Protein Phosphatase 1 binds strongly to the retinoblastoma protein but not to p107 or p130 in vitro and in vivo

Joshua L. Dunaief FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; Wilmer Eye Institute and Department of Molecular Biology & Genetics, and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Departments of Ophthalmology, Cell Biology and Anatomy, Cornell University School of Medicine, New York, NY, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

Ayala King FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; Wilmer Eye Institute and Department of Molecular Biology & Genetics, and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Departments of Ophthalmology, Cell Biology and Anatomy, Cornell University School of Medicine, New York, NY, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

Noriko Esumi FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; Wilmer Eye Institute and Department of Molecular Biology & Genetics, and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Departments of Ophthalmology, Cell Biology and Anatomy, Cornell University School of Medicine, New York, NY, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

Matthew Eagen FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; Wilmer Eye Institute and Department of Molecular Biology & Genetics, and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Departments of Ophthalmology, Cell Biology and Anatomy, Cornell University School of Medicine, New York, NY, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

Tzvete Dentchev FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; Wilmer Eye Institute and Department of Molecular Biology & Genetics, and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Departments of Ophthalmology, Cell Biology and Anatomy, Cornell University School of Medicine, New York, NY, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

Ching-Hwa Sung FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; Wilmer Eye Institute and Department of Molecular Biology & Genetics, and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Departments of Ophthalmology, Cell Biology and Anatomy, Cornell University School of Medicine, New York, NY, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

Shiming Chen FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; Wilmer Eye Institute and Department of Molecular Biology & Genetics, and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Departments of Ophthalmology, Cell Biology and Anatomy, Cornell University School of Medicine, New York, NY, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

Donald J. Zack FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; Wilmer Eye Institute and Department of Molecular Biology & Genetics, and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Departments of Ophthalmology, Cell Biology and Anatomy, Cornell University School of Medicine, New York, NY, USA; Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

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Abstract

Purpose. To identify and characterize retinoblastoma protein (pRb) binding proteins that may influence retinoblast proliferation and retinal pigment epithelial cell survival. Methods. The yeast two-hybrid system was used to screen a bovine retinal cDNA library and to characterize positive clones. DNA sequencing and site-directed mutagenesis were used for further analysis. Co-immunoprecipitation experiments were used to confirm the results of the two-hybrid system in vivo. Results. In the two-hybrid system, Protein Phosphatase 1a1 (PP1a1) binds the retinoblastoma protein. Unlike several other pRb binding proteins, PP1a1 binds only weakly to the Rb family member p107, and does not demonstrate detectable binding to p130. Confirming the two-hybrid results, endogenous PP1 in a human retinal pigment epithelial (RPE) cell line co-immunoprecipitates with endogenous pRb but not p107 or p130. Site directed mutagenesis of two pRb binding motifs in PP1a1 from LXSXE to LXCXE leads to slight increases in its two-hybrid interaction with pRb but does not alter its binding preference for pRb over the other family members. The complete sequence of bovine PP1a1 is reported. Conclusion. The strong two-hybrid interaction between PP1a1 and pRb, but not p107 or p130, suggests that the phosphorylation status of members of the pRb family may be regulated by different phosphatases, contributing to fine control of cell cycle progression. Conversely, PP1 activity may be specifically regulated by pRb and not p107 or p130. Mutagenesis studies suggest that PP1a1's LXSXE motif is not responsible for its binding preference for pRb over p107 and p130. Disruption of the PP1-pRb interaction may influence retinoblastoma tumorigenesis as well as RPE cell proliferation and survival.

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Protein Phosphatase 1 binds strongly to the retinoblastoma protein but not to p107 or p130 in vitro and in vivo

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Protein Phosphatase 1 binds strongly to the retinoblastoma protein but not to p107 or p130 in vitro and in vivo

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