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 p27Kip1. Here, we show that in LCLs, RA inhibits ubiquitination and proteasome-dependent 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 p45Skp2 and Cks1 proteins, two essential components of the SCF(Skp2) ubiquitin ligase complex that target p27Kip1 for degradation. Downregulation of p45Skp2)and Cks1 occurs before the onset of growth arrest and is due to enhanced proteasome-mediated proteolysis of these proteins. Moreover, overexpression of p45Skp2 in DG75 cells prevents p27Kip1 protein accumulation and promotes resistance to the antiproliferative effects of RA. Treatment with Leptomycin B (LMB) blocked the translocation of p27Kip1 to the cytoplasm and prevented its degradation, indicating that CRM1-dependent nuclear export is required for p27Kip1 degradation. The shuttle protein p38Jab1, however, does not accumulate in the nucleus upon LMB treatment, nor does it interact with p27Kip1. Conversely, p45Skp2 is associated with p27Kip1 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 p45Skp2 in CRM1-dependent nuclear export of p27Kip1. These results indicate that downregulation of p45Skp2 is a key element underlying RA-induced p27Kip1 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 p27Kip1.
Zancai
Paola
P
Immunovirology and Biotherapy Unit, Centro di Riferimento Oncologico, IRCCS--National Cancer Institute, Aviano, PN, Italy.
Dal Col
Jessica
J
Piccinin
Sara
S
Guidoboni
Massimo
M
Cariati
Roberta
R
Rizzo
Silvana
S
Boiocchi
Mauro
M
Maestro
Roberta
R
Dolcetti
Riccardo
R
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001402:B-Lymphocytes / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D049109:Cell Proliferation; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015536:Down-Regulation; D016147:Genes, Tumor Suppressor; D004854:Herpesvirus 4, Human; D006801:Humans; D046988:Proteasome Endopeptidase Complex; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000494:pharmacology
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D044786:S-Phase Kinase-Associated Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D014212:Tretinoin; D046988:Proteasome Endopeptidase Complex