Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells Piyamas Sumrejkanchanakij a,b , Kazuhiro Eto a , Masa-Aki Ikeda a, * a Section of Molecular Embryology, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8549, Japan b Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand Received 25 November 2005 Available online 12 December 2005 Abstract The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differenti- ated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16 INK4a , a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abroga- tion of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Cyclin D1; Neuroblastoma; Cell cycle; Cyclin-dependent kinase; Subcellular localization Progression of the mammalian cell cycle is regulated by a family of cyclins and cyclin-dependent kinases (cdks). During the G1 phase, cyclin D1 accumulates in response to mitogenic stimulation and assembles with its catalytic partners, cdk4 and cdk6. The cyclin D1-cdk4/cdk6 com- plex promotes G1-to-S phase progression by phosphorylat- ing retinoblastoma protein (Rb) together with its related p107 and p130 proteins, which bind to and suppress the transcription factor E2F, which regulates a number of genes required for DNA replication and cell cycle progres- sion. The phosphorylation of Rb releases E2F from Rb-mediated inhibition, thereby allowing E2F to activate its target genes (reviewed by [1,2]). The activity of cyclin D1-cdk4/cdk6 is specifically inhibited by the p16 INK4 fam- ily of cdk inhibitors, whereas the p21 Cip1 /p27 Kip1 family inhibits the activity of both cyclin D1-cdk4/cdk6 and cyclin A/cyclin E-cdk2 complexes. In addition to Rb phosphory- lation, the cyclin D1-cdk complex sequesters the p21 Cip1 / p27 Kip1 family of cdk inhibitors and thereby facilitates the activation of cyclin E/cyclin A-cdk2 kinase activities, leading to the entry into and progression of S phase. The subcellular localization of cyclin D1 plays a role in the regulation of the activity of the cyclin D-cdk complex during the cell cycle. Cyclin D1 accumulates in the nucleus during G1 phase and redistributes to the cytoplasm when cells enter into S phase. Cyclin D1 nuclear export is regu- lated by the phosphorylation of cyclin D1 via GSK-3b [3,4]. We recently showed that proliferating embryonic pro- genitors of neurons and cardiomyocytes lose the ability to import cyclin D1 during differentiation, and that nuclear accumulation of the cyclin D1-cdk4 complex is tightly inhibited in such terminally differentiated cells [5,6]. Because a distinctive property of terminally differentiated cells is permanent withdrawal from the cell cycle, the pre- vention of cyclin D1 nuclear import plays a critical role as a physical barrier to prevent cell proliferation. Indeed, targeting the expression of cyclin D1 to the nucleus effi- ciently promotes reentry of postmitotic cardiomyocytes 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.181 * Corresponding author. Fax: +81 3 5814 6789. E-mail address: mikeda.emb@tmd.ac.jp (M.-A. Ikeda). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 340 (2006) 302–308 BBRC