Direct Interaction Between the Catalytic Subunit of Protein Phosphatase 1 and PRb

Overview

Journal Cancer Cell Int

Publisher Biomed Central

Date 2006 Feb 10

PMID 16466572

Citations 17

Authors

Michele Vietri

Mariarita Bianchi

John W Ludlow

Sibylle Mittnacht

Emma Villa-Moruzzi

Affiliations

Soon will be listed here.

Abstract

Background: The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, alpha, gamma1 and delta, 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 PP1alpha 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.

Citing Articles

Engineered targeting OIP5 sensitizes bladder cancer to chemotherapy resistance via TRIP12-PPP1CB-YBX1 axis.

Wang X, Guo T, Niu L, Zheng B, Huang W, Xu H Oncogene. 2024; 43(38):2850-2867.

PMID: 39155295 DOI: 10.1038/s41388-024-03136-8.

microRNA-3129 promotes cell proliferation in gastric cancer cell line SGC7901 via positive regulation of pRb.

Yang S, Sheng N, Pan L, Cao J, Liu J, Ma R Braz J Med Biol Res. 2018; 51(6):e6452.

PMID: 29791595 PMC: 6002138. DOI: 10.1590/1414-431x20186452.

Overexpression of Protein Phosphatase 1γ (PP1γ) Is Associated with Enhanced Cell Proliferation and Poor Prognosis in Hepatocellular Carcinoma.

Li C, Wu M, Zong G, Wan C, Liu Q, Zhou H Dig Dis Sci. 2016; 62(1):133-142.

PMID: 27921263 DOI: 10.1007/s10620-016-4365-1.

Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits.

Verheyen T, Gornemann J, Verbinnen I, Boens S, Beullens M, Van Eynde A Nucleic Acids Res. 2015; 43(12):5771-84.

PMID: 25990731 PMC: 4499128. DOI: 10.1093/nar/gkv500.

Insights into the regulation of neuronal viability by nucleophosmin/B23.

Pfister J, DMello S Exp Biol Med (Maywood). 2015; 240(6):774-86.

PMID: 25908633 PMC: 4640450. DOI: 10.1177/1535370215579168.

References

Andreassen P, Lacroix F, Margolis R . Differential subcellular localization of protein phosphatase-1 alpha, gamma1, and delta isoforms during both interphase and mitosis in mammalian cells. J Cell Biol. 1998; 141(5):1207-15. PMC: 2137188. DOI: 10.1083/jcb.141.5.1207. View

Puntoni F . Association of protein phosphatase-1delta with the retinoblastoma protein and reversible phosphatase activation in mitotic HeLa cells and in cells released from mitosis. Biochem Biophys Res Commun. 1997; 235(3):704-8. DOI: 10.1006/bbrc.1997.6886. View

Tamrakar S, Ludlow J . The carboxyl-terminal region of the retinoblastoma protein binds non-competitively to protein phosphatase type 1alpha and inhibits catalytic activity. J Biol Chem. 2000; 275(36):27784-9. DOI: 10.1074/jbc.M004542200. View

Liu C, Wang R, Dohadwala M, Schonthal A, Berndt N . Inhibitory phosphorylation of PP1alpha catalytic subunit during the G(1)/S transition. J Biol Chem. 1999; 274(41):29470-5. DOI: 10.1074/jbc.274.41.29470. View

Chew Y, Ellis M, Wilkie S, Mittnacht S . pRB phosphorylation mutants reveal role of pRB in regulating S phase completion by a mechanism independent of E2F. Oncogene. 1998; 17(17):2177-86. DOI: 10.1038/sj.onc.1202443. View

Rubin E, Mittnacht S, Ludlow J . Site-specific and temporally-regulated retinoblastoma protein dephosphorylation by protein phosphatase type 1. Oncogene. 2001; 20(29):3776-85. DOI: 10.1038/sj.onc.1204518. View

Bollen M . Combinatorial control of protein phosphatase-1. Trends Biochem Sci. 2001; 26(7):426-31. DOI: 10.1016/s0968-0004(01)01836-9. View

Darman R, Flemmer A, Forbush B . Modulation of ion transport by direct targeting of protein phosphatase type 1 to the Na-K-Cl cotransporter. J Biol Chem. 2001; 276(37):34359-62. DOI: 10.1074/jbc.C100368200. View

Santoro M, Gaudino G, Villa-Moruzzi E . Protein phosphatase 1 binds to phospho-Ser-1394 of the macrophage-stimulating protein receptor. Biochem J. 2003; 376(Pt 3):587-94. PMC: 1223820. DOI: 10.1042/BJ20030391. View

10.

Ceulemans H, Bollen M . Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiol Rev. 2004; 84(1):1-39. DOI: 10.1152/physrev.00013.2003. View

11.

Berndt N, Ludlow J . Interaction between the retinoblastoma protein and protein phosphatase 1 during the cell cycle. Methods Mol Biol. 2004; 281:17-32. DOI: 10.1385/1-59259-811-0:017. View

12.

Bianchi M, De Lucchini S, Vietri M, Villa-Moruzzi E . Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase. Mol Cell Biochem. 2005; 272(1-2):85-90. DOI: 10.1007/s11010-005-7639-z. View

13.

Kaelin Jr W, Pallas D, DeCaprio J, Kaye F, Livingston D . Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell. 1991; 64(3):521-32. DOI: 10.1016/0092-8674(91)90236-r. View

14.

Chellappan S, Hiebert S, Mudryj M, Horowitz J, Nevins J . The E2F transcription factor is a cellular target for the RB protein. Cell. 1991; 65(6):1053-61. DOI: 10.1016/0092-8674(91)90557-f. View

15.

Sasaki K, Shima H, Kitagawa Y, Irino S, Sugimura T, Nagao M . Identification of members of the protein phosphatase 1 gene family in the rat and enhanced expression of protein phosphatase 1 alpha gene in rat hepatocellular carcinomas. Jpn J Cancer Res. 1990; 81(12):1272-80. PMC: 5918009. DOI: 10.1111/j.1349-7006.1990.tb02690.x. View

16.

Buchkovich K, Duffy L, Harlow E . The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell. 1989; 58(6):1097-105. DOI: 10.1016/0092-8674(89)90508-4. View

17.

Resink T, Hemmings B, Tung H, Cohen P . Characterisation of a reconstituted Mg-ATP-dependent protein phosphatase. Eur J Biochem. 1983; 133(2):455-61. DOI: 10.1111/j.1432-1033.1983.tb07485.x. View

18.

Weinberg R . The retinoblastoma protein and cell cycle control. Cell. 1995; 81(3):323-30. DOI: 10.1016/0092-8674(95)90385-2. View

19.

Dohadwala M, da Cruz e Silva E, Hall F, Williams R, Nairn A, Greengard P . Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases. Proc Natl Acad Sci U S A. 1994; 91(14):6408-12. PMC: 44211. DOI: 10.1073/pnas.91.14.6408. View

20.

Ludlow J, Glendening C, Livingston D, DeCarprio J . Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol Cell Biol. 1993; 13(1):367-72. PMC: 358916. DOI: 10.1128/mcb.13.1.367-372.1993. View