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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Mutational analysis of the Cy motif from p21 reveals sequence degeneracy and specificity for different

J A Wohlschlegel1, B T Dwyer, D Y Takeda

  • 1Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

Molecular and Cellular Biology
|July 5, 2001
PubMed

Insights

The cyclin-binding (Cy) motif, including the RXL sequence, is crucial for cyclin-dependent kinase (cdk) inhibitors like p21. However, this study reveals that RXL is not essential for p21 to inhibit cyclin E-cdk2, demonstrating functional non-RXL Cy motifs.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Cyclin-dependent kinases (cdks) regulate the cell cycle.
  • Cdk inhibitors (CKIs) bind to cyclin subunits via a cyclin-binding (Cy) motif, often containing an RXL sequence.
  • The CKI p21 interacts with cyclin E-cdk2 complexes.

Purpose of the Study:

  • To investigate the necessity of the RXL sequence within the Cy motif of p21 for inhibiting cyclin E-cdk2 activity.
  • To determine if non-RXL Cy motifs can maintain p21's inhibitory function and substrate targeting.
  • To explore the differential binding specificities of p21's Cy motif towards cyclin E and cyclin A.

Main Methods:

  • Alanine scanning mutagenesis of the p21 Cy motif.
  • Assays to measure p21's inhibition of cyclin E-cdk2 activity.
  • Mammalian cell growth inhibition assays.
  • Binding studies using p21 Cy mutants with cyclin E-cdk2 and cyclin A-cdk2.

Main Results:

  • Mutations within the RXL sequence did not abolish p21's inhibitory activity on cyclin E-cdk2; replacement with hydrophobic residues like valine preserved function.
  • Non-RXL Cy motifs were sufficient for inhibiting mammalian cell growth in vivo.
  • Variant Cy motifs could target substrates for phosphorylation by cyclin-cdk complexes.
  • The Cy motif was essential for p21 binding to cyclin E-cdk2 but not cyclin A-cdk2.
  • Cell growth inhibition by p21 derivatives was dependent on their ability to inhibit cyclin E-cdk2.

Conclusions:

  • The RXL sequence is not strictly necessary for a functional cyclin-binding motif in p21.
  • Non-RXL Cy motifs can effectively inhibit cyclin E-cdk2 activity and mediate cell growth arrest.
  • p21 exhibits differential binding specificity towards cyclin E versus cyclin A, which is critical for its cell growth inhibitory function.

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