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.