Elielson Souza‐Rodrígues1, Josep Marı́a Estanyol2, Erica Friedrich‐Heineken3, Eva María Olmedo Moreno1, Jorge Vera1, Núria Canela-Canela2, Sònia Brun1, Neus Agell1, Ulrich Hübscher3, Oriol Bachs1, Montserrat Jaumot1
1Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Universitat de Barcelona, Spain
2Unitat de Proteòmica, Serveis Científico-tècnics, Universitat de Barcelona, Spain
3Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich-Irchel, Zurich, Switzerland
Tóm tắt
AbstractThe 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.
