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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

Synchronized cells transfected with IMAGe-mutant CDKN1C show loss of oscillation of CDKN1C through the cell cycle. (A) Experimental outline for synchronization of HEK293T cells. Cells were synchronized with double thymidine block as on indicated. After the first block cells were transfected with a empty, wild-type CDKN1C, BWS-mutant CDKN1C (p.L42P) or IMAGE-mutant CDKN1C (p.K278E) and then synchronized again in early S phase with thymidine. After this process, a time was designated as “T0” and the cells were released and progressed through the S-phase (B) Upon release, cells were collected at the indicated times and CDKN1C was analyzed by immunoblotting. Beta-actin was used as a loading control. (C) Cell cycle analysis of double thymidine block synchronized cells transfected with empty plasmid, wild-type CDKN1C, BWS-mutant (p.L42P) or IMAGE-mutant (p.K278E) CDKN1C.
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Fig2: Synchronized cells transfected with IMAGe-mutant CDKN1C show loss of oscillation of CDKN1C through the cell cycle. (A) Experimental outline for synchronization of HEK293T cells. Cells were synchronized with double thymidine block as on indicated. After the first block cells were transfected with a empty, wild-type CDKN1C, BWS-mutant CDKN1C (p.L42P) or IMAGE-mutant CDKN1C (p.K278E) and then synchronized again in early S phase with thymidine. After this process, a time was designated as “T0” and the cells were released and progressed through the S-phase (B) Upon release, cells were collected at the indicated times and CDKN1C was analyzed by immunoblotting. Beta-actin was used as a loading control. (C) Cell cycle analysis of double thymidine block synchronized cells transfected with empty plasmid, wild-type CDKN1C, BWS-mutant (p.L42P) or IMAGE-mutant (p.K278E) CDKN1C.

Mentions: Regulation of protein turnover is required for normal progression through the cell cycle. Since loss of PCNA-binding led to increased stabilization of CDKN1C, we hypothesized that the turnover of CDKN1C during the cell cycle progression might be altered. To investigate this, we developed a protocol where HEK293T cells progress into S-phase in the presence of different CDKN1C proteins (wild-type, IMAGe-mutant (p.K278E) and BWS-mutant (p.L42P)). The BWS-mutant has a mutation in the cyclin dependent kinase (CDK) inhibitory domain, but expresses a normal PCNA binding domain, while the IMAGe-mutant p.K278E has been previously shown to have loss of PCNA binding [4]. Cultures of HEK293T cells were synchronized using an initial thymidine block (2 mM) at G1/S border phase and upon release of the thymidine block transfected with their respective plasmids. Cells were resynchronized with thymidine (Figure 2A) and after a second wash were harvested at various time points and processed for western blot and cell cycle analysis, as described [4].Figure 2


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)

Synchronized cells transfected with IMAGe-mutant CDKN1C show loss of oscillation of CDKN1C through the cell cycle. (A) Experimental outline for synchronization of HEK293T cells. Cells were synchronized with double thymidine block as on indicated. After the first block cells were transfected with a empty, wild-type CDKN1C, BWS-mutant CDKN1C (p.L42P) or IMAGE-mutant CDKN1C (p.K278E) and then synchronized again in early S phase with thymidine. After this process, a time was designated as “T0” and the cells were released and progressed through the S-phase (B) Upon release, cells were collected at the indicated times and CDKN1C was analyzed by immunoblotting. Beta-actin was used as a loading control. (C) Cell cycle analysis of double thymidine block synchronized cells transfected with empty plasmid, wild-type CDKN1C, BWS-mutant (p.L42P) or IMAGE-mutant (p.K278E) CDKN1C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4389716&req=5

Fig2: Synchronized cells transfected with IMAGe-mutant CDKN1C show loss of oscillation of CDKN1C through the cell cycle. (A) Experimental outline for synchronization of HEK293T cells. Cells were synchronized with double thymidine block as on indicated. After the first block cells were transfected with a empty, wild-type CDKN1C, BWS-mutant CDKN1C (p.L42P) or IMAGE-mutant CDKN1C (p.K278E) and then synchronized again in early S phase with thymidine. After this process, a time was designated as “T0” and the cells were released and progressed through the S-phase (B) Upon release, cells were collected at the indicated times and CDKN1C was analyzed by immunoblotting. Beta-actin was used as a loading control. (C) Cell cycle analysis of double thymidine block synchronized cells transfected with empty plasmid, wild-type CDKN1C, BWS-mutant (p.L42P) or IMAGE-mutant (p.K278E) CDKN1C.
Mentions: Regulation of protein turnover is required for normal progression through the cell cycle. Since loss of PCNA-binding led to increased stabilization of CDKN1C, we hypothesized that the turnover of CDKN1C during the cell cycle progression might be altered. To investigate this, we developed a protocol where HEK293T cells progress into S-phase in the presence of different CDKN1C proteins (wild-type, IMAGe-mutant (p.K278E) and BWS-mutant (p.L42P)). The BWS-mutant has a mutation in the cyclin dependent kinase (CDK) inhibitory domain, but expresses a normal PCNA binding domain, while the IMAGe-mutant p.K278E has been previously shown to have loss of PCNA binding [4]. Cultures of HEK293T cells were synchronized using an initial thymidine block (2 mM) at G1/S border phase and upon release of the thymidine block transfected with their respective plasmids. Cells were resynchronized with thymidine (Figure 2A) and after a second wash were harvested at various time points and processed for western blot and cell cycle analysis, as described [4].Figure 2

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