AbstractThe discovery of Cyclin-dependent 5 (Cdk5) added a new member to the mammalian Cdks, appearing uncharacteristic of its family members despite strong sequence homology. In contrast to the mitotic Cdks, Cdk5 exerts no apparent role in cell cycle regulation therefore its potential role in cancer is subject to much controversy.
Cdk5 is heavily depicted as a neuronal-specific kinase, due to its association with neuronal activators, and dysregulation of Cdk5 activity is thought to contribute to the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease. Nonetheless, the physiological functions of this kinase are being complicated by an upsurge in potential substrates that implicate Cdk5 activity in extra-neuronal functions and diseases such as cancer. However, the predicted substrates fail to meet a general consensus sequence and are yet to be fully validated in vivo. Furthermore, the involvement of Cdk5 in pathophysiological pathways is only partially understood. Adding further complexity, truncation of the Cdk5 cofactor, p35 is thought to increase the catalytic ability of the Cdk5 complex and alter its substrate specificity profile, although little evidence has been provided to support this claim.
In the present study, in vitro peptide analysis was performed in an attempt to identify a consensus sequence that regulates Cdk5 substrate phosphorylation, as a consensus for Cdk5 substrate selection is generally considered degenerative. Despite this theory, Cdk5 displayed high peptide selectivity that was shown to be distinct from other members of the CMGC family of proteins to which it belongs. While Cdk5 showed a preference for basic residues, interestingly, it was the presence of a proline residue located N-terminally (-2) relative to the phosphoacceptor residue that appeared to dictate substrate phosphorylation. Further in vitro analysis using full-length proteins confirmed a correlation between substrates possessing the optimal consensus residues and substrate phosphorylation; those containing these residues were phosphorylated to a greater extent in vitro compared to those lacking these residues. Furthermore, both Cdk5 complexes displayed similar substrate profiles and inherent catalytic ability, as determined by comparison of phosphorylation kinetic parameters between complexes, despite the notion of a hyperactive complex with altered substrate specificity following activator truncation.
Following manipulation of Cdk5 activity in cell lines and intact tissue, tau and CRMP2 were confirmed as Cdk5 targets. Subsequently, a panel of total and phospho-specific antibodies to each substrate was assessed for compatibility with the immunohistochemistry protocol to serve as a surrogate for examining Cdk5 activity in tumours. For the first time, this study presents abnormal CRMP2 phosphorylation at a Cdk5 sensitive site in the nuclei of lymphoma, lung, and breast tumour cells, implicating Cdk5 activity in tumourigenesis. Furthermore, this is the first implication of long-form CRMP2 (CRMP2A) in the nucleus and potentially the isoform associated with cancer.
Overall, the data presented in this study provides a platform in which to improve mechanistic insight into tumourigenesis in specific forms of cancer, which will potentially provide new diagnostic and therapeutic strategies for cancer in the near future.
|Date of Award||2014|
|Supervisor||Calum Sutherland (Supervisor)|