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PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF.

Kolupaeva V, Laplantine E, Basilico C - PLoS ONE (2008)

Bottom Line: To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex.Furthermore, an association between p107 and PP2A was induced by FGF treatment.Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, New York, USA.

ABSTRACT
FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.

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Effect of cyclin D1/cdk4 overexpression on p130 phosphorylation and cyclin E/cdk2 and cyclin A/cdk 2 kinase activities.Parental RCS, cyclin D1, cdk4 or cyclin D1/cdk4 cell lines were treated with FGF1 for the indicated times. (A) p130 dephosphorylation is prevented in cyclin D1/cdk4 expressed cells. 100 µg of total protein was analyzed by SDA-PAGE followed by immunoblotting with anti p130-antibodies. (B, D, E) Kinase activity of immunoprecipitated cyclin E/cdk2 and cyclin A/cdk 2 complexes as assayed in vitro. The cyclin E/cdk2 and cyclin A/cdk 2 complexes were isolated using anti-cyclin E and anti-cyclin A antibodies and histone H1 was used as a substrate. Antibody to mouse IgG was used as negative control. Equal amount of protein loading was confirmed by immunodetection of cdk 2 in immunoprecipitated complexes. (C, F) Summary of results from 3 independent experiments depicting relative levels of cyclinE/cdk2 and cyclin A/cdk2 activities upon FGF treatment in different cell lines. (G) 20 µg of total protein were analyzed by WB, with antibodies against cdk2 or p21. Equal amount of protein loading was confirmed by α-tubulin immunodetection.
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pone-0003447-g002: Effect of cyclin D1/cdk4 overexpression on p130 phosphorylation and cyclin E/cdk2 and cyclin A/cdk 2 kinase activities.Parental RCS, cyclin D1, cdk4 or cyclin D1/cdk4 cell lines were treated with FGF1 for the indicated times. (A) p130 dephosphorylation is prevented in cyclin D1/cdk4 expressed cells. 100 µg of total protein was analyzed by SDA-PAGE followed by immunoblotting with anti p130-antibodies. (B, D, E) Kinase activity of immunoprecipitated cyclin E/cdk2 and cyclin A/cdk 2 complexes as assayed in vitro. The cyclin E/cdk2 and cyclin A/cdk 2 complexes were isolated using anti-cyclin E and anti-cyclin A antibodies and histone H1 was used as a substrate. Antibody to mouse IgG was used as negative control. Equal amount of protein loading was confirmed by immunodetection of cdk 2 in immunoprecipitated complexes. (C, F) Summary of results from 3 independent experiments depicting relative levels of cyclinE/cdk2 and cyclin A/cdk2 activities upon FGF treatment in different cell lines. (G) 20 µg of total protein were analyzed by WB, with antibodies against cdk2 or p21. Equal amount of protein loading was confirmed by α-tubulin immunodetection.

Mentions: The maintenance of FGF-induced growth arrest is characterized by the accumulation of hypophosphorylated p130 by 10–16 hrs of FGF treatment, that follows a dramatic drop in cyclin E/cdk2 activity between 6 and 12 hrs [3]. We therefore investigated the phosphorylation status of p130 in the cells overexpressing both cdk4 and cyclin D1 after FGF treatment. p130 also remained phosphorylated in this cell line, while FGF caused dephosphorylation of p130 when cdk4 or cyclin D1 were overexpressed independently (Fig. 2A.) Several reports [16], [18], [19] indicate that the cyclin D1/cdk4 complex can phosphorylate both p107 and p130, but that inactivation of p130 requires the phosphorylation of additional sites by the cyclin E/cdk2 complex. We therefore studied the activity of the cyclin E/cdk2 complex in our cell lines.


PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF.

Kolupaeva V, Laplantine E, Basilico C - PLoS ONE (2008)

Effect of cyclin D1/cdk4 overexpression on p130 phosphorylation and cyclin E/cdk2 and cyclin A/cdk 2 kinase activities.Parental RCS, cyclin D1, cdk4 or cyclin D1/cdk4 cell lines were treated with FGF1 for the indicated times. (A) p130 dephosphorylation is prevented in cyclin D1/cdk4 expressed cells. 100 µg of total protein was analyzed by SDA-PAGE followed by immunoblotting with anti p130-antibodies. (B, D, E) Kinase activity of immunoprecipitated cyclin E/cdk2 and cyclin A/cdk 2 complexes as assayed in vitro. The cyclin E/cdk2 and cyclin A/cdk 2 complexes were isolated using anti-cyclin E and anti-cyclin A antibodies and histone H1 was used as a substrate. Antibody to mouse IgG was used as negative control. Equal amount of protein loading was confirmed by immunodetection of cdk 2 in immunoprecipitated complexes. (C, F) Summary of results from 3 independent experiments depicting relative levels of cyclinE/cdk2 and cyclin A/cdk2 activities upon FGF treatment in different cell lines. (G) 20 µg of total protein were analyzed by WB, with antibodies against cdk2 or p21. Equal amount of protein loading was confirmed by α-tubulin immunodetection.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2562983&req=5

pone-0003447-g002: Effect of cyclin D1/cdk4 overexpression on p130 phosphorylation and cyclin E/cdk2 and cyclin A/cdk 2 kinase activities.Parental RCS, cyclin D1, cdk4 or cyclin D1/cdk4 cell lines were treated with FGF1 for the indicated times. (A) p130 dephosphorylation is prevented in cyclin D1/cdk4 expressed cells. 100 µg of total protein was analyzed by SDA-PAGE followed by immunoblotting with anti p130-antibodies. (B, D, E) Kinase activity of immunoprecipitated cyclin E/cdk2 and cyclin A/cdk 2 complexes as assayed in vitro. The cyclin E/cdk2 and cyclin A/cdk 2 complexes were isolated using anti-cyclin E and anti-cyclin A antibodies and histone H1 was used as a substrate. Antibody to mouse IgG was used as negative control. Equal amount of protein loading was confirmed by immunodetection of cdk 2 in immunoprecipitated complexes. (C, F) Summary of results from 3 independent experiments depicting relative levels of cyclinE/cdk2 and cyclin A/cdk2 activities upon FGF treatment in different cell lines. (G) 20 µg of total protein were analyzed by WB, with antibodies against cdk2 or p21. Equal amount of protein loading was confirmed by α-tubulin immunodetection.
Mentions: The maintenance of FGF-induced growth arrest is characterized by the accumulation of hypophosphorylated p130 by 10–16 hrs of FGF treatment, that follows a dramatic drop in cyclin E/cdk2 activity between 6 and 12 hrs [3]. We therefore investigated the phosphorylation status of p130 in the cells overexpressing both cdk4 and cyclin D1 after FGF treatment. p130 also remained phosphorylated in this cell line, while FGF caused dephosphorylation of p130 when cdk4 or cyclin D1 were overexpressed independently (Fig. 2A.) Several reports [16], [18], [19] indicate that the cyclin D1/cdk4 complex can phosphorylate both p107 and p130, but that inactivation of p130 requires the phosphorylation of additional sites by the cyclin E/cdk2 complex. We therefore studied the activity of the cyclin E/cdk2 complex in our cell lines.

Bottom Line: To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex.Furthermore, an association between p107 and PP2A was induced by FGF treatment.Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, New York, USA.

ABSTRACT
FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.

Show MeSH
Related in: MedlinePlus