Related Experiment Video
Updated: Jul 10, 2026
Mapping Dysfunctional Protein-Protein Interactions in Disease
Published on: October 24, 2025
Proteomic analysis of p16ink4a-binding proteins
Elielson Souza-Rodrígues1, Josep M Estanyol, Erica Friedrich-Heineken
1Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Universitat de Barcelona, Spain.
Abstract:
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 Sepharose 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 delta accessory protein PCNA and thereby inhibits the polymerase activity.
Insights
The p16(ink4a) tumor suppressor protein interacts with new partners, including PCNA, a key DNA replication factor. This interaction inhibits DNA polymerase activity, revealing novel functions beyond cell cycle control.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The p16(ink4a) protein is a crucial tumor suppressor involved in cell cycle control, tumorogenesis, and senescence.
- Its known function involves inhibiting CDK4 and CDK6 kinases, essential for cell cycle progression.
Purpose of the Study:
- To identify novel p16(ink4a)-interacting proteins and elucidate new functions of p16(ink4a).
Main Methods:
- Affinity chromatography using p16(ink4a) coupled to Sepharose 4B.
- Mass spectrometry (MALDI-TOF) for protein identification.
- Western blotting, immunoprecipitation, and immunofluorescence for validation.
Main Results:
- Thirty-one p16(ink4a)-interacting proteins were identified, grouped into functional clusters.
- Proliferating cell nuclear antigen (PCNA) and minichromosome maintenance protein 6 (MCM6) were identified and validated.
- p16(ink4a) directly interacts with PCNA, inhibiting its DNA polymerase activity.
Conclusions:
- p16(ink4a) possesses novel functions beyond cell cycle regulation, including direct interaction with and inhibition of PCNA's polymerase activity.
- These findings expand our understanding of p16(ink4a)'s role in cellular processes and cancer.
