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Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase.

Borges KS, Arboleda VA, Vilain E - Cell Div (2015)

Bottom Line: Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase.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.

View Article: PubMed Central - PubMed

Affiliation: Department 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.

No MeSH data available.


Related in: MedlinePlus

IMAGe-mutant CDKN1C stabilizes CDKN1C protein. Cells were transiently transfected with plasmids expressing CDKN1C wild-type or IMAGE-mutants (p.K278E and p.F276V). After addition of cycloheximide (CHX) at 100 μg/mL to the culture medium, cells were harvested at various times and processed by Western blot analysis of CDKN1C protein.
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Fig1: IMAGe-mutant CDKN1C stabilizes CDKN1C protein. Cells were transiently transfected with plasmids expressing CDKN1C wild-type or IMAGE-mutants (p.K278E and p.F276V). After addition of cycloheximide (CHX) at 100 μg/mL to the culture medium, cells were harvested at various times and processed by Western blot analysis of CDKN1C protein.

Mentions: Mutations in the PCNA binding domain of the related protein CDKN1A, which shares sequence homology with CDKN1C, prevents its degradation [5]. Therefore, we decided to investigate whether the loss of direct binding of PCNA alters the degradation of CDKN1C. To address this, we transiently overexpressed wild-type or IMAGe-mutant (p.K278E and p.F276V) CDKN1C into HEK293T cells and examined the protein degradation rate of CDKN1C in the presence of the de novo protein synthesis inhibitor cycloheximide (100 μg/mL). pcDNA3.1-FLAG-CDKN1C constructs used here were described previously [4]. The following antibodies were used in our western blot experiments: anti-CDKN1C (Santa Cruz Biotechnology, sc-1040), anti-Cyclin A (Santa Cruz Biotechnology, sc-271682) and anti-B-actin (Abcam, mAbcam 8226). The secondary antibodies used were goat anti-mouse HRP (Bio-rad, 170–5047) and goat anti-rabbit HRP (Santa Cruz Biotechnology, sc-2030). IMAGe-mutant CDKN1C (p.K278E and p.276 V) led to increased stabilization of CDKN1C compared to wild-type CDKN1C (Figure 1). This is in accordance with recently published results, where IMAGE-associated proteins also demonstrated increased stability compared to wild-type [6,7].Figure 1


Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase.

Borges KS, Arboleda VA, Vilain E - Cell Div (2015)

IMAGe-mutant CDKN1C stabilizes CDKN1C protein. Cells were transiently transfected with plasmids expressing CDKN1C wild-type or IMAGE-mutants (p.K278E and p.F276V). After addition of cycloheximide (CHX) at 100 μg/mL to the culture medium, cells were harvested at various times and processed by Western blot analysis of CDKN1C protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389716&req=5

Fig1: IMAGe-mutant CDKN1C stabilizes CDKN1C protein. Cells were transiently transfected with plasmids expressing CDKN1C wild-type or IMAGE-mutants (p.K278E and p.F276V). After addition of cycloheximide (CHX) at 100 μg/mL to the culture medium, cells were harvested at various times and processed by Western blot analysis of CDKN1C protein.
Mentions: Mutations in the PCNA binding domain of the related protein CDKN1A, which shares sequence homology with CDKN1C, prevents its degradation [5]. Therefore, we decided to investigate whether the loss of direct binding of PCNA alters the degradation of CDKN1C. To address this, we transiently overexpressed wild-type or IMAGe-mutant (p.K278E and p.F276V) CDKN1C into HEK293T cells and examined the protein degradation rate of CDKN1C in the presence of the de novo protein synthesis inhibitor cycloheximide (100 μg/mL). pcDNA3.1-FLAG-CDKN1C constructs used here were described previously [4]. The following antibodies were used in our western blot experiments: anti-CDKN1C (Santa Cruz Biotechnology, sc-1040), anti-Cyclin A (Santa Cruz Biotechnology, sc-271682) and anti-B-actin (Abcam, mAbcam 8226). The secondary antibodies used were goat anti-mouse HRP (Bio-rad, 170–5047) and goat anti-rabbit HRP (Santa Cruz Biotechnology, sc-2030). IMAGe-mutant CDKN1C (p.K278E and p.276 V) led to increased stabilization of CDKN1C compared to wild-type CDKN1C (Figure 1). This is in accordance with recently published results, where IMAGE-associated proteins also demonstrated increased stability compared to wild-type [6,7].Figure 1

Bottom Line: Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase.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.

View Article: PubMed Central - PubMed

Affiliation: Department 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.

No MeSH data available.


Related in: MedlinePlus