Retinoic acid stabilizes p27Kip1 in EBV-immortalized lymphoblastoid B cell lines through enhanced proteasome-dependent degradation of the p45Skp2 and Cks1 proteins

Retinoic acid (RA) arrests the growth of EBV-immortalized lymphoblastoid B cell lines (LCLs) by upregulating the cyclin-dependent kinase inhibitor p27(Kip1). Here, we show that in LCLs, RA inhibits ubiquitination and proteasomedependent degradation of p27Kip1, a phenomenon that is associated with downregulation of Thr187 phosphorylation of the protein, whereas the phosphorylation on Ser10 is unaffected. Furthermore, we demonstrate that RA downregulates the expression of the p45(Skp2) and Cks1 proteins, two essential components of the SCFSkp2 ubiquitin ligase complex that target p27(Kip1) for degradation. Downregulation of p45(Skp2) and Cks1 occurs before the onset of growth arrest and is due to enhanced proteasome-mediated proteolysis of these proteins. Moreover, overexpression of p45(Skp2) in DG75 cells prevents p27(Kip1) protein accumulation and promotes resistance to the antiproliferative effects of RA. Treatment with Leptomycin B (LMB) blocked the translocation of p27(Kip1) to the cytoplasm and prevented its degradation, indicating that CRM1-dependent nuclear export is required for p27(Kip1) degradation. The shuttle protein p38(Jab1), however, does not accumulate in the nucleus upon LMB treatment, nor does it interact with p27(Kip1). Conversely, p45(Skp2) is associated with p27(Kip1) both in the nucleus and in the cytoplasm, accumulating within the nuclei after exposure to LMB and co-localizing with the exportin CRM1, suggesting a possible involvement of p45(Skp2) in CRM1-dependent nuclear export of p27(Kip1). These results indicate that downregulation of p45(Skp2) is a key element underlying RA-induced p27(Kip1) stabilization in B cells, resulting in an impaired targeting of the protein to the ubiquitin-proteasome pathway and probably contributing to the nuclear accumulation of p27(Kip1).