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Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase

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Abstract

Protein phosphatase 1δ (PP1δ) localizes to focal adhesions and associates with the focal adhesion kinase (FAK). In the present work we used deletion mutants of PP1δ and FAK to detect their reciprocally interacting domains. Dissection of PP1δ indicated 194–260 as the shortest FAK-interacting domain among those tested. Domain 194–260 encompasses several sites involved in catalysis, indirectly confirming that FAK is a PP1 substrate. Mutation of one of these sites, R220 (R220S or R220Q), did not abolish but on the contrary increased the ability of 194–260 to pull-down FAK. Such property might be exploited to detect new potential PP1 substrates. Among the FAK deletion mutants, only the C-terminal domain (684–1053, also known as FRNK) pulled-down a significant amount of PP1. The PP1 eluted from a GST-FRNK affinity column displayed Mr of 35,000 when analyzed by gel-filtration on FPLC Superose 12, indicating the presence of an isolated PP1 catalytic subunit.

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Abbreviations

FAK:

focal adhesion kinase

FRNK:

FAK-related nonkinase C-terminal domain of FAK

PP1:

type-1 protein serine/threonine phosphatase

LB-broth:

Luria–Bertani broth

IPTG:

isopropyl b-D-thiogalactopyranoside

GST:

glutathione-S-tranferase

DMEM:

Dulbecco’s modified Eagle’s medium

PBS:

phosphate-buffered saline

TRIS:

Tris-(hydroxymethyl)aminomethane

EDTA:

Ethylenediaminetetracetic acid

TPCK:

L-1-p-tosylamino-2-phenylethyl chloromethyl ketone

PMSF:

phenylmethyl sulfonyl fluoride

BSA:

bovine serum albumin

SDS:

sodium dodecyl sulfate

TCA:

trichloroacetic acid

References

  1. Clark EA, Brugge JS: Integrins and signal transduction pathways: The road taken. Science 268: 233–239, 1995

    CAS  PubMed  Google Scholar 

  2. Schaller MD, Parsons JT: Focal adhesion kinase and associated proteins. Curr Opin Cell Biol 6: 705–710, 1994

    Article  CAS  PubMed  Google Scholar 

  3. Schaller MD, Parsons JT: Focal adhesion kinase: An integrin-linked protein tyrosine kinase. Trends Cell Biol 3: 258–262, 1993

    Article  CAS  PubMed  Google Scholar 

  4. Ma A, Richardson A, Schaefer EM, Parsons JT: Serine phosphorylation of Focal Adhesion Kinase in interphase and mitosis: A possible role in modulating binding to p130cas. Mol Biol Cell 12: 1–12, 2001

    CAS  PubMed  Google Scholar 

  5. Vuori K, Ruoslahti E: Activation of protein kinase C precedes α6β1 integrin-mediated cell spreading on fibronectin. J Biol Chem 268: 21459–21462, 1993

    CAS  PubMed  Google Scholar 

  6. Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva G, Filmus J, Bell JC, Dedhar S: Regulation of cell adhesion and anchorage-dependent growth by a new beta1-integrin-linked protein kinase. Nature 379: 91–96, 1996

    CAS  PubMed  Google Scholar 

  7. Villa-Moruzzi E, Tognarini M, Cecchini G, Marchisio PC: Protein phosphatase 1 δ is associated with focal adhesions. Cell Adh Commun 5: 297–305, 1998

    CAS  Google Scholar 

  8. Fresu M, Bianchi M, Parsons JT, Villa-Moruzzi E: Cell cycle-dependent association of protein phosphatase 1 and focal adhesion kinase. Biochem J 358: 407–414, 2001

    CAS  PubMed  Google Scholar 

  9. Ceulemans H, Bollen M: Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiol Rev ([0-9]+): 1–39, 2004

    CAS  PubMed  Google Scholar 

  10. Bollen M: Combinatorial control of protein phosphatase-1. Trends Biochem Sci 26: 426–431, 2001

    CAS  PubMed  Google Scholar 

  11. Hubbard M, Cohen P: On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem Sci 18: 172–177, 1993

    CAS  PubMed  Google Scholar 

  12. 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 α gene in rat hepatocellular carcinomas. Jpn J Cancer 81: 1272–1280, 1990

    CAS  Google Scholar 

  13. Tognarini M, Villa-Moruzzi E: Analysis of the isoforms of protein phosphatase 1 (PP1) with polyclonal peptide antibodies. In: J.W. Ludlow (ed). Methods in Molecular Biology, Vol 93, The Humana Press, Totowa, NJ, 1998, pp 169–183

    Google Scholar 

  14. Rubin E, Mittnacht S, Villa-Moruzzi E, Ludlow JW: Site-specific and temporally-regulated retinoblastoma protein dephosphorylation by protein phosphatase type 1. Oncogene 20, 3776–3786, 2001

    CAS  PubMed  Google Scholar 

  15. Bianchi M, Villa-Moruzzi E: Binding of protein phosphatase-1 δ to the retinoblastoma protein pRb involves domains that include substrate recognition residues an a pRb binding motif. Biochem Biophys Res Commun 280: 1–3, 2001

    CAS  PubMed  Google Scholar 

  16. Zhang L, Lee EYC: Mutational analysis of substrate recognition by protein phosphatase 1. Biochemistry 36: 8209–8214, 1997

    CAS  PubMed  Google Scholar 

  17. Huang H-B, Horiuchi A, Goldberg J, Greengard P, Nairn AC: Site-directed mutagenesis of aminoacid residues of protein phosphatase 1 involved in catalysis and inhibitor binding. Proc Natl Acad Sci USA 94: 3530–3535, 1997

    CAS  PubMed  Google Scholar 

  18. Wakula P, Beullens M, Ceulemans H, Stalmans W, Bollen M: Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1. J Biol Chem 278: 18817–18823, 2003

    CAS  PubMed  Google Scholar 

  19. Santoro MM, Gaudino G, Villa-Moruzzi E: Protein phosphatase 1 binds to phospho-Ser-1394 of the macrophage-stimulating protein receptor. Biochem J 376: 587–594, 2003

    CAS  PubMed  Google Scholar 

  20. Villa-Moruzzi E: Purification and inactivation-reactivation of phosphorylase phosphatase from the protein-glycogen complex. Arch Biochem Biophys 15: 155–164, 1986

    Google Scholar 

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Authors and Affiliations

  1. Department of Experimental Pathology, University of Pisa, Pisa, Italy

    Mariarita Bianchi, Michele Vietri & Emma Villa-Moruzzi

  2. Cell and Developmental Biology Laboratories, Department of Physiology and Biochemistry, University of Pisa, Pisa, Italy

    Stefania de Lucchini

  3. Scuola Normale Superiore, Pisa, Italy

    Stefania de Lucchini

  4. Dipartimento di Patologia Sperimentale, Sezione di Patologia Generale, Via Roma 55, 56126, Pisa, Italy

    Emma Villa-Moruzzi

Authors
  1. Mariarita Bianchi
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  2. Stefania de Lucchini
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  3. Michele Vietri
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  4. Emma Villa-Moruzzi
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Correspondence to Emma Villa-Moruzzi.

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Bianchi, M., de Lucchini, S., Vietri, M. et al. Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase. Mol Cell Biochem 272, 85–90 (2005). https://doi.org/10.1007/s11010-005-7639-z

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  • DOI: https://doi.org/10.1007/s11010-005-7639-z

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