Identification of the Active Region of the DNA Synthesis Inhibitory Gene p21Sdi1/CIP1/WAF1

EMBO J. 1995 Feb 1;14(3):555-63.

Abstract

The cloning of the negative growth regulatory gene, p21Sdi1, 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 (SD11) 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 p21Sdi1, 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 p27Kip1 protein, another Cdk inhibitor. Point mutations made in p21Sdi1 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 p21Sdi1 were substituted with amino acids 60-76 of p27Kip1, had almost the same DNA synthesis inhibitory activity as the wild-type protein. The results indicate that the region of sequence similarity between p21Sdi1 and p27Kip1 encodes an inhibitory motif characteristic of this family of Cdk inhibitors.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • CDC2-CDC28 Kinases*
  • Cell Compartmentation
  • Cell Cycle Proteins*
  • Cell Nucleus / metabolism
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclin-Dependent Kinase Inhibitor p27
  • Cyclin-Dependent Kinases / antagonists & inhibitors*
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclins / genetics
  • Cyclins / metabolism*
  • Cytoplasm / metabolism
  • DNA Mutational Analysis
  • DNA Replication / physiology
  • Fibroblasts
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Humans
  • Male
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Molecular Sequence Data
  • Point Mutation
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Sequence Deletion
  • Skin / cytology
  • Structure-Activity Relationship
  • Tumor Suppressor Proteins*

Substances

  • CDKN1A protein, human
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • Fungal Proteins
  • Microtubule-Associated Proteins
  • Tumor Suppressor Proteins
  • Cyclin-Dependent Kinase Inhibitor p27
  • Protein-Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases