Retinoic acid induces P27Kip1 nuclear accumulation by modulating its phosphorylation

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 p27(Kipl), a cyclin-dependent kinase (CDK) inhibitor (CDK). Here, we investigate the mechanisms by which ATHA regulates p27(Kipl) level in LAN-5, a neuroblastoma cell line. When added to the cells, ATRA causes a rapid nuclear increase of p27(Kipl), 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 p27(Kipl) 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 p27(Kipl). Immunologic studies established that this isoform corresponds to p27(Kipl) phosphorylated on S10. The buildup of phospho(SIO)p27(Kipl) precedes the CDKI accumulation and increases its half-life. Finally, ATRA-treated nuclear LAN-5 extracts showed an enhanced capability of phosphorylating p27(Kipl) on SIO, 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 p27(Kipl) on S10. In turn, this event causes the stabilization of p27(Kipl) and its accumulation in the nuclear compartment.