Preferences help
enabled [disable] Abstract
Number of results
2005 | 52 | 3 | 713-719
Article title

Advantage of a baculovirus expression system for protein-protein interaction studies. Involvement of posttranslational phosphorylation in the interaction between wt p53 protein and poly(ADP-ribose) polymerase-1

Title variants
Languages of publication
We recently observed an interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 protein. However, more extensive studies on both proteins, especially those on characterization of their domains involved in the interaction were difficult due to very low expression levels of p53 in mammalian cells. Therefore, we generated recombinant proteins for such studies. To clarify which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction, we generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 each were sufficient to confer binding to PARP-1, whereas the amino-terminal part harbouring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were both necessary for complex formation with p53 protein. Since the most important features of p53 protein are regulated by phosphorylation, we addressed the question whether its phosphorylation is essential for the binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomers were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1, indicating that complex formation between the two proteins was regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export.
Physical description
  • Cell Cycle Regulation Group, Division: Institute of Cancer Research, Department of Medicine I, Vienna Medical University, Vienna, Austria
  • Cell Cycle Regulation Group, Division: Institute of Cancer Research, Department of Medicine I, Vienna Medical University, Vienna, Austria
  • Cell Cycle Regulation Group, Division: Institute of Cancer Research, Department of Medicine I, Vienna Medical University, Vienna, Austria
  • Apella E, Anderson CW (2001) Post-translational modifications and activation of p53 by genotoxic stresses. Eur J Biochem 268: 2764-2772.
  • Bargonetti J, Manfredi JJ (2002) Multiple roles of the tumor suppressor p53. Curr Opin Oncol 14: 86-91.
  • Berger NA (1985) Poly(ADP-ribose) in the cellular response to DNA damage. Radiat Res 101: 4-15.
  • Blagosklonny MV (2002) P53: An ubiquitous target of anticancer drugs. Int J Cancer 98: 161-166.
  • D'Amours D, Desnoyers S, D'Silvia I, Poirier GG (1999) Poly(ADP-ribosyl)ation reactions in the regulations of nuclear functions. Biochem J 342: 249-268.
  • de Murcia G, Menissier-de Murcia J (1994) Poly(ADP-ribose) polymerase: a molecular nick-sensor. Trends Biochem Sci 19: 172-176.
  • Donehower LA (2002) Does p53 affect organismal aging? J Cell Physiol 192: 23-33.
  • el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75: 817-825.
  • Fuchs SY, Adler V, Buschmann T, Yin Z, Wu X, Jones SN, Ronai Z (1998) JNK targets p53 ubiquitination and degradation in nonstressed cells. Genes Dev 12: 2658-2663.
  • Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid degradation of p53. Nature 387: 296-299.
  • Kubbutat MH, Jones SN, Vousden KH (1997) Regulation of p53 stability by Mdm2. Nature 387: 299-303.
  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: 680-685.
  • Maier B, Gluba W, Bernier B, Turner T, Mohammad K, Guise T, Sutherland A, Thorner M, Scrable H (2004) Modulation of mammalian life span by a short isoform of p53. Genes Dev 18: 306-319.
  • Momand J, Zambetti G, Olson D, George D, Levine AJ (1992) The Mdm2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69: 1237-1245.
  • Mowat MR (1998) p53 in tumor progression: life, death, and everything. Adv Cancer Res 74: 25-48.
  • O'Farell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007-4021.
  • O'Reilly DR, Miller LK, Luckow VA (1994) Baculovirus Expression Vectors: A Laboratory Manual. Freeman, New York.
  • Shaulsky G, Ben-Ze'ev A, Rotter V (1990) Subcellular distribution of the p53 protein during the cell cycle of Balb/c 3T3 cells. Oncogene 5: 1707-1711.
  • Stommel JM, Marchenko ND, Jimenez GS, Moll UM, Hope TJ, Wahl GM (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J 18: 1660-1672.
  • Wesierska-Gadek J, Schmid G (2000) Overexpressed poly(ADP-ribose) polymerase delays the release of rat cells from p53-mediated G1 checkpoint. J Cell Biochem 80: 85-103.
  • Wesierska-Gadek J, Schmid G (2001) Poly(ADP-ribose) polymerase-1 regulates the stability of the wild-type p53 protein. Cell Mol Biol Lett 6: 117-140.
  • Wesierska-Gadek J, Bugajska-Schretter A, Cerni C (1996a) ADP-ribosylation of p53 tumor suppressor protein: mutant but not wild-type p53 is modified. J Cell Biochem 62: 90-101.
  • Wesierska-Gadek J, Schmid G, Cerni C (1996b) ADP-ribosylation of wild-type p53 in vitro: binding of p53 protein to specific p53 consensus sequence prevents its modification. Biochem Biophys Res Commun 224: 96-102.
  • Wesierska-Gadek J, Wang ZQ, Schmid G (1999) Reduced stability of regularly spliced but not alternatively spliced p53 protein in PARP-deficient mouse fibroblasts. Cancer Res 59: 28-34.
  • Wesierska-Gadek J, Bohrn E, Herceg Z, Wang Z-Q, Wurzer G (2000) Differential susceptibility of normal and PARP knock-out mouse fibroblasts to proteasome inhibitors. J Cell Biochem 78: 681-696.
  • Wesierska-Gadek J, Wojciechowski J, Schmid G (2003a) Central and carboxy-terminal regions of human p53 protein are essential for interaction and complex formation with PARP-1. J Cell Biochem 89: 220-232.
  • Wesierska-Gadek J, Wojciechowski J, Schmid G (2003b) Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP-1. J Cell Biochem 89: 1260-1284.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.