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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: FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations.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.

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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FGF-induced dephosphorylation of p107 is prevented by protein phosphatase inhibitors and by SV40 small t-antigen (st SV40).(A) RCS cells were pretreated with either okadaic acid (OA) or Calyculin A 2 hours before FGF treatment. 20 µg of total protein were subjected to SDS-PAGE following inmmunodetection of p107. (B–G) RCS cells were infected with adenoviruses expressing GFP, st SV40 or st SV40 mutant C97S following FGF1 treatment as indicated. (B,C) 20 µg of total protein were used for immmunodetection of (B) st SV40 and (C) p107. Equal amount of protein loading was confirmed by α-actin immunodetection. (D–G) BrdU incorporation assay of FGF treated or untreated RCS cells expressing either GFP, st SV40, or st SV40 C97S mutant. BrdU was detected by using BrdU antibodies (red). Nuclei were stained with DAPI (blue). (D) Summary of results in panels E–G.
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pone-0003447-g003: FGF-induced dephosphorylation of p107 is prevented by protein phosphatase inhibitors and by SV40 small t-antigen (st SV40).(A) RCS cells were pretreated with either okadaic acid (OA) or Calyculin A 2 hours before FGF treatment. 20 µg of total protein were subjected to SDS-PAGE following inmmunodetection of p107. (B–G) RCS cells were infected with adenoviruses expressing GFP, st SV40 or st SV40 mutant C97S following FGF1 treatment as indicated. (B,C) 20 µg of total protein were used for immmunodetection of (B) st SV40 and (C) p107. Equal amount of protein loading was confirmed by α-actin immunodetection. (D–G) BrdU incorporation assay of FGF treated or untreated RCS cells expressing either GFP, st SV40, or st SV40 C97S mutant. BrdU was detected by using BrdU antibodies (red). Nuclei were stained with DAPI (blue). (D) Summary of results in panels E–G.

Mentions: We then determined whether inhibition of phosphatase activity affected the p107 status in FGF-treated RCS cells, using okadaic acid (OA) or calyculin A which are potent inhibitors of protein phosphatases [24]. As shown in Fig. 3A, p107 dephosphorylation was significantly prevented in the presence of OA, or calyculin A.


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)

FGF-induced dephosphorylation of p107 is prevented by protein phosphatase inhibitors and by SV40 small t-antigen (st SV40).(A) RCS cells were pretreated with either okadaic acid (OA) or Calyculin A 2 hours before FGF treatment. 20 µg of total protein were subjected to SDS-PAGE following inmmunodetection of p107. (B–G) RCS cells were infected with adenoviruses expressing GFP, st SV40 or st SV40 mutant C97S following FGF1 treatment as indicated. (B,C) 20 µg of total protein were used for immmunodetection of (B) st SV40 and (C) p107. Equal amount of protein loading was confirmed by α-actin immunodetection. (D–G) BrdU incorporation assay of FGF treated or untreated RCS cells expressing either GFP, st SV40, or st SV40 C97S mutant. BrdU was detected by using BrdU antibodies (red). Nuclei were stained with DAPI (blue). (D) Summary of results in panels E–G.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2562983&req=5

pone-0003447-g003: FGF-induced dephosphorylation of p107 is prevented by protein phosphatase inhibitors and by SV40 small t-antigen (st SV40).(A) RCS cells were pretreated with either okadaic acid (OA) or Calyculin A 2 hours before FGF treatment. 20 µg of total protein were subjected to SDS-PAGE following inmmunodetection of p107. (B–G) RCS cells were infected with adenoviruses expressing GFP, st SV40 or st SV40 mutant C97S following FGF1 treatment as indicated. (B,C) 20 µg of total protein were used for immmunodetection of (B) st SV40 and (C) p107. Equal amount of protein loading was confirmed by α-actin immunodetection. (D–G) BrdU incorporation assay of FGF treated or untreated RCS cells expressing either GFP, st SV40, or st SV40 C97S mutant. BrdU was detected by using BrdU antibodies (red). Nuclei were stained with DAPI (blue). (D) Summary of results in panels E–G.
Mentions: We then determined whether inhibition of phosphatase activity affected the p107 status in FGF-treated RCS cells, using okadaic acid (OA) or calyculin A which are potent inhibitors of protein phosphatases [24]. As shown in Fig. 3A, p107 dephosphorylation was significantly prevented in the presence of OA, or calyculin A.

Bottom Line: FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations.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.

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