LATE DEPHOSPHORYLATION OF THE RB PROTEIN IN G2 DURING THE PROCESS OF INDUCED CELL-DIFFERENTIATION

The cell cycle phase-specific phosphorylation status of the RB protein (retinoblastoma tumor suppressor gene product) during an elicited cel lular program of G0 arrest and cell differentiation was characterized. The RB protein phosphorylation state is presumed to be an important d eterminant of cell proliferation or arrest. The cell cycle phase speci ficity of RB protein phosphorylation and dephosphorylation during HL-6 0 human leukemia cell proliferation and differentiation was determined using DNA-based fluorescence-activated cell sorting and Western analy sis. The RB protein in proliferating G1 cells was phosphorylated, but at a relatively low level. The extent of phosphorylation increased in S phase cells and was maximum in G2 + M cells. After the cells were tr eated with retinoic acid or 1,25-dihydroxy vitamin D3, they began to a ccumulate in G1/0 and phenotypically convert. Significant unphosphoryl ated RB protein did not appear until after the first cells had arreste d and differentiated. Dephosphorylation of the RB protein was first ap parent at the beginning of G2 in the remaining cycling cells after ons et of arrest and differentiation had already occurred. By the time the remaining cycling cells had divided and arrested, resulting in G0 cel ls, a majority of RB protein was dephosphorylated, but some remained p hosphorylated. The data indicate that dephosphorylation of RB does not determine residence in G1/0. Rather dephosphorylation appears as one relatively late-occurring component of the metabolic cascade culminati ng in G0-arrested, phenotypically differentiated cells. Dephosphorylat ed RB appears as a feature of differentiated cells. The data are consi stent with a role for hypophosphorylated RB not so much in deriving, b ut in possibly sustaining the differentiated state. (C) 1994 Academic Press, Inc.