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

Paola Zancai, Jessica Dal Col, Sara Piccinin, Massimo Guidoboni, Roberta Cariati, Silvana Rizzo, Mauro Boiocchi, Roberta Maestro, Riccardo Dolcetti

Research output: Contribution to journalArticle

Abstract

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 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 SCFSkp2 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 p27 Kip1 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 p27 Kip1.

Original languageEnglish
Pages (from-to)2483-2494
Number of pages12
JournalOncogene
Volume24
Issue number15
DOIs
Publication statusPublished - Apr 7 2005

    Fingerprint

Keywords

  • B lymphocytes
  • Cks1
  • EBV
  • P27
  • P45
  • Proteasome
  • Retinoic acid

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research
  • Genetics

Cite this