004.09.029)

Figure 3 p27 Plays an Active Role in Rb-Mediated G1 Cell Cycle Arrest (A) Rb inducible cells were first transfected with the indicated oligonucleotides (C indicates buffer control) and then induced or uninduced as indicated. Total extracts were analyzed by Western blot. Parallel samples were harvested for FACS analysis. Percentages of S phase cells are indicated (representative of three similar experiments). (B) Cdk2-associated kinase activities (toward histone H1) in extracts were determined after various mixings as indicated. Ratio of mixing was 1:3 (Tet+ extract:Tet− extract). All samples were brought to a final volume of 500 μl for Cdk2 immunoprecipitation. Heating of the extract was performed at 100°C for 3 min followed by centrifugation for 3 min in a microcentrifuge. Molecular Cell 2004 16, 47-58DOI: (10.1016/j.molcel.2004.09.029)

Figure 4 p27 Is Degraded with a Faster Kinetics after Serum Restimulation of Rb−/− MEFs (A) Western blot of total cell extracts harvested at the indicated times after serum stimulation. (B) Cdk2 IP-kinase assays of the same extracts. (C) [3H]-thymidine incorporation at the indicated time points after serum restimulation. Counts presented are averages of triplicate samples. Molecular Cell 2004 16, 47-58DOI: (10.1016/j.molcel.2004.09.029)

Figure 5 Rb Inhibits p27 Ubiquitination (A) Western blot of Rb and p27 levels in total extracts before and after induction of Rb. (B) Same cell samples were subjected to Northern blot with p27 and GAPDH probes. (C) After 16 hr of Rb induction, CHX (100 μg/ml) was added, and aliquots of cell samples were harvested at the indicated time points for Western blot analysis. The results were plotted after quantitation. (D) Rb-inducible cells were treated without or with LLnL (50 μM) for 20 hr either in the presence or absence (24 hr) of tetracycline. Total extracts were immunoprecipitated with regular anti-p27 antibody, and the immunoprecipitates Western blotted. Two identical blots were each probed with a phospho-T187-specific anti-p27 antibody or a regular anti-p27 antibody, as indicated. A duplicate set of immunoprecipitates was treated with lamda phosphatases before electrophoresis. Bacteria-produced GST-p27 phosphorylated with cyclin E/Cdk2 and further treated with phosphatases were included in the same gel. (E) Extracts of Rb-inducible cells treated as indicated were generated with conditions to more efficiently detect polyubiquitinated p27, as described in Experimental Procedures. Two identical blots were each probed with anti-p27 or anti-ubiquitin antibodies. The bottom panel shows a Western blot with anti-Skp2 of the same extracts. (F) In vitro p27 ubiquitination with purified proteins. p27 was first phosphorylated by cyclin E/Cdk2 and then the kinase activity was inactivated with staurosporine prior to the ubiquitination reaction in the presence of Rb or NRP (nonrelevant protein). The asterisk refers to a nonspecific band. Molecular Cell 2004 16, 47-58DOI: (10.1016/j.molcel.2004.09.029)

Figure 6 Rb Binds the N Terminus of Skp2 to Dissociate Skp2-p27 Interaction, Inhibit p27 Ubiquitination, and Cause G1 Arrest (A) Saos-2-TetRb cell extracts with or without induction for 12 hr were analyzed by Western blot as indicated either straight (Total) or first immunoprecipitated with anti-Rb. U2OS cell extracts were analyzed in the same way with normal IgG as control. (B) Purified Rb and Skp2/Skp1 were incubated together for 1 hr at 4°C, immunoprecipitated, and blotted as indicated. (C) 293T cells were transfected with indicated plasmids. Total cell extracts and immunoprecipitates with indicated antibodies were analyzed with Western blot. The abbreviation L.P. refers to longer exposure. (D) A schematic drawing of Skp2 domain structure and various mutants and their abilities for Rb binding. (E) Skp2 and Rb were cotransfected with either Skp1 or p27, and interactions between Skp2-Skp1 and Skp2-p27 were analyzed by immunoprecipitation Western blot as indicated. (F) Saos-2-TetRb cell extracts harvested at different time points after induction were immunoprecipitated with anti-p27 antibody. The immunoprecipitates and total extracts were Western blotted as indicated. (G) As in (E), except Skp2ΔN was used. (H) SCFSkp2-Roc1 complexes were immunopurified with FLAG tag on Skp2 from transfected 293T cells and incubated in a p27 ubiquitination reaction. Purified Rb was added in lanes as marked. Ubiquitinated p27 was determined by immunoprecipitation-Western blot with anti-p27. The asterisk refers to a nonspecific band and Rosc. to roscovitine. (I) Saos-2-TetRb cells were infected, or mock infected, with lentivirus expressing the indicated proteins. Cells were then induced for 20 hr and labeled with BrdU for the last 2 hr. Cell extracts were Western blotted as indicated. The asterisk indicates a nonspecific band. (J) In the same experiment, BrdU-positive cells were revealed by indirect immunofluorescence staining and counted. Molecular Cell 2004 16, 47-58DOI: (10.1016/j.molcel.2004.09.029)

Figure 7 Binding to Skp2 and Dissociating Skp2-p27 Are Separate Activities of Rb; the Partial Penetrance Mutant R661W Retains Both and Can Induce p27 Accumulation and G1 Arrest with Wild-Type Kinetics (A) A schematic drawing of Rb and various mutations and their abilities to bind Skp2 and to dissociate Skp2-p27 interaction. (B) 293T cells were transfected with the indicated plasmids to study Rb-Skp2 and Skp2-p27 interaction as in Figure 6. (C) Expression of wild-type and mutant Rb in various inducible cell lines. (D) IP-DOC E2F gel shift experiments. Extracts were immunoprecipitated with anti-Rb. (E) Northern blot analysis as in Figure 1. (F) Time course experiments after induction of wt and R661W-inducible cells as in Figure 1. (G) In the same time course, a fraction of cells was analyzed by FACS to determine cell cycle profile. S phase fractions were plotted. Molecular Cell 2004 16, 47-58DOI: (10.1016/j.molcel.2004.09.029)