Peptide arrays for kinome analysis: New opportunities and remaining challenges

Abstract

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 ultimately 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 investigations, 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 considerable 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.

Tumor phosphatidylinositol 3-kinase signaling in therapy resistance and metastatic dissemination of rectal cancer: Opportunities for signaling-adapted therapies

“…Ideally, to become clinically useful, technologies must provide rapid and high-fidelity analysis of small-quantity samples. Kinase substrate array technologies are high-throughput tools for global profiling of kinase activities in tissue samples without prior knowledge of which signaling pathways are activated [33][34][35][36]. Using such arrays, each tumor sample will generate an individual phosphosubstrate signature, theoretically portraying the state of composite information flow through signaling cascades.…”

Divergent Immune Responses to Mycobacterium avium subsp. paratuberculosis Infection Correlate with Kinome Responses at the Site of Intestinal Infection

“…Monitoring global responses at the level of cellular kinase ac-tivity (the kinome) is an effective approach to understand complex biology as well as to identify therapeutic targets and biomarkers (10). Many methods have been used to assay kinase activity under different conditions, each with advantages and disadvantages (11).…”

Dynamic Protein Pathway Activation Mapping of Adipose-Derived Stem Cell Differentiation Implicates Novel Regulators of Adipocyte Differentiation

“…One study utilizing human primary preadipocytes, with a comparable amount of inhibitor applied, noted a complete disruption of differentiation (13). An additional study examining 3T3-L1 cells treated with rapamycin demonstrated a substantial decrease in lipid formation and moreover, a reduction in the gene expression of the adipocytic markers PPAR␥, adipsin, adducin 1/sterol regulatory element binding transcription factor 1 (ADD1/SREBP1c), and TNF receptor superfamily, member 6 (FAS) (30).…”

“…These reversible kinasedriven phosphorylation events alter protein confirmation, ultimately affecting enzymatic activity and protein-protein in-teractions leading to an array of cellular events from differentiation to gene expression, thus encompassing signal transduction. Characterization of this broad-scale signaling architecture is necessary to provide a more finite depiction of the complex signaling events directing a given cellular phenotype and aid in the understanding of the repercussions of alterations in regulation (13,14). Presently, phosphoproteomic analysis of cellular differentiation processes such as with mass spectrometry has been performed in a very limited number of independent time points that span the cellular differentiation process, and important dynamic changes in the cellular signaling architecture may have been missed (12,15,16).…”