title: Forskolin-mediated G1 arrest in acute lymphoblastic leukaemia cells: phosphorylated pRB sequesters E2Fs.
abstract: Increased intracellular levels of cAMP, induced by forskolin, lead to permanent G1 arrest of Reh cells. As expected, we observed a rapid dephosphorylation of the retinoblastoma protein (pRB) within 2 hours of forskolin treatment concomitant with reduced activity of the pRB-specific kinases. Interestingly, however, the dephosphorylation of pRB, as well as the inhibition of the kinase activities, was only transient, despite the permanent arrest of cells in G1. Importantly, although the pRB-specific kinases were fully active after 48 hours, pRB became only partially rephosphorylated. The transient dephosphorylation of pRB could be explained by the transient decrease in the activities of the pRB-specific kinases, but to understand why pRB became only partially rephosphorylated, despite fully activated kinases, we postulated that cAMP could activate a pRB-directed phosphatase. It was therefore interesting to find that the phosphatase inhibitor, tautomycin, was able to abolish the forskolin-mediated dephosphorylation of pRB, without increasing the activities of the pRB-specific kinases. To understand how Reh cells expressing hyperphosphorylated forms of pRB can remain arrested in G1, we used three different methods to test for the ability of pRB to form functional complexes with the family of E2F transcription factors. As expected, we observed an increased complex formation between E2F-1, E2F-4 and pRB after 2 hours when pRB was in its most dephosphorylated state. Surprisingly, however, prolonged treatment with forskolin, which induced partial rephosphorylation of pRB, in fact further increased the complex formation between the E2Fs and pRB, and this also resulted in reduced E2F-promoter activity in vivo. These data imply that in Reh cells, partially phosphorylated forms of pRB retain the ability to inhibit E2F-promoter activity, and thereby prevent cells from entering into S-phase.
authors.affiliation: Institute of Medical Biochemistry, University of Oslo, PO Box 1112, Blindern, N-0317, Oslo, Norway.
authors.forename: Kristine Bjerve
authors.initials: KB
authors.lastname: Gützkow
authors.forename: Soheil
authors.initials: S
authors.lastname: Naderi
authors.forename: Heidi K
authors.initials: HK
authors.lastname: Blomhoff
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology*; D018844:Cyclin-Dependent Kinases / Q000187:drug effects / Q000378:metabolism; D016213:Cyclins / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050704:E2F4 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D011506:Proteins; D011714:Pyrans; D016160:Retinoblastoma Protein / Q000187:drug effects / Q000378:metabolism*; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D013141:Spiro Compounds; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000935:Antifungal Agents; D018797:Cell Cycle Proteins; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D050704:E2F4 Transcription Factor; C493762:E2F4 protein, human; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D011714:Pyrans; C493837:RBL2 protein, human; D016160:Retinoblastoma Protein; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D013141:Spiro Compounds; D014157:Transcription Factors; C053079:tautomycin; D005576:Colforsin; D018844:Cyclin-Dependent Kinases