Establishment of Irreversible Growth Arrest in Myogenic Differentiation Requires the RB LXCXE-Binding Function

Abstract

The crystal structure of the A-B domain of RB has defined the binding pocket for the LXCXE peptide motif. Using the crystal structure as a guide, we have inactivated the LXCXE-binding pocket by replacing N757 with Phe [to obtain RB(N757F)]. RB(N757F) does not bind to viral oncoproteins but retains the ability to bind and inhibit E2F. RB(N757F) is less effective than the wild-type RB [RB(WT)] in repressing E2F-regulated transcription, and its repression activity is not affected by trichostatin A, an inhibitor of histone deacetylases. However, RB(N757F) is as effective as RB(WT) in suppressing cell growth. Interestingly, RB(N757F) cannot establish an irreversible growth arrest in differentiated myocytes. Differentiated myocytes with RB(WT) become refractory to serum. By contrast, differentiated myocytes with RB(N757F) undergo DNA synthesis and phosphorylate RB(N757F) in response to serum, despite a high level of p21Cip1 expression. Mutation of the phosphorylation sites in RB(N757F) rescued its defect and allowed myocytes to permanently withdraw from the cell cycle. These results demonstrate that it is possible to inactivate the LXCXE-binding pocket without compromising the overall integrity of RB. Moreover, the LXCXE-binding pocket is dispensable for the intrinsic growth suppression function of RB. However, the LXCXE-binding function is essential for RB to establish the serum-refractory state in differentiated myocytes.

ACKNOWLEDGMENTS

We thank Annick Harel-Bellan for the HDAC1-expressing plasmids and Geoff Wahl for the H2B-GFP expression plasmid. We are grateful to Pier Lorenzo Puri for sharing critical reagents, Irina Hunton for the isolation of p21^−/− and Rb^−/− fibroblasts, and other members of the Wang laboratory for their critical comments on the manuscript.

This study was supported by a grant from the National Institutes of Health (CA58320) to J.Y.J.W.