title: Retinoic acid induces p27Kip1 nuclear accumulation by modulating its phosphorylation.
abstract: All-trans-retinoic acid (ATRA), the most biologically active metabolite of vitamin A, controls cell proliferation, apoptosis, and differentiation depending on the cellular context. These activities point to ATRA as a candidate for cancer therapy. A pivotal effect of the molecule is the modulation of p27Kip1, a cyclin-dependent kinase (CDK) inhibitor (CDKI). Here, we investigate the mechanisms by which ATRA regulates p27Kip1 level in LAN-5, a neuroblastoma cell line. When added to the cells, ATRA causes a rapid nuclear increase of p27Kip1, which clearly precedes growth arrest. The early buildup is not due to impairment of the CDKI degradation, in contrast to previous observations. Particularly, we did not detect the down-regulation of Skp2 and Cks1, two proteins involved in the nuclear ubiquitin-dependent p27Kip1 removal. Moreover, the morphogen does not impair the CDKI nuclear export and does not cause CDK2 relocalization. The characterization of CDKI isoforms by two-dimensional PAGE/immunoblotting showed that ATRA induces an early nuclear up-regulation of monophosphorylated p27Kip1. Immunologic studies established that this isoform corresponds to p27Kip1 phosphorylated on S10. The buildup of phospho(S10)p27Kip1 precedes the CDKI accumulation and increases its half-life. Finally, ATRA-treated nuclear LAN-5 extracts showed an enhanced capability of phosphorylating p27Kip1 on S10, thus explaining the nuclear up-regulation of the isoform. In conclusion, our data suggest a novel mechanism of ATRA antiproliferative activity, in which the morphogen rapidly up-regulates a nuclear kinase activity that phosphorylates p27Kip1 on S10. In turn, this event causes the stabilization of p27Kip1 and its accumulation in the nuclear compartment.
authors.affiliation: Department of Biochemistry and Biophysics F. Cedrangolo, Second University of Naples, Italy.
authors.forename: Adriana
authors.initials: A
authors.lastname: Borriello
authors.forename: Valeria
authors.initials: V
authors.lastname: Cucciolla
authors.forename: Maria
authors.initials: M
authors.lastname: Criscuolo
authors.forename: Stefania
authors.initials: S
authors.lastname: Indaco
authors.forename: Adriana
authors.initials: A
authors.lastname: Oliva
authors.forename: Alfonso
authors.initials: A
authors.lastname: Giovane
authors.forename: Debora
authors.initials: D
authors.lastname: Bencivenga
authors.forename: Achille
authors.initials: A
authors.lastname: Iolascon
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Zappia
authors.forename: Fulvio
authors.initials: F
authors.lastname: Della Ragione
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000096:biosynthesis / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D020033:Protein Isoforms; D044786:S-Phase Kinase-Associated Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D014212:Tretinoin