Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb, p107, and p130.
Oxidative stress induces cell death and growth arrest. In this study, 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 hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas 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. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation 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.
Cicchillitti
Lucia
L
Laboratorio di Biologia Vascolare e Terapia Genica, Istituto Cardiologico Fondazione I. Monzino, Istituto di Ricovero e Cura a Carattere Scientifico, Via Parea 4, Milan, Italy.
Fasanaro
Pasquale
P
Biglioli
Paolo
P
Capogrossi
Maurizio C
MC
Martelli
Fabio
F
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000032:analysis; D016213:Cyclins / Q000032:analysis; D004247:DNA / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008387:Marine Toxins; D009687:Nuclear Proteins / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D011506:Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism*; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D016196:S Phase; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D000952:Antigens, Polyomavirus Transforming; D016213:Cyclins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D009687:Nuclear Proteins; D010080:Oxazoles; D010750:Phosphoproteins; D011506:Proteins; C493844:RBL1 protein, human; C493837:RBL2 protein, human; D016160:Retinoblastoma Protein; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D019319:Okadaic Acid; C059041:calyculin A; D004247:DNA; D006861:Hydrogen Peroxide; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2