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Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb

BACKGROUND: The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported...

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Autores principales: Vietri, Michele, Bianchi, Mariarita, Ludlow, John W, Mittnacht, Sibylle, Villa-Moruzzi, Emma
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1382259/
https://www.ncbi.nlm.nih.gov/pubmed/16466572
http://dx.doi.org/10.1186/1475-2867-6-3
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author Vietri, Michele
Bianchi, Mariarita
Ludlow, John W
Mittnacht, Sibylle
Villa-Moruzzi, Emma
author_facet Vietri, Michele
Bianchi, Mariarita
Ludlow, John W
Mittnacht, Sibylle
Villa-Moruzzi, Emma
author_sort Vietri, Michele
collection PubMed
description BACKGROUND: The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1α binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1. RESULTS: The requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal. CONCLUSION: The work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct.
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spelling pubmed-13822592006-02-25 Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb Vietri, Michele Bianchi, Mariarita Ludlow, John W Mittnacht, Sibylle Villa-Moruzzi, Emma Cancer Cell Int Primary Research BACKGROUND: The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1α binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1. RESULTS: The requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal. CONCLUSION: The work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct. BioMed Central 2006-02-08 /pmc/articles/PMC1382259/ /pubmed/16466572 http://dx.doi.org/10.1186/1475-2867-6-3 Text en Copyright © 2006 Vietri et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Primary Research
Vietri, Michele
Bianchi, Mariarita
Ludlow, John W
Mittnacht, Sibylle
Villa-Moruzzi, Emma
Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb
title Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb
title_full Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb
title_fullStr Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb
title_full_unstemmed Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb
title_short Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb
title_sort direct interaction between the catalytic subunit of protein phosphatase 1 and prb
topic Primary Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1382259/
https://www.ncbi.nlm.nih.gov/pubmed/16466572
http://dx.doi.org/10.1186/1475-2867-6-3
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