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Molecular and Cellular Biology, August 2000, p. 5571-5580, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Tung-Ti Chen and Jean Y. J. Wang*

Department of Biology and the Cancer Center, University of California, San Diego, La Jolla, California 92093-0322

Received 20 December 1999/Returned for modification 16 February 2000/Accepted 25 April 2000

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.

* Corresponding author. Mailing address: Department of Biology, 3326 Bonner Hall, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0322. Phone: (858) 534-6253. Fax: (858) 534-2821. E-mail: jywang{at}ucsd.edu.

Molecular and Cellular Biology, August 2000, p. 5571-5580, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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