Cks1 Promotion of S Phase Entry and Proliferation Is Independent of p27^Kip1 Suppression

Abstract

Cks1 is an activator of the SCF Skp2 ubiquitin ligase complex that targets the cell cycle inhibitor p27 Kip1 for degradation. The loss of Cks1 results in p27 Kip1 accumulation and decreased proliferation and inhibits tumorigenesis. We identify here a function of Cks1 in mammalian cell cycle regulation that is independent of p27 Kip1 . Specifically, Cks1 −/− ; p27 Kip1−/− mouse embryonic fibroblasts retain defects in the G 1 -S phase transition that are coupled with decreased Cdk2-associated kinase activity and defects in proliferation that are associated with Cks1 loss. Furthermore, concomitant loss of Cks1 does not rescue the tumor suppressor function of p27 Kip1 that is manifest in various organs of p27 Kip1−/− mice. In contrast, defects in mitotic entry and premature senescence manifest in Cks1 −/− cells are p27 Kip1 dependent. Collectively, these findings establish p27 Kip1 -independent functions of Cks1 in regulating the G 1 -S transition.

Cks1 is a critical regulator of hematopoietic stem cell quiescence and cycling, operating upstream of Cdk inhibitors

“…In this context it also has to be noted that loss of Cks1, but not loss of Skp2, results in a significant delay in tumorigenesis in the same Myc lymphoma model, 35,39 and that Cks1 has cell cycle regulatory functions independent of p27 suppression. 19 We also found that under cell culture conditions which stimulate HSC to be recruited into the cell cycle, Cks1 deficiency promotes cell death of MPs upon growth factor withdrawal. One possible explanation could be the accumulation of p27, as p27 Figure 2c).…”

“…12 We observed a strong reduction of the total cell numbers in the fetal liver of E14 embryo and the BM of 8-12-week-old Cks1 − / − mice compared with their wt (Cks1 +/+ ) counterparts (Figure 2a). Compared with the 10-20% reduction in body size, 12,19 the distinct reduction in fetal liver (36.0%) and BM (37.7%) cells suggests possible alterations in early hematopoiesis. However, the populations of mature blood cells and the bone structure of Cks1 − / − mice were unchanged, indicating unaltered differentiation of Cks1-deficient HSCs ( Supplementary Figures S2b and c).…”

“…Hematoxilin-eosin stain was performed as described previously. 19 All animal experiments were performed in accordance with the regional animal ethics committee approvals.…”

“…The sorted cells were lysed and immunoblotting was performed with the antibodies listed in Supplementary Table S2b as described previously. 19 In vitro cell culture and CFC assays Lineage-deleted BM cells were cultured in medium (MyeloCult 5300, STEMCELL Technologies, Vancouver, BC, Canada) supplemented with stem cell factor (100 ng/ml), thrombopoietin (20 ng/ml) and Flt3 Ligand (50 ng/ ml) (all from R&D Systems, Minneapolis, MN, USA) at 37°C with 5% CO 2 . After cytokine withdrawal cells were collected, fixed with 4% paraformaldehyde in 1X phosphate buffered saline and stained with Annexin V-FITC (BioLegend) and propidium iodide (BioLegend).…”

“…Skp2 knockout models have demonstrated a hematopoietic phenotype. 16,17 Considering Cks1 shows features that are not necessarily connected to this adaptor function, in particular, regulation of Cdk1 18 and Cdk2 19 activity, Cks1 could be involved in HSC regulation through the SCF Skp2 complex, but also through other mechanisms. Here we studied the hematopoiesis in mice deficient for Cks1.…”