As phosphorylation represents the pivotal mechanism for regulation of biological processes, kinases belong to one of the most biologically significant enzyme classes. The development of analytical techniques for characterization of kinase activity, in particular at a global scale, is a central priority for proteomic and cell biology researchers. In order to facilitate global analysis of cellular phosphorylation, a new paradigm of microarray technology which focuses on analysis of total cellular kinase activity, kinome, has emerged in the past few years. As the specificity of many kinases is dictated primarily by recognition of residues immediately surrounding the site of phosphorylation a logical methodology is to employ peptides representing these immediate sequences as experimental substrates. Microarray chips carrying hundreds of such substrate targets have been developed for human kinome analysis, however, lack of similar tools for species outside research mainstream has limited kinome analysis in these species.<p> Based on sequence alignment of orthologous phosphoproteins from mammalian species, conservation of amino acid identity is reported to be 80 %. Accordingly, the potential exists to utilize phosphorylation sequence databases to extrapolate phosphorylation sites in other species based on their genomic sequence information. Peptides representing these proposed phosphorylation sites can then be utilized as substrates to quantify the activity of the corresponding kinase. Based on these principles, a bovine microarray of 300 unique peptide targets was constructed. The bovine phosphorylation targets were selected to represent a spectrum of cellular events but with focus on processes related to innate immunity.
Initial application and validation of the bovine peptide arrays was carried out for kinome analysis of bovine blood monocytes stimulated with either lipopolysaccharide (LPS) or CpG-ODNs; ligands for Toll-like receptors (TLR) 4 and 9, respectively. The arrays confirmed activation of the known TLR signaling pathway as well as identifying receptor-specific phosphorylation events. Phosphorylation events not previously attributed to TLR activation were also identified and validated by independent bioassays. This investigation offers insight into the complexity of TLR signaling and more importantly verifies the potential to use bioinformatics approaches to create tools for species-specific kinome analysis based on genomic information.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-09152008-153026 |
| Date | 22 September 2008 |
| Creators | Jalal, Shakiba |
| Contributors | Napper, Scott, Misra, Vikram, Khandelwal, Ramji L., Griebel, Philip J., Geyer, C. Ronald, Warrington, Rob C. |
| Publisher | University of Saskatchewan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-09152008-153026/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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