Evidence for Direct Cross-Talk between Two Essential Nutrient-Sensing Enzymes

O-linked b-N-acetylglucosamine (O-GlcNAc) is a highly dynamic Ser/Thr specific post-translational modification of cytoplasmic and nuclear proteins, and is regulated by the concerted actions of two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation is a nutrient-sensitive process involved in the regulation of various signaling pathways mediating transcription, cellular metabolism, growth and proliferation. Activation of AMP-activated kinase (AMPK), a vital monitor of cellular energy homeostasis, is also a nutrient-sensitive process that regulates cellular metabolism, growth and proliferation. AMPK and OGT share many downstream targets with the net effect to prolong cell viability under metabolic stress, suggesting significant overlap in both upstream and downstream regulation of AMPK and OGT signaling pathways. Despite this, no studies have investigated whether AMPK and OGT are capable of directly regulating each other. We demonstrate that acute inhibition of O-GlcNAc cycling disrupts pharmacological and physiological activation of AMPK and that both alpha (catalytic) and all 3 gamma (AMP/ADP-sensing regulatory) subunits of AMPK are O-GlcNAcylated. Furthermore, O-GlcNAcylation of the gamma1 subunit directly correlates with both the time and extent of AMPK activation. To further assess the possibility of cross-talk between OGT and AMPK, we demonstrate that acute pharmacological activation of AMPK increases nuclear localization of OGT and alters OGT's substrate selectivity. AMPK also phosphorylates OGT on Thr 444 in vitro; a residue that lies in close proximity to regions that may facilitate OGT's nuclear localization and substrate selectivity. Collectively, these results demonstrate significant cross-talk between two essential nutrient sensing processes, possibly via direct signaling between AMPK and OGT. (Supported by NIH R01 DK61671, R01 CA42486 and R24 DK084949. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHU).