Abstract 2007 Evaluation of the changes in proteome and phosphome upon O-GlcNAc mediated disruption of ERK signaling

Pharmacological and genetic disruptions to O-GlcNAc increase ERK activation, which is involved in the pathogenesis of several diseases like Alzheimer's disease. O-GlcNAc is a single sugar post-translational modification that dynamically cycles on and off proteins in response to nutrient changes by the action of the enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, O-GlcNAc quickly returns to a baseline level after stimulus removal (called O-GlcNAc homeostasis). Disruption of O-GlcNAc homeostasis increases ERK activation via the regulation of upstream kinase MEK and downstream phosphatase DUSP4. However, O-GlcNAc mediated regulation of other upstream kinases like RAS and RAF, and downstream targets like cFOS and MAPKAPK kinases in the ERK signaling cascade is unknown. Hence, to identify the changes in the proteome and phosphome upon O-GlcNAc mediated disruption of ERK signaling, we performed quantitative mass spectrometry. We used shRNAi mediated OGT knockdown (KD) SH- SY5Y neuroblastoma cells harvested at time 0 (control with no ERK activation) and 10 minutes (shows robust ERK activation) after serum stimulation in triplicate. After harvesting and digestion, peptides were labeled with tandem mass tags (TMT) for quantitation. Phosphorylated peptides were enriched via sequential enrichment using metal affinity columns. Mass spectrometry analysis was done using Thermo Finnigan Orbitrap Fusion: Lumos mass spectrometer at the KUMC Proteomics Core. ERK phosphorylation increased, OGT and OGA decreased as expected in the OGT KD samples harvested for mass spectrometry. A total of 9831 high-confidence proteins were identified in the OGT KD samples. Out of these, 492 proteins were kinases including 30 mitogen associated protein kinases (MAPKs) and 181 were phosphatases. Several proteins involved in the ERK pathway including MRAS and KRAS GTPases that activate RAF kinase, and ARAF kinase which activates MEK kinase were up regulated with OGT KD. Phosphatases including DUSP4 and DUSP3 that inactivate ERK were down regulated. Interestingly, MAPKAPK2 – a downstream kinase of ERK increased with OGT KD. The differentially expressed proteins and PTMs will be further analyzed using the AMEND - Active Module identification using Experimental data and Network Diffusion - R package. The AMEND algorithm uses the equivalent change index (ECI) - the weighted ratio of effect sizes between any two experiments (transcriptome, proteome or metabolome) to find a subset of connected molecules in a protein - protein interaction (PPI) network. Next, we will validate the O-GlcNAc mediated changes in proteome and phosphome within the ERK signaling cascade as predicted by the bioinformatic analysis using western blot analysis. Together, these experiments will validate the changes in proteome and phosphome and help to comprehend the molecular mechanism including the upstream regulators and downstream targets of the ERK signaling cascade that are regulated by O-GlcNAc. I would like to thank the Bio-medical Research Training Program (BRTP) Fellowship, Woodyard Fellowship (funding for SJE) and the National Institute of Aging (NIA) (funding for CS). I would also like to thank the KU Endowment, the Biochemistry and Molecular Biology Department and the Institute for Neurological Discoveries of the University of Kansas Medical Center.