Identification of the active region of the DNA synthesis inhibitory gene p21(Sdi1/CIP1/WAF1)

M. Nakanishi, R. S. Robetorye, G. R. Adami, O. M. Pereira-Smith, J. R. Smith

Research output: Contribution to journalArticle

168 Scopus citations

Abstract

The cloning of the negative growth regulatory gene, p21(Sdi1), has led to the convergence of the fields of cellular senescence, cell cycle regulation and tumor suppression. This gene was first cloned as an inhibitor of DNA synthesis that was overexpressed in terminally non-dividing senescent human fibroblasts (SDI1) and later as a p53 transactivated gene (WAF1) and a Cdk-interacting protein (CIP1, p21) that inhibited cyclin-dependent kinase activity. To identify the active region(s) of p21(Sdi1), cDNA constructs encoding various deleted forms of the protein were analyzed. Amino acids 22-71 were found to be the minimal region required for DNA synthesis inhibition. Amino acids 49-71 were involved in binding to Cdk2, and constructs deleted in this region expressed proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amino acids 60-76 of the p27(Kip1) protein, another Cdk inhibitor. Point mutations made in p21(Sdi1) in this region confirmed that amino acids common to both proteins were involved in DNA synthesis inhibition. Additionally, a chimeric protein, in which amino acids 49-65 of p21(Sdi1) were substituted with amino acids 60-76 of p27(Kip1), had almost the same DNA synthesis inhibitory activity as the wild-type protein. The results indicate that the region of sequence similarity between p21(Sdi1) and p27(Kip1) encodes an inhibitory motif characteristic of this family of Cdk inhibitors.

Original languageEnglish (US)
Pages (from-to)555-563
Number of pages9
JournalEMBO Journal
Volume14
Issue number3
DOIs
StatePublished - 1995

Keywords

  • Cdk
  • Cell cycle
  • Proliferation
  • Sdi1
  • p21

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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    Nakanishi, M., Robetorye, R. S., Adami, G. R., Pereira-Smith, O. M., & Smith, J. R. (1995). Identification of the active region of the DNA synthesis inhibitory gene p21(Sdi1/CIP1/WAF1). EMBO Journal, 14(3), 555-563. https://doi.org/10.1002/j.1460-2075.1995.tb07031.x