A. Yen et al., CONCENTRATION OF RB PROTEIN IN NUCLEUS VS CYTOPLASM IS STABLE AS PHOSPHORYLATION OF RB CHANGES DURING THE CELL-CYCLE AND DIFFERENTIATION, European journal of cell biology, 72(2), 1997, pp. 159-165
Unphosphorylated RE (retinoblastoma tumor suppressor) protein is known
to bind isolated nuclear matrix in vitro, whereas phosphorylated RE h
as a lower affinity, suggesting a mechanism which might contribute to
differential nuclear/cytoplasmic localization as part of its regulator
y activity, This motivates interest in the in vivo localization of the
endogenous RE protein as its phosphorylation state changes during the
cell cycle and cell differentiation. It is known that in proliferatin
g HL-60 cells all the RE protein is phosphorylated, but the extent of
phosphorylation increases with progression from G1 to S to G2+M. It ha
s also been previously shown that retinoic acid and 1,25-dihydroxy vit
amin D3 shift the RE protein to the unphosphorylated state with cell d
ifferentiation (Yen, A., S. Varvayanis, Exp, Cell Res. 214, 250-257 (1
994)). The dependence of cell cycle progression and differentiation on
RE nuclear versus cytoplasmic localization, as well as the dependence
of RE localization on phosphorylation state can thus be tested, Confo
cal image analysis of the RE protein in vivo shows that the ratio of t
he concentration of the RE tumor suppressor gene protein in the nucleu
s versus the cytoplasm remains stable as the RE protein undergoes eith
er phosphorylation during cell cycle progression or dephosphorylation
during cell differentiation induced by retinoic acid or 1,25-dihydroxy
vitamin D3, For the cell cycle analysis, HL-60 human promyelocytic le
ukemia cells were fluorescently stained for DNA and for the RE protein
, G1, S, and G2+M subpopulations were isolated by fluorescence-activat
ed cell sorting. For each subpopulation, the relative concentration of
RE protein in the nucleus and the cytoplasm was measured by laser con
focal image analysis, To determine the effect of retinoic acid-induced
myeloid differentiation or 1,25-dihydroxy vitamin D3-induced monocyti
c differentiation, the same cell sorting and image analysis was perfor
med on cells treated with these inducers, In all cases the concentrati
on of the RE protein in the nucleus was approximately 2 times that in
the cytoplasm, Thus, the ratio of nuclear versus cytoplasmic RE protei
n concentration is stable and independent of phosphorylation or dephos
phorylation of RE during both the cell cycle and cell differentiation.