Targeted in vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction.
β-O-N-acetyl-D-glucosamine (O-GlcNAc) is a post-translational modification involved in a plethora of biological systems ranging from cellular stress to insulin signaling. This modification shares many hallmarks with phosphorylation, including its dynamic cycling onto a host of proteins such as transcription factors, kinases, and phosphatases, and regulation of cellular functions, including cell signaling. Herein, we report the development of an improved genetically based O-GlcNAc FRET sensor and compartmentalized targeted variants for the characterization of the spatiotemporal dynamics of O-GlcNAc. During serum-stimulated signal transduction, rapid increases in O-GlcNAc activity were observed at both the plasma membrane and the nucleus, with a concomitant decrease detected in the cytoplasm. These findings suggest the existence of compartment specific dynamics for O-GlcNAc in response to signal-inducing stimuli, pointing to complex regulation of this modification. In addition, inhibition of the PI3K pathway by wortmannin abolished the O-GlcNAc response, suggesting that the activity observed is modulated downstream of the PI3K pathway. Taken together, our data argues that O-GlcNAc is a rapidly induced component of signaling and that the interplay between O-GlcNAc and kinase signaling may be more akin to the complex relationship between kinase pathways.
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Luz D
LD
Carrillo
Joshua A
JA
Froemming
Lara K
LK
Mahal
D000117:Acetylglucosamine / Q000235:genetics / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D000077191:Wortmannin
D000117:Acetylglucosamine; D000730:Androstadienes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D000077191:Wortmannin