Multiple mechanisms regulate subcellular localization of human CDC6

Citation
Lm. Delmolino et al., Multiple mechanisms regulate subcellular localization of human CDC6, J BIOL CHEM, 276(29), 2001, pp. 26947-26954
Citations number
30
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
276
Issue
29
Year of publication
2001
Pages
26947 - 26954
Database
ISI
SICI code
0021-9258(20010720)276:29<26947:MMRSLO>2.0.ZU;2-#
Abstract
CDC6 is a protein essential for DNA replication, the expression and abundan ce of which are cell cycle-regulated in Saccharomyces cerevisiae. We have d emonstrated previously that the subcellular localization of the human CDC6 homolog, HsCDC6, is cell cycle-dependent: nuclear during G(1) phase and cyt oplasmic during S phase. Here we demonstrate that endogenous HsCDC6 is phos phorylated during the G(1)/S transition. The N-terminal region contains put ative cyclin-dependent kinase phosphorylation sites adjoining nuclear local ization sequences (NLSs) and a cyclin-docking motif, whereas the C-terminal region contains a nuclear export signal (NES). In addition, we show that t he observed regulated subcellular localization depends on phosphorylation s tatus, NLS, and NES. When the four putative substrate sites (serines 45, 54 , 74, and 106) for cyclin-dependent kinases are mutated to alanines, the re sulting HsCDC6A4 protein is localized predominantly to the nucleus. This lo calization depends upon two functional NLSs, because expression of HsCDC6 c ontaining mutations in the two putative NLSs results in predominantly cytop lasmic distribution. Furthermore, mutation of the four serines to phosphate -mimicking aspartates (HsCDC6D4) results in strictly cytoplasmic localizati on. This cytoplasmic localization depends upon the C-terminal NES. Together these results demonstrate that HsCDC6 is phosphorylated at the G(1)/S phas e of the cell cycle and that the phosphorylation status determines the subc ellular localization.