Peptide arrays for kinome analysis: New opportunities and remaining challenges

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<ul><li><p>REVIEW</p><p>Peptide arrays for kinome analysis: New opportunities</p><p>and remaining challenges</p><p>Ryan Arsenault1,2, Philip Griebel2,3 and Scott Napper1,2</p><p>1 Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada2 Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan,</p><p>Canada3 School of Public Health, University of Saskatchewan, Saskatoon, Saskatchewan, Canada</p><p>Received: May 31, 2011</p><p>Revised: September 28, 2011</p><p>Accepted: October 4, 2011</p><p>Phosphorylation is the predominant mechanism of post-translational modification for</p><p>regulation of protein function. With central roles in virtually every cellular process, and</p><p>strong linkages with many diseases, there is a considerable interest in defining, and ulti-</p><p>mately controlling, kinase activities. Investigations of human cellular phosphorylation events,</p><p>which includes over 500 different kinases and tens of thousands of phosphorylation targets,</p><p>represent a daunting challenge for proteomic researchers and cell biologists alike. As such,</p><p>there is a priority to develop tools that enable the evaluation of cellular phosphorylation events</p><p>in a high-throughput, and biologically relevant, fashion. Towards this objective, two distinct,</p><p>but functionally related, experimental approaches have emerged; phosphoproteome investi-</p><p>gations, which focus on the sub-population of proteins which undergo phosphorylation and</p><p>kinome analysis, which considers the activities of the kinase enzymes mediating these</p><p>phosphorylation events. Within kinome analysis, peptide arrays have demonstrated consid-</p><p>erable potential as a cost-effective, high-throughput approach for defining phosphorylation-</p><p>mediated signal transduction activity. In particular, a number of recent advances in the</p><p>application of peptide arrays for kinome analysis have enabled researchers to tackle</p><p>increasingly complex biological problems in a wider range of species. In this review, recent</p><p>advances in kinomic analysis utilizing peptides arrays including several of the biological</p><p>questions studied by our group, as well as outstanding challenges still facing this technology,</p><p>are discussed.</p><p>Keywords:</p><p>Kinase / Kinome / Peptide array / Phosphoproteome / Phosphorylation /</p><p>Protein arrays</p><p>1 Background</p><p>In the late 1950s, Krebs and Fischer were the first to</p><p>describe the role of reversible protein phosphorylation for</p><p>the regulation of enzymatic activity [1, 2]. For this pivotal</p><p>contribution to science they were awarded the Nobel Prize.</p><p>Protein kinases, which catalyze the transfer of the g phos-phate group from ATP to specific serine, threonine or</p><p>tyrosine hydroxyl groups in a target protein substrate, are</p><p>now recognized as one of the largest and most important</p><p>enzyme classes. Consisting of over 500 members, human</p><p>protein kinases are responsible for modifying an estimated</p><p>one-third of the human proteome [3, 4] with many members</p><p>of the proteome undergoing complex patterns of kinase</p><p>modification at multiple sites to generate distinct isoforms</p><p>with unique functional characteristics. The presence and</p><p>dynamic nature of these phosphorylation isoforms adds a</p><p>daunting layer of complexity to characterizing and under-</p><p>standing the proteome. While much is unknown of how</p><p>Colour Online: See the article online to view Figs. 1, 35 in colour.</p><p>Abbreviations: Bregs, regulatory B cells; LPS, lipopolysaccharide;</p><p>ODN, oligodeoxynucleotide; PP, Peyers Patch; TLR, Toll-like</p><p>receptor</p><p>Correspondence: Dr. Scott Napper, Department of Biochemistry,</p><p>University of Saskatchewan, 120 Veterinary Road, University of</p><p>Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3 Canada</p><p>E-mail: scott.napper@usask.ca</p><p>Fax: 11-306-966-7478</p><p>&amp; 2011 WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheim www.proteomics-journal.com</p><p>Proteomics 2011, 11, 45954609 4595DOI 10.1002/pmic.201100296</p></li><li><p>these modifications, even within a static infrastructure of</p><p>proteins, achieve complex biological responses, there is a</p><p>growing appreciation of the importance of kinases in</p><p>controlling cellular responses and of the potential for char-</p><p>acterizations of global kinase activity (the kinome) to offer</p><p>critical insight into biology.</p><p>There is a considerable debate as to the most appropriate</p><p>level at which to define cell responses. Transcriptional</p><p>analysis, based largely on availability and maturity of the</p><p>approach, remains the most widely applied technique for</p><p>global analysis of cellular responses. However, due to a</p><p>multitude of post-transcriptional regulatory events, there are</p><p>concerns that descriptions of patterns of gene expression, no</p><p>matter how comprehensive, do not accurately describe or</p><p>predict cellular phenotypes. Specifically, a major criticism of</p><p>genetic approaches is their inability to consider post-</p><p>transcription regulatory events such as gene silencing,</p><p>mRNA stability, unique translational efficiencies, protein</p><p>turnover, sequestration of enzymes away from substrates,</p><p>and activation and deactivation of proteins by any number of</p><p>post-translational modifications. Intuitively, characteriza-</p><p>tions of host responses that occur closer to the functional</p><p>phenotype should have greater potential to circumvent these</p><p>complicating factors and offer a clearer picture of cellular</p><p>response. From this perspective, protein kinases are at the</p><p>core of signal transduction with central roles in regulation of</p><p>virtually every aspect of cellular behavior. Through their</p><p>ability to modulate protein conformation and functional</p><p>characteristics, kinases control diverse processes such as</p><p>metabolism, transcription, cell cycle progression, cytoskeletal</p><p>rearrangement and cell movement, apoptosis, and differ-</p><p>entiation. As such, characterizations of host cellular</p><p>responses at the level of phosphorylation-mediated signal</p><p>transduction have the potential to offer important, and</p><p>predictive, insight in cellular mechanisms of phenotypes.</p><p>Investigations of cellular response at the level of protein</p><p>phosphorylation are also important and appropriate as the</p><p>disruption of kinase-mediated signaling cascades are asso-</p><p>ciated with a spectrum of diseases including cancer,</p><p>inflammation, neurological disorders and diabetes [5].</p><p>Indeed within the human genome, over 250 protein kinase</p><p>genes map to disease loci [6]. The involvement of kinases in</p><p>disease typically results from improper levels of expression/</p><p>localization/activity or mutations in the protein sequences</p><p>that alter these activities.</p><p>The role of kinases in many diseases, as well as their</p><p>regulatory role in many central pathways, makes them</p><p>logical targets for drug therapy [7]. Fortuitously, the</p><p>conserved catalytic cleft of the kinases is highly attractive for</p><p>drug design making the kinases highly druggable [8].</p><p>Kinase inhibitors have also been proposed as a more precise</p><p>mechanism for therapeutic intervention than other strate-</p><p>gies such as the targeted down-regulation of particular</p><p>genes. Not surprisingly, the central role of kinases in many</p><p>diseases, cancer in particular, and the potential to treat</p><p>complex phenotypes by targeting specific biomolecules,</p><p>have prompted drug companies to invest considerable effort</p><p>into the development of kinase inhibitors. There are esti-</p><p>mates that approximately half of the current Research and</p><p>Development budget of the pharmaceutical industry is</p><p>focused on kinases and their inhibitors. Kinases are the</p><p>most frequently targeted gene class in cancer therapeutics,</p><p>and are second only to G protein-coupled receptors across all</p><p>therapeutic areas [7, 8]. In addition to the immediate value</p><p>of these emerging molecules as therapeutics, these inhibi-</p><p>tors also represent a valuable resource with the potential for</p><p>utilization in research for hypothesis validation. Given the</p><p>magnitude of effort devoted towards their development it is</p><p>certain that additional kinase inhibitors, of greater range</p><p>and improved specificity, will be developed.</p><p>There are a number of licensed, and soon-to-be licensed,</p><p>kinase inhibitors that emphasize the potential of these</p><p>targets. Gleevac (imatinib), a potent inhibitor of the consti-</p><p>tutively active breakpoint cluster region-Abelson murine</p><p>leukemia viral oncogene homolog 1 (BCR-ABL) fusion</p><p>protein, is approved for the treatment of leukemia and</p><p>gastrointestinal stromal tumors [9, 10]. Other protein kinase</p><p>inhibitors, such as the epidermal growth factor receptor</p><p>(EGFR) inhibitors (Tarceva, Genetech) and getinib (Iressa</p><p>AstraZeneca, London UK), have either received FDA</p><p>approval or are in the late stage clinical development to treat</p><p>different cancers [11, 12]. The potential therapeutic value of</p><p>kinase inhibitors is not limited to the treatment of cancers.</p><p>For example, ruboxistaurin to treat diabetic retinopathy,</p><p>safingol, a protein kinase C inhibitor, for treatment of</p><p>atopical dermatitis and fasudil, which has received approval</p><p>in Japan, for treatment of cerebral ischemia. Therapeutic</p><p>modulation of kinase activity can also have anti-inflamma-</p><p>tory and immunosuppressive effects. For example, two</p><p>critical immunosuppressive drugs, cyclosporine A and</p><p>rapamycin, function through modulation of the phosphor-</p><p>ylation status of the cell; cyclosporine A through inhibition</p><p>of a phosphatase [13] and rapamycin through inhibition of a</p><p>kinase [14]. Other anti-inflammatory drugs that suppress</p><p>tumor necrosis factor (TNF)-a and interleukin (IL)-1bexpression also function through kinase inhibition [15].</p><p>1.1 Phosphoproteome and kinome analysis</p><p>A number of experimental approaches are available for the</p><p>analysis of phosphorylation based cellular signaling, these</p><p>can be divided into two groups, phosphoproteome and</p><p>kinome analysis [16]. The difference is dependent on</p><p>whether the consideration is on the protein kinases that</p><p>phosphorylate proteins, the kinome, or the targets of these</p><p>enzymes, the phosphoproteome. While these types of</p><p>analysis are strongly linked, representing the same biologi-</p><p>cal phenomena, and are at times considered interchange-</p><p>able, the experimental approaches are distinct. We suggest a</p><p>strict delineation where phosphoproteome analysis consid-</p><p>ers only the proteins containing phosphoryl groups and the</p><p>4596 R. Arsenault et al. Proteomics 2011, 11, 45954609</p><p>&amp; 2011 WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheim www.proteomics-journal.com</p></li><li><p>kinome refers to the enzymatic activities of the kinase</p><p>compliment of a cell, regardless of substrate target. This</p><p>distinction between the two should not be exaggerated,</p><p>however, as the phosphoproteome represents the net action</p><p>of the kinome, as well as that of active phosphatases.</p><p>Phosphoproteome analysis seeks to define the sub-</p><p>population of the proteome with respect to the identities and</p><p>points of phosphorylation. Phosphoproteome analysis</p><p>shares the same challenges that are associated with standard</p><p>proteomic analysis but is complicated experimentally by: the</p><p>relative scarcity of phosphoproteome members, the dynamic</p><p>nature of protein phosphorylation and the proteome size</p><p>overwhelming, or suppressing, the ability to detect the</p><p>phosphoproteome [1719]. The small concentration of</p><p>phosphoproteins relative to the entire proteome is a signif-</p><p>icant challenge in phosphoproteome analysis. While a large</p><p>fraction of the proteome undergoes phosphorylation, many</p><p>of the proteins involved in signal transduction are expressed</p><p>at very low levels. Thus, the proteins of most interest when</p><p>studying cellular signaling are those that are the most</p><p>difficult to isolate. This is exacerbated by the fact that these</p><p>proteins, in addition to being found in low abundance, are</p><p>often phosphorylated at sub-stoichiometric levels. This</p><p>means that a small fraction of a given signaling protein is</p><p>phosphorylated at any one time; only 12% of the total</p><p>individual protein compliment of a cell is found in the</p><p>phosphorylated form [1619]. When considering all these</p><p>experimental limitations, a promising alternative for char-</p><p>acterization of cellular phosphorylation is to focus instead</p><p>on the kinome. Investigations of enzymatic activities offer</p><p>greater potential for targeted, and perhaps mostly impor-</p><p>tantly, sensitive, analysis. Specifically, the well-defined and</p><p>highly conserved chemistry of enzymatic phosphorylation</p><p>permits rapid characterization of kinase activity, provided an</p><p>appropriate substrate is available.</p><p>1.2 Kinome Analysis through peptide arrays</p><p>A central obstacle for global kinome analysis is the nature of</p><p>the substrates to be employed. While proteins are the</p><p>physiological substrates for the kinases they are problematic</p><p>to mass produce and relatively unstable on array format. An</p><p>alternative is to use peptides that represent sequences</p><p>surrounding a site of phosphorylation. Many protein kina-</p><p>ses recognize phosphoacceptor sites determined by residues</p><p>surrounding the phosphorylated amino acid, as opposed to</p><p>higher order secondary or tertiary structures. Specifically,</p><p>the target specificity of many kinases is a function of the</p><p>residues in the 14 and 4 flanking positions of the phos-phoacceptor site [20]. Synthetic peptides modeled on the site</p><p>of phosphorylation have been shown to be appropriate</p><p>substrates with Vmax and Km values approaching that of theintact protein [21]. Relative to the complete protein, peptides</p><p>are easily synthesized, inexpensive, highly stable and</p><p>amenable to array technology [22]. Construction of arrays</p><p>representing hundreds to thousands of immobilized</p><p>peptides allows the profiling of cellular signaling activities</p><p>by determining the activities of hundreds of kinases in a</p><p>single experiment.</p><p>The basic premise of kinome analysis through peptide</p><p>arrays is that peptides representing the phosphorylation</p><p>target sites of proteins are synthesized and spotted onto an</p><p>array surface [23]. Detailed review of the commonly</p><p>employed methodologies for peptide synthesis and array</p><p>spotting are available elsewhere [16]. Following production</p><p>of the array a sample containing active kinase, or kinases,</p><p>such as a cellular lysate, is applied for the array and these</p><p>enzymes phosphorylate their respective target sequences</p><p>using ATP as the phosphate source. This phosphorylation</p><p>event is visualized using one of a number of methods</p><p>including phosphorylation-specific antibodies, radioactivity</p><p>or phospho-specific stains (Fig. 1). Quantification of the</p><p>extent of phosphorylation of a given peptide by lysates</p><p>representing different experimental conditions (control</p><p>versus for treatment) enable evaluation of relative kinase</p><p>activity. It is also possible to infer the extent of cellular</p><p>phosphorylation of the protein that is represented by the</p><p>peptide.</p><p>A large number of peptide arrays for kinome analysis are</p><p>commercially available. These arrays range in size from</p><p>dozens to thousands of peptides, representing defined</p><p>phosphorylation sites. A partial listing of the peptide arrays</p><p>that are currently available for kinome analysis is presented</p><p>in (Table 1).</p><p>2 Success stories</p><p>While still an emerging technology, the literature contains</p><p>numerous examples of the successful application of peptide</p><p>arrays for kinome analysis. The following studies carried out</p><p>by...</p></li></ul>

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