Proteomic analysis of p16ink4a‐binding proteins

The p16 ink4a tumor suppressor protein plays a critical role in cell cycle control, tumorogenesis and senescence. The best known activity for p16 ink4a 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 p16 ink4a functions we used affinity chromatography and MS techniques to identify new p16 ink4a -interacting proteins. We generated p16 ink4a columns by coupling the protein to activated Sephar-ose 4B. The proteins from MOLT-4 cell line that bind to p16 ink4a affinity columns were resolved by SDS-PAGE and identified by MS using a MALDI-TOF. Thirty-one p16 ink4a -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 p16 ink4a were demonstrated by immunoprecipitation and immunofluorescence studies. Results also revealed that p16 ink4a interacts directly with the DNA polymerase δ accessory protein PCNA and thereby inhibits the polymerase activity.