In vitro phosphorylation and acetylation of the murine pocket protein Rb2/p130

• Published in: PloS one 2012-09, Vol.7 (9), p.e46174-e46174
• Main Authors: Saeed, Muhammad, Schwarze, Florian, Loidl, Adele, Meraner, Joachim, Lechner, Markus, Loidl, Peter
• Format: Article
• Language: English
• Subjects: Acetylation; Acetyltransferase; Animals; Arginine - genetics; Arginine - metabolism; Biological activity; Biology; Cancer; Cell cycle; Cell Division - genetics; Chromatography; Cloning; Cyclin-dependent kinase 4; Cyclin-Dependent Kinase 4 - genetics; Cyclin-Dependent Kinase 4 - metabolism; Cyclin-dependent kinases; Deacetylation; Deoxyribonucleic acid; DNA; E1A-Associated p300 Protein - genetics; E1A-Associated p300 Protein - metabolism; E2F protein; E7 protein; Event-related potentials; Gene Expression Regulation; Genetic aspects; Glutamine - genetics; Glutamine - metabolism; Human papillomavirus; Humans; Immunoglobulins; Kinases; Lysine; Lysine - genetics; Lysine - metabolism; Mice; Molecular biology; Mutation; NIH 3T3 Cells; Papillomavirus E7 Proteins - genetics; Papillomavirus E7 Proteins - metabolism; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation; Physiological aspects; Protein kinase; Protein Structure, Tertiary; Proteins; Regulators; Residues; Retina; Retinoblastoma; Retinoblastoma protein; Retinoblastoma-Like Protein p130 - genetics; Retinoblastoma-Like Protein p130 - metabolism; Risk factors; S Phase; Senescence; Signal Transduction - genetics; Tumor proteins
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0046174

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.