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  • Original Paper
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Glutamic acid mutagenesis of retinoblastoma protein phosphorylation sites has diverse effects on function

Oncogene volume 19, pages 562–570 (2000)Cite this article

Abstract

The retinoblastoma tumor suppressor gene (Rb) has many functions within the cell including regulation of transcription, differentiation, apoptosis, and the cell cycle. Regulation of these functions is mediated by phosphorylation at as many as 16 cyclin-dependent kinase (CDK) phosphorylation sites in vivo. The contribution of these sites to the regulation of the various Rb functions is not well understood. To characterize the effect of phosphorylation at these sites, we systematically mutagenized the serines or threonines to glutamic acid. Thirty-five mutants with different combinations of modified phosphorylation sites were assayed for their ability to arrest the cell cycle and for their potential to induce differentiation. Only the most highly substituted mutants failed to arrest cell cycle progression. However, mutants with as few as four modified phosphorylation sites were unable to promote differentiation. Other mutants had increased activity in this assay. We conclude that modification of Rb phosphorylation sites can increase or decrease protein activity, that different Rb functions can be regulated independently by distinct combinations of sites, and that the effects of modification at any one site are context dependent.

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Acknowledgements

We thank Dr Wen-Hwa Lee and for Rb antibodies and the pCMV vector. We also thank Dr J Wade Harper for the N-terminal Rb M13 clone used for mutagenesis. We acknowledge other members of the Goodrich laboratory for helpful discussions. This work was supported by the National Institutes of Health (CA-70292-01 to DW Goodrich) and the MD Anderson Physicians Referral Service (DW Goodrich).

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  1. Department of Cancer Biology, Box 108, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, 77030, Texas, TX, USA

    Sylvia Barrientes, Carolyn Cooke & David W Goodrich

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Barrientes, S., Cooke, C. & Goodrich, D. Glutamic acid mutagenesis of retinoblastoma protein phosphorylation sites has diverse effects on function. Oncogene 19, 562–570 (2000). https://doi.org/10.1038/sj.onc.1203332

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