Concentration of RB protein in nucleus vs. cytoplasm is stable as phosphorylation of RB changes during the cell cycle...

Unphosphorylated RB (retinoblastoma tumor suppressor) protein is known to bind isolated nuclear matrix in vitro, whereas phosphorylated RB has a lower affinity, suggesting a mechanism which might contribute to differential nuclear/cytoplasmic localization as part of its regulatory activity. This motivates interest in the in vivo localization of the endogenous RB protein as its phosphorylation state changes during the cell cycle and cell differentiation. It is known that in proliferating HL-60 cells all the RB protein is phosphorylated, but the extent of phosphorylation increases with progression from G1 to S to G2 + M. It has also been previously shown that retinoic acid and 1,25-dihydroxy vitamin D3 shift the RB protein to the unphosphorylated state with cell differentiation (Yen, A., S. Varvayanis, Exp. Cell Res. 214, 250-257 (1994)). The dependence of cell cycle progression and differentiation on RB nuclear versus cytoplasmic localization, as well as the dependence of RB localization on phosphorylation state can thus be tested. Confocal image analysis of the RB protein in vivo shows that the ratio of the concentration of the RB tumor suppressor gene protein in the nucleus versus the cytoplasm remains stable as the RB protein undergoes either 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 leukemia cells were fluorescently stained for DNA and for the RB protein. G1, S, and G2 + M subpopulations were isolated by fluorescence-activated cell sorting. For each subpopulation, the relative concentration of RB protein in the nucleus and the cytoplasm was measured by laser confocal image analysis. To determine the effect of retinoic acid-induced myeloid differentiation or 1,25-dihydroxy vitamin D3-induced monocytic differentiation, the same cell sorting and image analysis was performed on cells treated with these inducers. In all cases the concentration of the RB protein in the nucleus was approximately 2 times that in the cytoplasm. Thus, the ratio of nuclear versus cytoplasmic RB protein concentration is stable and independent of phosphorylation or dephosphorylation of RB during both the cell cycle and cell differentiation.