title: Phosphorylation of the Retinoblastoma protein (Rb) on serine-807 is required for association with Bax.
abstract: The recent finding that the Retinoblastoma protein (Rb) is able to regulate apoptosis in a non-transcriptional manner directly at the mitochondria by interaction with the pro-apoptotic protein Bax prompted this investigation of the complex formed between Rb and Bax. Because the function of Rb in the cellular processes of proliferation, apoptosis, senescence and differentiation is regulated by phosphorylation we endeavored to elucidate the phosphorylation status of Rb with respect to its association with Bax and its role in apoptosis. In this study we found that Rb phosphorylated on at least 4 C-terminal phosphorylation sites (S608, S795, S807/S811, and T821) is present at the mitochondria under non-stressed cellular conditions. An in vitro binding assay showed that Bax binds to Rb phosphorylated at S807/S811 in 3 cancer cell types. Physiologically relevant association between Bax and Rb phosphorylated on S807/S811 was demonstrated by reciprocal co-immunoprecipitation experiments using antibodies specific for Rb phosphorylated on S807/S811 and Bax. Mutant Rb proteins expressed in Rb-null C33A cells showed that phosphorylation of S807 of Rb promotes association with Bax and that mimicking phosphorylation at S807 of Rb can block the induction of apoptosis due to PNUTS downregulation. Finally using siRNA to activate phosphatase activity in MCF7 cells, Rb is dephosphorylated at several sites including S807/S811, dissociates from Bax and apoptosis is triggered. These studies show that phosphorylation of Rb regulates its association with Bax and its role in apoptosis.
authors.affiliation: a Department of Biology and Health Science ; Pace University ; Pleasantville , NY USA.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Antonucci
authors.forename: Jacklynn V
authors.initials: JV
authors.lastname: Egger
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Krucher
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D001665:Binding Sites; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D006801:Humans; D061986:MCF-7 Cells; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics*; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; D051028:bcl-2-Associated X Protein; D012694:Serine
keywords: Bax; Rb phosphorylation; Retinoblastoma protein; apoptosis; phosphatase