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Rb inhibits the intrinsic kinase activity of TATA-binding protein-associated factor TAFII250

J L Siegert1, P D Robbins

  • 1Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

Molecular and Cellular Biology
|December 22, 1998
PubMed

Insights

The retinoblastoma tumor suppressor protein (Rb) inhibits the kinase activity of TAFII250, a key transcription factor. This interaction, mediated by the Rb pocket, suggests a new way Rb regulates gene transcription.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Transcriptional Regulation

Background:

  • The retinoblastoma tumor suppressor protein (Rb) is crucial for cell cycle control.
  • TAFII250 is a large subunit of the TFIID complex, involved in transcription initiation.
  • Rb and TAFII250 interact, but the functional significance is unclear.

Purpose of the Study:

  • To investigate whether Rb regulates the kinase activity of TAFII250.
  • To elucidate the mechanism and functional domains involved in Rb-TAFII250 interaction.

Main Methods:

  • Immunopurification and gel purification of recombinant TAFII250.
  • In vitro kinase assays to measure TAFII250 autophosphorylation and RAP74 phosphorylation.
  • Site-directed mutagenesis of Rb to identify functional domains.

Main Results:

  • Rb inhibits the kinase activity of TAFII250 in a dose-dependent manner.
  • Rb's inhibitory function resides in its pocket domain (aa 379-928), not the N-terminus.
  • Rb specifically inhibits TAFII250's N-terminal kinase domain via direct interaction.
  • Rb pocket mutants (C706F, Deltaex22) are defective in kinase inhibition despite binding TAFII250.

Conclusions:

  • Rb directly inhibits TAFII250 kinase activity, suggesting a novel regulatory mechanism.
  • This inhibition involves specific interaction with TAFII250's kinase domain.
  • Rb's tumor suppressor function may involve modulating TAFII250's phosphorylation of itself and other targets like RAP74.

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