peptide arrays for kinome analysis: new opportunities and remaining challenges

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  • REVIEW

    Peptide arrays for kinome analysis: New opportunities

    and remaining challenges

    Ryan Arsenault1,2, Philip Griebel2,3 and Scott Napper1,2

    1 Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada2 Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan,

    Canada3 School of Public Health, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

    Received: May 31, 2011

    Revised: September 28, 2011

    Accepted: October 4, 2011

    Phosphorylation is the predominant mechanism of post-translational modification for

    regulation of protein function. With central roles in virtually every cellular process, and

    strong linkages with many diseases, there is a considerable interest in defining, and ulti-

    mately controlling, kinase activities. Investigations of human cellular phosphorylation events,

    which includes over 500 different kinases and tens of thousands of phosphorylation targets,

    represent a daunting challenge for proteomic researchers and cell biologists alike. As such,

    there is a priority to develop tools that enable the evaluation of cellular phosphorylation events

    in a high-throughput, and biologically relevant, fashion. Towards this objective, two distinct,

    but functionally related, experimental approaches have emerged; phosphoproteome investi-

    gations, which focus on the sub-population of proteins which undergo phosphorylation and

    kinome analysis, which considers the activities of the kinase enzymes mediating these

    phosphorylation events. Within kinome analysis, peptide arrays have demonstrated consid-

    erable potential as a cost-effective, high-throughput approach for defining phosphorylation-

    mediated signal transduction activity. In particular, a number of recent advances in the

    application of peptide arrays for kinome analysis have enabled researchers to tackle

    increasingly complex biological problems in a wider range of species. In this review, recent

    advances in kinomic analysis utilizing peptides arrays including several of the biological

    questions studied by our group, as well as outstanding challenges still facing this technology,

    are discussed.

    Keywords:

    Kinase / Kinome / Peptide array / Phosphoproteome / Phosphorylation /

    Protein arrays

    1 Background

    In the late 1950s, Krebs and Fischer were the first to

    describe the role of reversible protein phosphorylation for

    the regulation of enzymatic activity [1, 2]. For this pivotal

    contribution to science they were awarded the Nobel Prize.

    Protein kinases, which catalyze the transfer of the g phos-phate group from ATP to specific serine, threonine or

    tyrosine hydroxyl groups in a target protein substrate, are

    now recognized as one of the largest and most important

    enzyme classes. Consisting of over 500 members, human

    protein kinases are responsible for modifying an estimated

    one-third of the human proteome [3, 4] with many members

    of the proteome undergoing complex patterns of kinase

    modification at multiple sites to generate distinct isoforms

    with unique functional characteristics. The presence and

    dynamic nature of these phosphorylation isoforms adds a

    daunting layer of complexity to characterizing and under-

    standing the proteome. While much is unknown of how

    Colour Online: See the article online to view Figs. 1, 35 in colour.

    Abbreviations: Bregs, regulatory B cells; LPS, lipopolysaccharide;

    ODN, oligodeoxynucleotide; PP, Peyers Patch; TLR, Toll-like

    receptor

    Correspondence: Dr. Scott Napper, Department of Biochemistry,

    University of Saskatchewan, 120 Veterinary Road, University of

    Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3 Canada

    E-mail: scott.napper@usask.ca

    Fax: 11-306-966-7478

    & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

    Proteomics 2011, 11, 45954609 4595DOI 10.1002/pmic.201100296

  • these modifications, even within a static infrastructure of

    proteins, achieve complex biological responses, there is a

    growing appreciation of the importance of kinases in

    controlling cellular responses and of the potential for char-

    acterizations of global kinase activity (the kinome) to offer

    critical insight into biology.

    There is a considerable debate as to the most appropriate

    level at which to define cell responses. Transcriptional

    analysis, based largely on availability and maturity of the

    approach, remains the most widely applied technique for

    global analysis of cellular responses. However, due to a

    multitude of post-transcriptional regulatory events, there are

    concerns that descriptions of patterns of gene expression, no

    matter how comprehensive, do not accurately describe or

    predict cellular phenotypes. Specifically, a major criticism of

    genetic approaches is their inability to consider post-

    transcription regulatory events such as gene silencing,

    mRNA stability, unique translational efficiencies, protein

    turnover, sequestration of enzymes away from substrates,

    and activation and deactivation of proteins by any number of

    post-translational modifications. Intuitively, characteriza-

    tions of host responses that occur closer to the functional

    phenotype should have greater potential to circumvent these

    complicating factors and offer a clearer picture of cellular

    response. From this perspective, protein kinases are at the

    core of signal transduction with central roles in regulation of

    virtually every aspect of cellular behavior. Through their

    ability to modulate protein conformation and functional

    characteristics, kinases control diverse processes such as

    metabolism, transcription, cell cycle progression, cytoskeletal

    rearrangement and cell movement, apoptosis, and differ-

    entiation. As such, characterizations of host cellular

    responses at the level of phosphorylation-mediated signal

    transduction have the potential to offer important, and

    predictive, insight in cellular mechanisms of phenotypes.

    Investigations of cellular response at the level of protein

    phosphorylation are also important and appropriate as the

    disruption of kinase-mediated signaling cascades are asso-

    ciated with a spectrum of diseases including cancer,

    inflammation, neurological disorders and diabetes [5].

    Indeed within the human genome, over 250 protein kinase

    genes map to disease loci [6]. The involvement of kinases in

    disease typically results from improper levels of expression/

    localization/activity or mutations in the protein sequences

    that alter these activities.

    The role of kinases in many diseases, as well as their

    regulatory role in many central pathways, makes them

    logical targets for drug therapy [7]. Fortuitously, the

    conserved catalytic cleft of the kinases is highly attractive for

    drug design making the kinases highly druggable [8].

    Kinase inhibitors have also been proposed as a more precise

    mechanism for therapeutic intervention than other strate-

    gies such as the targeted down-regulation of particular

    genes. Not surprisingly, the central role of kinases in many

    diseases, cancer in particular, and the potential to treat

    complex phenotypes by targeting specific biomolecules,

    have prompted drug companies to invest considerable effort

    into the development of kinase inhibitors. There are esti-

    mates that approximately half of the current Research and

    Development budget of the pharmaceutical industry is

    focused on kinases and their inhibitors. Kinases are the

    most frequently targeted gene class in cancer therapeutics,

    and are second only to G protein-coupled receptors across all

    therapeutic areas [7, 8]. In addition to the immediate value

    of these emerging molecules as therapeutics, these inhibi-

    tors also represent a valuable resource with the potential for

    utilization in research for hypothesis validation. Given the

    magnitude of effort devoted towards their development it is

    certain that additional kinase inhibitors, of greater range

    and improved specificity, will be developed.

    There are a number of licensed, and soon-to-be licensed,

    kinase inhibitors that emphasize the potential of these

    targets. Gleevac (imatinib), a potent inhibitor of the consti-

    tutively active breakpoint cluster region-Abelson murine

    leukemia viral oncogene homolog 1 (BCR-ABL) fusion

    protein, is approved for the treatment of leukemia and

    gastrointestinal stromal tumors [9, 10]. Other protein kinase

    inhibitors, such as the epidermal growth factor receptor

    (EGFR) inhibitors (Tarceva, Genetech) and getinib (Iressa

    AstraZeneca, London UK), have either received FDA

    approval or are in the late stage clinical development to treat

    different cancers [11, 12]. The potential therapeutic value of

    kinase inhibitors is not limited to the treatment of cancers.

    For example, ruboxistaurin to treat diabetic retinopathy,

    safingol, a protein kinase C inhibitor, for treatment of

    atopical dermatitis and fasudil, which has received approval

    in Japan, for treatment of cerebral ischemia. Therapeutic

    modulation of kinase activity can also have anti-inflamma-

    tory and immunosuppressive effects. For example, two

    critical immunosuppressive drugs, cyclosporine A and

    rapamycin, function through modulation of the phosphor-

    ylation s

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