Related Experiment Video
Updated: Jun 27, 2026
12:26
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Conformational switch upon phosphorylation: human CDK inhibitor p19INK4d between the native and partially folded
Christian Löw1, Nadine Homeyer, Ulrich Weininger
1Institut für Physik, Biophysik, Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle (Saale), Germany.
ACS Chemical Biology
|December 10, 2008
Summary
Phosphorylation of p19INK4d at Ser76 destabilizes its native structure, leading to an intermediate state. This modification may signal p19INK4d for degradation via ubiquitination.
Area of Science:
- Cell biology
- Molecular biology
- Protein biochemistry
Background:
- p19INK4d regulates the human cell cycle by inhibiting cyclin D-dependent kinases 4 and 6.
- Posttranslational phosphorylation occurs at Ser66 and Ser76 of p19INK4d.
Purpose of the Study:
- To investigate the impact of mimicking phosphorylation at Ser76 on p19INK4d stability and structure.
- To explore the role of phosphorylation in p19INK4d degradation pathways.
Main Methods:
- Site-directed mutagenesis to mimic phosphorylation (glutamate substitution at position 76).
- Kinetic and equilibrium analysis to assess protein stability.
- High-resolution Nuclear Magnetic Resonance (NMR) spectroscopy.
- Molecular dynamic simulations.
- In vitro ubiquitination assays.
Main Results:
- Mimicking phosphorylation at Ser76 dramatically decreased the stability of the native p19INK4d conformation.
- At body temperature, p19INK4d adopted an intermediate state where the C-terminal repeats remained folded, but the N-terminal repeats lost native chemical shifts.
- Molecular dynamics revealed large-scale motions in phosphorylated p19INK4d, destabilizing the interface between ankyrin repeats 2 and 3.
- Doubly phosphorylated p19INK4d mimics showed increased ubiquitination susceptibility in vitro.
Conclusions:
- Phosphorylation at Ser76 is critical for p19INK4d stability, promoting a transition to a less stable intermediate state.
- The structural destabilization induced by phosphorylation may facilitate protein degradation.
- Increased ubiquitination of doubly phosphorylated p19INK4d suggests a mechanism for its in vivo degradation.
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