Exploiting the MDM2-CK1α protein-protein interface to develop novel biologics that induce UBL-kinase-modification and inhibit cell growth.
Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α) forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2) oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i) ELISA with recombinant MDM2; (ii) cell lysate pull-down towards endogenous MDM2; (iii) MDM2-CK1α complex-based competition ELISA; and (iv) MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i) function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii) be used as a tool to study NEDDylation of CK1α, and (iii) reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross-talk between NEDDylation, protein kinase signalling, and cell survival.
p53 Signal Transduction Group, Edinburgh Cancer Research Centre in the Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.
Anne-Sophie
AS
Huart
Nicola J
NJ
MacLaine
Vikram
V
Narayan
Ted R
TR
Hupp
D015153:Blotting, Western; D048128:Casein Kinase Ialpha / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology
D034741:RNA, Small Interfering; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D048128:Casein Kinase Ialpha