title: In vitro phosphorylation and acetylation of the murine pocket protein Rb2/p130.
abstract: The retinoblastoma protein (pRb) and the related proteins Rb2/p130 and 107 represent the "pocket protein" family of cell cycle regulators. A key function of these proteins is the cell cycle dependent modulation of E2F-regulated genes. The biological activity of these proteins is controlled by acetylation and phosphorylation in a cell cycle dependent manner. In this study we attempted to investigate the interdependence of acetylation and phosphorylation of Rb2/p130 in vitro. After having identified the acetyltransferase p300 among several acetyltransferases to be associated with Rb2/p130 during S-phase in NIH3T3 cells in vivo, we used this enzyme and the CDK4 protein kinase for in vitro modification of a variety of full length Rb2/p130 and truncated versions with mutations in the acetylatable lysine residues 1079, 128 and 130. Mutation of these residues results in the complete loss of Rb2/p130 acetylation. Replacement of lysines by arginines strongly inhibits phosphorylation of Rb2/p130 by CDK4; the inhibitory effect of replacement by glutamines is less pronounced. Preacetylation of Rb2/p130 strongly enhances CDK4-catalyzed phosphorylation, whereas deacetylation completely abolishes in vitro phosphorylation. In contrast, phosphorylation completely inhibits acetylation of Rb2/p130 by p300. These results suggest a mutual interdependence of modifications in a way that acetylation primes Rb2/p130 for phosphorylation and only dephosphorylated Rb2/p130 can be subject to acetylation. Human papillomavirus 16-E7 protein, which increases acetylation of Rb2/p130 by p300 strongly reduces phosphorylation of this protein by CDK4. This suggests that the balance between phosphorylation and acetylation of Rb2/p130 is essential for its biological function in cell cycle control.
authors.affiliation: Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Saeed
authors.forename: Florian
authors.initials: F
authors.lastname: Schwarze
authors.forename: Adele
authors.initials: A
authors.lastname: Loidl
authors.forename: Joachim
authors.initials: J
authors.lastname: Meraner
authors.forename: Markus
authors.initials: M
authors.lastname: Lechner
authors.forename: Peter
authors.initials: P
authors.lastname: Loidl
mesh_terms: D000107:Acetylation; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics / Q000378:metabolism; D050881:E1A-Associated p300 Protein / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D050725:Papillomavirus E7 Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D050717:Retinoblastoma-Like Protein p130 / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D015398:Signal Transduction / Q000235:genetics
chemical_list: D050725:Papillomavirus E7 Proteins; D010750:Phosphoproteins; C493838:Rbl2 protein, mouse; D050717:Retinoblastoma-Like Protein p130; D005973:Glutamine; D001120:Arginine; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D008239:Lysine