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Papers In Press, published online ahead of print March 5, 2003
Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata-IRCCS, Rome 00167
Corresponding Author: f.martelli{at}idi.it
Oxidative stress induces cell death and growth arrest. In this report, the regulation and the functional role of the retinoblastoma family proteins, pRb, p107 and p130, in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypo-phosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi and was not associated with Cyclin/CDK down-modulation. Four lines of evidence indicate that H2O2-induced hypo-phosphorylation of pRb, p107 and p130 was due to the activity of protein phosphatase 2A (PP2A): First, cell treatment with two phosphatase inhibitors, okadaic acid and Calyculin-A, prevented the hypo-phosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, that binds and inhibits PP2A, when overexpressed, prevented H2O2-induced de-phosphorylation of the retinoblastoma family proteins, while a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2 treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Since DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was assessed whether the PP2A-mediated de-phosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed it was found that inhibition of PP2A by SV40 small t over-expression, prevented DNA synthesis inhibition induced by H2O2.
J. Biol. Chem, 10.1074/jbc.M300511200
Submitted on January 16, 2003
Revised on March 3, 2003
Accepted on March 4, 2003
Oxidative stress induces PP2A-dependent de-phosphorylation of the pocket proteins pRb, p107 and p130
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