Proteomic analysis of p16ink⁴a-binding proteins

• Published in: Proteomics (Weinheim) 2007-11, Vol.7 (22), p.4102-4111
• Main Authors: Souza-Rodrígues, Elielson, Estanyol, Josep M, Friedrich-Heineken, Erica, Olmedo, Eva, Vera, Jorge, Canela, Nuria, Brun, Sonia, Agell, Neus, Hübscher, Ulrich, Bachs, Oriol, Jaumot, Montserrat
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Blotting, Western; Cell Line, Tumor; Chromatography, Affinity - methods; Cyclin-Dependent Kinase Inhibitor p16 - chemistry; Cyclin-Dependent Kinase Inhibitor p16 - metabolism; Cyclin-Dependent Kinase Inhibitor p16 - pharmacology; DNA Polymerase III - antagonists & inhibitors; Electrophoresis, Polyacrylamide Gel; Enzyme Activation - drug effects; Fluorescent Antibody Technique - methods; Fundamental and applied biological sciences. Psychology; HeLa Cells; Humans; Immunoprecipitation; MALDI-TOF MS; MCM6; Mice; Miscellaneous; p16ink4a-binding proteins; PCNA; Proliferating Cell Nuclear Antigen - metabolism; Proliferating Cell Nuclear Antigen - pharmacology; Proteins; Proteomics; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Tumor Cells, Cultured
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200700133

The p16ink⁴a tumor suppressor protein plays a critical role in cell cycle control, tumorogenesis and senescence. The best known activity for p16ink⁴a is the inhibition of the activity of CDK4 and CDK6 kinases, both playing a key role in cell cycle progression. With the aim to study new p16ink⁴a functions we used affinity chromatography and MS techniques to identify new p16ink⁴a-interacting proteins. We generated p16ink⁴a columns by coupling the protein to activated Sepharose 4B. The proteins from MOLT-4 cell line that bind to p16ink⁴a affinity columns were resolved by SDS-PAGE and identified by MS using a MALDI-TOF. Thirty-one p16ink⁴a -interacting proteins were identified and grouped in functional clusters. The identification of two of them, proliferating cell nuclear antigen (PCNA) and minichromosome maintenance protein 6 (MCM6), was confirmed by Western blotting and their in vivo interactions with p16ink⁴a were demonstrated by immunoprecipitation and immunofluorescence studies. Results also revealed that p16ink⁴a interacts directly with the DNA polymerase δ accessory protein PCNA and thereby inhibits the polymerase activity.