Novel peptide-based approaches to study the activity and substrate specificity of protein kinases

Protein phosphorylation, catalyzed by protein kinases, is the predominant post-translational modification made to proteins. This process is readily reversible, with protein phosphatases catalyzing removal of the installed phosphate groups. Together, protein kinases and phosphatases modulate the structure and activity of many cellular proteins, providing exquisite regulation of many physiological processes. In fact, phosphorylation/dephosphorylation is the predominant mechanism via which cellular proteins are regulated, with the activities of about 30% of all proteins encoded in the human genome controlled by this process. It is not surprising, therefore, that aberrant activation and inactivation of protein kinases have been found to contribute to the pathogenesis and the progression of many diseases, such as cancers and neurodegenerative diseases. This thesis sought to develop new peptide-based approaches to studying the activity and other properties of kinases, with an ultimate view towards improving our understanding of the mechanism of aberrant regulation of protein kinases and how dys-regulated protein kinases direct disease formation and progression, as well as providing screening tools for identifying new kinase inhibitors. The first part of this work saw the successful development of peptide-based chemosensors for monitoring the activity of Src-family kinases (SFKs) in vitro and in lysates of cancer cells and primary mammalian cells via fluorescence. Click chemistry was first used to install a series of fluorophores into the optimal phosphorylation sequence of SFKs and then experiments conducted to establish which fluorophores would respond the phosphorylation state of the peptide. Two fluorophores – pyrene and naphthalimide – were found to provide an increase in fluorescence upon phosphorylation, enabling SFK activity to be conveniently measured in real-time. One of the chemosensors, pyrene Src optimal peptide, was successfully employed to monitor the activity of endogenous SFKs expressed in chronic myelogenous leukemia (CML) cell lines and human platelets. In an attempt to develop a kinase assay more suitable for high-throughput screening applications, a number of new assay designs based on the use of luminescent lanthanide complexes were also explored. The simplest of these exploited the high affinity of a specially designed terbium complex for phosphorylated tyrosine residues. Upon phosphorylation of a tyrosine kinase peptide substrate (Src optimal peptide), the complex was found to bind to the peptide and undergo sensitization via the phosphotyrosine, leading to an increase in luminescence and thus providing a direct measure of kinase activity. To overcome the problem of competitive binding by ATP, however, it was found to be necessary to add a large excess of the complex, which led to a high degree of enzyme “poisoning” if attempts were made to monitor enzyme activity in real-time. Instead, efforts focused on the development of a two-step assay format, with the complex added only after a specified incubation period. To overcome the ATP out-competition issue, the use of a biotinylated peptide substrate with streptavidin plates was also explored. Whilst this allowed for a washing step to remove ATP at the end of the incubation period, the loading of peptide proved insufficient to provide a measurable change in luminescence upon subsequent addition of complex to the plate. A biotinylated Multiple Antigenic Peptide System (MAPS)-style substrate featuring four linked peptides was therefore synthesized, with the ultimate goal of providing an enhanced peptide loading. Studies revealed that this substrate was more efficiently phosphorylated than a single copy of the Src optimal peptide, presumably because phosphorylation of one of the tyrosine residues within the structure provides a “docking” site for SFKs, which are then able to phosphorylate the remaining tyrosines more readily. However, further studies are required to investigate the use of this substrate in combination with streptavidin plates to see if a functioning two-step assay can be developed. A somewhat more sophisticated and generally applicable kinase assay was successfully developed by employing a phosphate-binding luminescent europium complex in combination with kinase peptide substrates bearing a fluorophore (ATTO-647) capable of acting as an “acceptor” for the energy of the europium excited state. Upon phosphorylation, the europium complex was found to bind phosphorylated residues, leading to enhanced emission from the ATTO-647 fluorophore after excitation of the europium complex followed by Luminescence Resonance Energy Transfer (LRET). This method could be used to monitor both the activity of a tyrosine kinase (SFK) as well as a serine kinase (cAMP-dependent protein kinase; PKA), although the limited water-solubility of the europium complex required the use of 20% DMSO solution, which impacted on enzyme activity. Future work will therefore need to focus on the development of a water-soluble analogue more suited to biological applications. A further aspect of this thesis was concerned with the development of an optimal peptide substrate sequence for the tyrosine kinase, C-terminal Src kinase homologous kinase (CHK). In addition to phosphorylating SFKs, CHK is known to perform other non-SFK-related cellular functions. Identifying the protein substrates of CHK that mediate its non-SFK function is an avenue to elucidate the molecular basis of the non-SFK function of CHK. In this project, a combinatorial peptide library approach was used to define the optimal phosphorylation sequence recognized by the active site of CHK. A synthetic peptide referred to as CHK optimal peptide, modeled after this optimal phosphorylation sequence, was phosphorylated by CHK with high efficiency and specificity. Finally, a peptide-based approach was used to investigate a unique post-translational modification of Src, a member of SFK in neurons. In neurons that undergo excitotoxic neuronal death due to overstimulation of glutamate receptors, Src kinase was found to be cleaved by a calcium-activated protease calpain. In this study, the calpain cleavage site in Src kinase was mapped and a cell-permeable peptide substrate corresponding to this region was demonstrated to have the ability to block calpain cleavage and prevent excitotoxic neuronal death. This cell-permeable peptide could provide the basis of a potential new therapy to prevent excitotoxic neuronal death occurring in ischemic stroke.

Author dc:creator

• Kamaruddin, Mohd Aizuddin

• protein kinases
• fluorescence
• phosphorescence
• lanthanides
• Src
• CHK
• excitotoxicity

Language dc:language

eng

Handle dc:identifier.uri

http://hdl.handle.net/11343/38062

OAI identifier oai:identifier

oai:jupiter.its.unimelb.edu.au:11343/38062