Abstract
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.
Highlights
Casein Kinase 1 (CK1) human isoforms - a, c1, c2, c3, d and e - represent a unique group within the superfamily of serine/threonine specific protein kinases that function as monomeric and constitutively active enzymes [1,2]
Ubiquitin and NEDDylation-like modifications were assessed after FLAG-tagged peptide 35 co-transfection with STrEP-tagged Casein Kinase 1 a (CK1a) sp1 or 2 in H1299 cells. This experimental approach revealed significant increase of these post-translational modifications for CK1a splice variant 2 but not sp1 (Figure 10A), emphasizing novel interactions/modifications of CK1a following its release from the E3 ligase murine double minute chromosome 2 (MDM2). These data highlight how an approach to develop a Biologic tool based on a protein-protein interface can give rise to novel information about cellular signalling; in this case we have identified a peptide that can induce growth arrest and reduce cell viability in a p53independent manner, and co-ordinately induce ubiquitination and novel NEDDylation signals to CK1a that might explain in part alterations in growth responses
Previous published data indicated that blocking MDM2 with Nutlin-3a or depleting/ inhibiting CK1a generated the same genetic effects: an increase of p53, MDM2 and p21 protein levels ([16], Figure 1A)
Summary
CK1 human isoforms - a, c1, c2, c3, d and e - represent a unique group within the superfamily of serine/threonine specific protein kinases that function as monomeric and constitutively active enzymes [1,2]. They differ significantly in the length and primary structure of their C-terminal non-catalytic domain which is an extended tail in the case of d/e as opposed to a which has a limited C-terminal domain, but CK1c isoforms on the other hand vary in a longer N-terminal head [3]. A novel stimulator of CK1a has been identified named pyrvinium that opens the door to pharmacological induction of CK1a flux in cells [22,23]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
