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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...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2006
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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. |
format | Text |
id | pubmed-1382259 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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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