Profiling the O‐GlcNAcylation of the kinome using kinase microarrary (739.8)

Mutations and dysregulation of kinases play causal roles in human disease development, signaling, and metabolism. Understanding the function of kinases is of an outstanding interest for biomarker discovery and also necessary for the development of agonists and antagonists for the use in disease therapy. O‐linked beta‐N‐acetylglucosamine (O‐GlcNAcylation) is a post‐translational modification known to regulate different aspects of a proteins function including localization, activity, and stability. Like phosphorylation, O‐GlcNAcylation, which modifies serine and threonine residues on nuclear and cytoplasmic proteins, is a ubiquitous, reversible process that regulates numerous cellular processes. Recent evidence indicates that site‐specific crosstalk between O‐GlcNAcylation and phosphorylation and the O‐GlcNAcylation of kinases play an important role in regulating cell signaling. Hence, it is very important to investigate the O‐GlcNAcylation of the kinome. Previously, we identified 42 kinases that are substrates for O‐GlcNAcylation using an in vitro OGT assay with [H3] radiolabel on a functional kinase array. However, using [H3] has serve senstitive limiations suggesting a more sensitive approach is needed to probe large libraries of proteins. Herein, using a novel kinase microarray coupled with an in vitro OGT labeling assay with immuno fluorophore detection technique, we report a simple, yet sensitive, strategy by which this method can be used to profile the entire kinome for O‐GlcNAcylated proteins. Grant Funding Source : NIH R01CA42486, R01DK61671; N01‐HV‐00240; P01HL107153, R24DK084949