Abstract
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.
Highlights
O-GlcNAc cycles onto cytoplasmic and nuclear serine/ threonine sites through the actions of only two enzymes
These findings suggest the existence of compartment specific dynamics for O-GlcNAc in response to serum stimulation, arguing a role for localization in the regulation of this modification
The change in FRET values observed for the OS2 reporter were 2-fold higher than those seen for the original sensor when subjected to our test conditions, i.e. HeLa cells treated with 100 M PUGNAc and 4 mM glucosamine (15–30% for OS2 compared with 7–15% for O-GlcNAc sensor (OS), Fig. 1B)
Summary
Gene Construction of Improved Sensor and Its Targeted Variants—The improved O-GlcNAc sensor (OS2) was composed of an enhanced cyan fluorescent protein (CFP) fused to the fimbrial adhesin lectin domain GafD [19, 20], a known substrate peptide domain for O-GlcNAc from casein kinase II placed between two flexible linkers (GGSGG) followed by a variant of the yellow fluorescent protein Venus. CFP and FRET images were obtained either every 2 min (HeLa PUGNAc, GlcNH2 experiments) or every 30 s (all Cos experiments) through an S430/25ϫ filter, a 86022v2bs dichroic mirror, and two emission filters (S470/30m and S535/30m), respectively. A YFP image was obtained as a control through S500/20ϫ excitation filter, a 86022v2bs dichroic mirror, and a S535/ 30m emission filter. Changes in the average intensity of YFP between the first and second frame did not allow us to include the initial time of the experiment (t ϭ 0) in the calculation of the FRET response. Statistical Analysis—p values were obtained by a paired Student’s t test (two-tailed) analysis of the normalized average FRET ratios of the OS2 and its control sensor after 30 min of serum treatment. Nuclear and cytoplasmic normalized average FRET ratios of seven cells for each sensor were examined. The GraphPad QuickCalcs Software was utilized to perform the calculations
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