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Retinoblastoma Protein is Rapidly Dephosphorylated by Elevated Cyclic Adenosine Monophosphate Levels in Human B-Lymphoid Cells¹

Departments of Immunology [J. C., E. B. S., K. B. A., H. K. B.] and Biophysics [T. S.], Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, and Institute for Medical Biochemistry, University of Oslo [K. T.], Oslo, Norway

Elevated cyclic AMP levels induce a rapid block in the mid-G₁ phase of the cell cycle in B-lymphoid Reh cells, accompanied by a transient block in G₂. The retinoblastoma (Rb) gene product has been implicated as a key regulator of eukaryotic cell growth. The Rb protein enforces its growth-suppressive effect in early G₁, where it is underphosphorylated and firmly bound in the nucleus. A possible link between the cyclic AMP-mediated growth arrest and regulation of Rb protein phosphorylation was explored by Western blot analysis. We found that both forskolin and 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate induced a rapid (within 3 h) dephosphorylation of Rb protein. These data were confirmed by flow-cytometric analysis of isolated nuclei costained with anti-Rb antibodies and propidium iodide. The percentage of cells containing underphosphorylated Rb protein (i.e., G₁ nuclei with bound Rb protein) increased from 9 to 87% after 4 h of forskolin treatment.

During the first 4 h of forskolin treatment, the cells were transiently blocked in the G₂ phase of the cell cycle, and virtually no cells had passed through mitosis. The increased level of dephosphorylated Rb protein at 4 h was therefore not due to an accumulation in early G₁ of cells containing underphosphorylated Rb protein. Instead, our data indicated that dephosphorylation of Rb protein occurred in cells that had already passed the point in G₁ of Rb protein phosphorylation.

Dephosphorylation of Rb protein was prevented by high concentrations of the protein phosphatase inhibitor okadaic acid, indicating that activation of a phosphatase is involved in the cyclic AMP-mediated dephosphorylation of Rb protein. We suggest that the dephosphorylation of Rb protein is required for the forskolin-mediated arrest of the Reh cells in mid-G₁.

¹ We thank the Norwegian Cancer Society for financially supporting the project.

² To whom requests for reprints should be addressed, at Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.

Received 9/16/93. Accepted 2/15/94.

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