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 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.
Souza-Rodrígues
Elielson
E
Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Universitat de Barcelona, Spain.
Estanyol
Josep M
JM
Friedrich-Heineken
Erica
E
Olmedo
Eva
E
Vera
Jorge
J
Canela
Nuria
N
Brun
Sonia
S
Agell
Neus
N
Hübscher
Ulrich
U
Bachs
Oriol
O
Jaumot
Montserrat
M
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004258:DNA Polymerase III / Q000037:antagonists & inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005455:Fluorescent Antibody Technique / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism / Q000494:pharmacology; D040901:Proteomics; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014407:Tumor Cells, Cultured
D019941:Cyclin-Dependent Kinase Inhibitor p16; D018809:Proliferating Cell Nuclear Antigen; D004258:DNA Polymerase III