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Dual cyclin-binding domains are required for p107 to function as a kinase inhibitor

E Castaño1, Y Kleyner, B D Dynlacht

  • 1Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

Molecular and Cellular Biology
|August 26, 1998
PubMed

Insights

The retinoblastoma (pRB) protein family members p107 and p130 inhibit cyclin-dependent kinases (cdks), unlike pRB. Their N-terminal domains are crucial for binding and inhibiting cdks, impacting cell growth.

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • Cancer Biology

Background:

  • The retinoblastoma (pRB) protein family comprises three growth-suppressing proteins in mammalian cells.
  • Previous research indicated that p107 and p130 might suppress growth by inhibiting cyclin-dependent kinases (cdks).
  • The precise mechanism of cdk inhibition by p107 and p130 remained unclear.

Purpose of the Study:

  • To investigate the mechanism by which p107 and p130 inhibit cdks.
  • To determine if p107 and p130 function as genuine cdk inhibitors.
  • To elucidate the role of specific domains within p107 and p130 in cdk inhibition and growth suppression.

Main Methods:

  • In vivo and in vitro assays were employed to study protein interactions and kinase activity.
  • Site-directed mutagenesis was used to map functional domains within p107 and p130.
  • Peptide inhibition assays were performed to assess the role of specific regions in cdk binding and inhibition.

Main Results:

  • p107 was identified as a potent inhibitor of cyclin A-cdk2 and cyclin E-cdk2, with inhibitory potency comparable to p21/WAF1.
  • pRB did not exhibit inhibitory activity against cdks.
  • A second cyclin-binding site was mapped to the N-terminal regions of p107 and p130, which mediated cdk inhibition.
  • The C-terminal domain of p107, while a kinase substrate, did not inhibit cdk activity.
  • Mutations in the N-terminal domain impaired cyclin binding and growth suppression.
  • Peptides from the N-terminal cyclin-binding region interfered with p107-cdk2 complex formation and kinase inhibition.

Conclusions:

  • p107 and p130 function as true inhibitors of cyclin-cdk2 complexes, distinguishing them from mere substrates.
  • The N-terminal domain of p107 and p130 is critical for cdk inhibition and mediating growth suppression.
  • These findings clarify the mechanism of cdk inhibition by the pRB family, with implications for understanding cell cycle control and cancer.

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