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Identification of a PP2A-interacting protein that functions as a negative regulator of phosphatase activity in the ATM/ATR signaling pathway

Oncogene volume 26, pages 6021–6030 (2007)Cite this article

Abstract

Protein serine/threonine phosphatase 2A (PP2A) activity must be tightly controlled to maintain cell homeostasis. Here, we report the identification of a previously uncharacterized mammalian protein, type 2A-interacting protein (TIP), as a novel regulatory protein of PP2A and the PP2A-like enzymes PP4 and PP6. TIP is a ubiquitously expressed protein and parallels the distribution of the PP2A catalytic subunit. Unlike its role in yeast, TIP does not interact with the mammalian homolog of type 2A-associated protein of 42 kDa (Tap42), α4, but instead associates with PP2A, PP4 and PP6 catalytic subunits independently of mammalian target of rapamycin kinase activity. Interestingly, the 20 kDa TIP splice variant TIP_i2, which lacks amino acids 173–272 of TIP's C-terminus, does not interact with PP2A; this finding indicates that residues 173–272 are important for the assembly of the TIP·phosphatase complex. In contrast to purified PP2A holoenzymes, TIP·PP2A complexes are devoid of phosphatase activity. Furthermore, alterations in the cellular levels of TIP influence the phosphorylation state of a specific protein substrate of ataxia-telangiectasia mutated (ATM)/ATM- and Rad3-related (ATR) kinases. Elevated levels of TIP result in an increase in the phosphorylation state of this protein substrate, whereas TIP-depleted cells exhibit a significant decrease in this protein's phosphorylation state, which is reversed by treatment with the PP2A inhibitor okadaic acid. These results indicate TIP is a novel inhibitory regulator of PP2A and implicate a role for TIP·PP2A complexes within the ATM/ATR signaling pathway controlling DNA replication and repair.

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Abbreviations

PP2A:

protein serine/threonine phosphatase 2A

PP4:

protein serine/threonine phosphatase 4

PP6:

protein serine/threonine phosphatase 6

PP1:

protein serine/threonine phosphatase 1

TIP:

type 2A-interacting protein

TIP_i2:

20 kDa splice variant of TIP

Tap42:

type 2A-associated protein of 42 kDa

mTOR:

mammalian target of rapamycin

ATM:

ataxia-telangiectasia mutated

ATR:

ATM- and Rad3-related

References

  • Abraham RT . (2001). Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 15: 2177–2196.

    Article  CAS  PubMed  Google Scholar 

  • Adams DG, Coffee Jr RL, Zhang H, Pelech S, Strack S, Wadzinski BE . (2005). Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes. J Biol Chem 280: 42644–42654.

    Article  CAS  PubMed  Google Scholar 

  • Beck T, Hall MN . (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402: 689–692.

    Article  CAS  PubMed  Google Scholar 

  • Bryant JC, Westphal RS, Wadzinski BE . (1999). Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Bα subunit. Biochem J 339 (Part 2): 241–246.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cayla X, Goris J, Hermann J, Hendrix P, Ozon R, Merlevede W . (1990). Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes. Biochemistry 29: 658–667.

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Martin BL, Brautigan DL . (1992). Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257: 1261–1264.

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Peterson RT, Schreiber SL . (1998). α4 associates with protein phosphatases 2A, 4, and 6. Biochem Biophys Res Commun 247: 827–832.

    Article  CAS  PubMed  Google Scholar 

  • Di Como CJ, Arndt KT . (1996). Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev 10: 1904–1916.

    Article  CAS  PubMed  Google Scholar 

  • Dozier C, Bonyadi M, Baricault L, Tonasso L, Darbon JM . (2004). Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A via its B′ regulatory subunit. Biol Cell 96: 509–517.

    Article  CAS  PubMed  Google Scholar 

  • Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415: 141–147.

    Article  CAS  PubMed  Google Scholar 

  • Gingras AC, Caballero M, Zarske M, Sanchez A, Hazbun TR, Fields S et al. (2005). A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol Cell Proteomics 4: 1725–1740.

    Article  CAS  PubMed  Google Scholar 

  • Goodarzi AA, Jonnalagadda JC, Douglas P, Young D, Ye R, Moorhead GB et al. (2004). Autophosphorylation of ataxia-telangiectasia mutated is regulated by protein phosphatase 2A. EMBO J 23: 4451–4461.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Inui S, Sanjo H, Maeda K, Yamamoto H, Miyamoto E, Sakaguchi N . (1998). Ig receptor binding protein 1 (α4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A. Blood 92: 539–546.

    CAS  PubMed  Google Scholar 

  • Jacinto E, Guo B, Arndt KT, Schmelzle T, Hall MN . (2001). TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. Mol Cell 8: 1017–1026.

    Article  CAS  PubMed  Google Scholar 

  • Janssens V, Goris J . (2001). Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353: 417–439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang Y, Broach JR . (1999). Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J 18: 2782–2792.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kloeker S, Reed R, McConnell JL, Chang D, Tran K, Westphal RS et al. (2003). Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A(C), PP4(C), and PP6(C)) and analysis of the interaction of PP2A(C) with α4 protein. Protein Expr Purif 31: 19–33.

    Article  CAS  PubMed  Google Scholar 

  • Kloeker S, Wadzinski BE . (1999). Purification and identification of a novel subunit of protein serine/threonine phosphatase 4. J Biol Chem 274: 5339–5347.

    Article  CAS  PubMed  Google Scholar 

  • Li M, Guo H, Damuni Z . (1995). Purification and characterization of two potent heat-stable protein inhibitors of protein phosphatase 2A from bovine kidney. Biochemistry 34: 1988–1996.

    Article  CAS  PubMed  Google Scholar 

  • Luke MM, Della Seta F, Di Como CJ, Sugimoto H, Kobayashi R, Arndt KT . (1996). The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase. Mol Cell Biol 16: 2744–2755.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McCright B, Rivers AM, Audlin S, Virshup DM . (1996). The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem 271: 22081–22089.

    Article  CAS  PubMed  Google Scholar 

  • Murata K, Wu J, Brautigan DL . (1997). B cell receptor-associated protein α4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc Natl Acad Sci USA 94: 10624–10629.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nanahoshi M, Nishiuma T, Tsujishita Y, Hara K, Inui S, Sakaguchi N et al. (1998). Regulation of protein phosphatase 2A catalytic activity by α4 protein and its yeast homolog Tap42. Biochem Biophys Res Commun 251: 520–526.

    Article  CAS  PubMed  Google Scholar 

  • Petersen P, Chou DM, You Z, Hunter T, Walter JC, Walter G . (2006). Protein phosphatase 2A antagonizes ATM and ATR in a Cdk2- and Cdc7-independent DNA damage checkpoint. Mol Cell Biol 26: 1997–2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM et al. (1999). Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 59: 4375–4382.

    CAS  PubMed  Google Scholar 

  • Scheidtmann KH, Mumby MC, Rundell K, Walter G . (1991). Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen. Mol Cell Biol 11: 1996–2003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schmidt A, Beck T, Koller A, Kunz J, Hall MN . (1998). The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. EMBO J 17: 6924–6931.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sontag E, Nunbhakdi-Craig V, Lee G, Bloom GS, Mumby MC . (1996). Regulation of the phosphorylation state and microtubule-binding activity of Tau by protein phosphatase 2A. Neuron 17: 1201–1207.

    Article  CAS  PubMed  Google Scholar 

  • Strack S, Zaucha JA, Ebner FF, Colbran RJ, Wadzinski BE . (1998). Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J Comp Neurol 392: 515–527.

    Article  CAS  PubMed  Google Scholar 

  • Trockenbacher A, Suckow V, Foerster J, Winter J, Krauss S, Ropers HH et al. (2001). MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nat Genet 29: 287–294.

    Article  CAS  PubMed  Google Scholar 

  • Van Hoof C, Martens E, Longin S, Jordens J, Stevens I, Janssens V et al. (2005). Specific interactions of PP2A and PP2A-like phosphatases with the yeast PTPA homologues, Ypa1 and Ypa2. Biochem J 386: 93–102.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wada T, Miyata T, Inagi R, Nangaku M, Wagatsuma M, Suzuki D et al. (2001). Cloning and characterization of a novel subunit of protein serine/threonine phosphatase 4 from mesangial cells. J Am Soc Nephrol 12: 2601–2608.

    CAS  PubMed  Google Scholar 

  • Walsh AH, Cheng A, Honkanen RE . (1997). Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 (PP1) and 2A (PP2A), is highly selective for PP2A. FEBS Lett 416: 230–234.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr Joy Marlo and Matthew Mazalouskas for expert technical assistance and analysis. We are grateful to Dr Roger Colbran, Dr David Cortez and our laboratory colleagues for helpful discussions. This work was supported by grants from the National Institutes of Health (GM51366 and DK070787 to BEW), the Vanderbilt Diabetes Research & Training Center (DK20593), the Vanderbilt-Ingram Cancer Center (CA68485) and the Center for Molecular Neuroscience (MH19732).

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Authors and Affiliations

  1. Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA

    J L McConnell, R J Gomez, L R A McCorvey & B E Wadzinski

  2. Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA

    B K Law

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  1. J L McConnell
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  2. R J Gomez
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  3. L R A McCorvey
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Correspondence to B E Wadzinski.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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McConnell, J., Gomez, R., McCorvey, L. et al. Identification of a PP2A-interacting protein that functions as a negative regulator of phosphatase activity in the ATM/ATR signaling pathway. Oncogene 26, 6021–6030 (2007). https://doi.org/10.1038/sj.onc.1210406

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