Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase
Kleiton S Borges1, Valerie A Arboleda2, Eric Vilain3
1Department of Human Genetics, David Geffen School of Medicine at UCLA, University of California, Los Angeles, 695 Charles E. Young Drive, Los Angeles, CA 90095 USA ; Department of Genetics, Ribeirão Preto Medical School, University of São, Ribeirão Preto, Av. Bandeirantes 3900, CEP 14049-900 Ribeirão Preto, SP Brazil.
Abstract:
CDKN1C (also known as P57 (kip2) ) is a cyclin-dependent kinase inhibitor that functions as a negative regulator of cell proliferation through G1 phase cell cycle arrest. Recently, our group described gain-of-function mutations in the PCNA-binding site of CDKN1C that result in an undergrowth syndrome called IMAGe Syndrome (Intrauterine Growth Restriction, Metaphyseal dysplasia, Adrenal hypoplasia, and Genital anomalies), with life-threatening consequences. Loss-of-function mutations in CDKN1C have been identified in 5-10% of individuals with Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with features that are the opposite of IMAGe syndrome. Here, we investigate the effects of IMAGe-associated mutations on protein stability, cell cycle progression and cell proliferation. Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase. Overexpression of either wild-type or BWS-mutant CDKN1C inhibited cell proliferation. However, the IMAGe-mutant CDKN1C protein decreased cell growth significantly more than both the wild-type or BWS protein. These findings bring new insights into the molecular events underlying IMAGe syndrome.
Insights
Gain-of-function mutations in CDKN1C cause IMAGe syndrome by increasing protein stability and inhibiting cell proliferation. These IMAGe-mutant CDKN1C proteins decrease cell growth more than wild-type or BWS-mutant forms.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- CDKN1C (P57/kip2) is a cell cycle inhibitor.
- Gain-of-function mutations in CDKN1C cause IMAGe syndrome.
- Loss-of-function mutations in CDKN1C are linked to Beckwith-Wiedemann syndrome (BWS).
Purpose of the Study:
- Investigate the molecular effects of IMAGe-associated CDKN1C mutations.
- Analyze the impact on protein stability, cell cycle, and proliferation.
- Compare IMAGe-mutant CDKN1C with wild-type and BWS-mutant forms.
Main Methods:
- Site-directed mutagenesis to create IMAGe-associated mutations.
- Western blotting to assess protein stability.
- Cell cycle analysis (e.g., flow cytometry) to evaluate cell cycle progression.
- Cell proliferation assays to measure cell growth.
Main Results:
- IMAGe mutations in the PCNA-binding site increase CDKN1C protein stability.
- Mutant CDKN1C prevents cell cycle progression into S phase.
- IMAGe-mutant CDKN1C significantly inhibits cell proliferation more than wild-type or BWS-mutant CDKN1C.
Conclusions:
- IMAGe syndrome mutations enhance CDKN1C stability and cell cycle arrest.
- Altered CDKN1C function underlies the pathogenesis of IMAGe syndrome.
- These findings provide molecular insights into IMAGe syndrome and CDKN1C regulation.
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