U.V.C.-Induction of p53 activation and accumulation is dependent on cell cycle and pathways involving protein synthesis and phosphorylation

Kimmo Pitkänen, Tarja Haapajärvi, Marikki Laiho

Research output: Contribution to journalArticle

Abstract

Transcriptional activation and stabilization of p53 is a major response of mammalian cells to U.V.-light induced genetic damages, and possibly responsible for cell damage control. We have studied here by gel mobility shift and immunoblotting assays the activation and accumulation of p53 by U.V.C. and its dependency on cell cycle, protein synthesis and protein phosphorylation. In G0/G1 synchronized cells U.V.C.-induced p53 DNA-binding activity, but not its accumulation, whereas both events took place in G1/S and S-phase cells. The kinetics of p53 activation by U.V.C. were slow requiring at least 1 h and slowly increasing thereafter with full activation observed at 6 h. Treatment of cells with cycloheximide (CHX) prevented the activation of p53 in all phases of the cell cycle and its accumulation in G1/S and S. However, removing CHX-block allowed full activation and accumulation of p53 with fast kinetics even if 4 h had lapsed since the initial U.V.C. insult. This suggests that the protein synthesis-dependent signal initiating p53 activation by U.V.C. remains continuous in the cells. The requirement of protein phosphorylation as mediator of p53 activation by U.V.C. was studied by using chemical protein kinase inhibitors. Of the tested inhibitors, only staurosporine, a known inhibitor of protein kinase C (PKC) and various other kinases, inhibited both p53 activation and accumulation, whereas specific PKC inhibitors, tyrosine kinase inhibitors and a serine/threonine kinase inhibitor did not. PKC-mediation of the p53 U.V.-response was further ruled out by the reactivity of the activated p53 to C-terminal antibody PAb 421. Kinetic studies showed that staurosporine-mediated inhibition of p53 function is an early event in cell damage response. Thus dual, kinetically different events, de novo protein synthesis and staurosporine-inhibited protein phosphorylation are required for p53 activation and accumulation in all phases of the cell cycle. Notably, in the absence of U.V.-induced accumulation in G0/G1 cells, p53 activation is still subject to inhibition of protein synthesis.

Original languageEnglish (US)
Pages (from-to)459-469
Number of pages11
JournalOncogene
Volume16
Issue number4
DOIs
StatePublished - Jan 29 1998
Externally publishedYes

Keywords

  • DNA damage
  • Post-transcriptional modification
  • Staurosporine
  • Transcriptional regulation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cancer Research

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