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. 2012 Jun 1;422(2):224-8.
doi: 10.1016/j.bbrc.2012.04.124. Epub 2012 Apr 30.

O-GlcNAcylation of kinases

Wagner B Dias  1 , Win D Cheung, Gerald W Hart
Affiliations

O-GlcNAcylation of kinases

Wagner B Dias et al. Biochem Biophys Res Commun. .

Abstract

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. However, relatively few kinases have been analyzed for O-GlcNAcylation. Here, we identify additional kinases that are substrates for O-GlcNAcylation using an in vitro OGT assay on a functional kinase array. Forty-two kinases were O-GlcNAcylated in vitro, representing 39% of the kinases on the array. In addition, we confirmed the in vivo O-GlcNAcylation of three identified kinases. Our results suggest that O-GlcNAcylation may directly regulate a substantial number of kinases and illustrates the increasingly complex relationship between O-GlcNAcylation and phosphorylation in cellular signaling.

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Figures

Figure 1
Figure 1
Strategy to identify O-GlcNAcylation of human kinases using recombinant OGT. (A) All kinases were fused to a carrier protein and present a myc-tag for quantifying protein levels. (B) The protein array containing 152 kinases spotted in quadruplicate was incubated with recombinant human OGT and UDP-[3H]-GlcNAc. After washing, the array was incubated with anti myc-Cy3 antibody to quantify the protein levels and then exposed to a 3H-sensitive storage phosphor screen to quantify the O-GlcNAcylation levels.
Figure 2
Figure 2
OGT modifies several kinases in vitro. The left panel shows the protein levels after scanning the array for Cy3 labeling. The right panel shows the O-GlcNAcylation levels after scanning the array for 3H labeling. The enlarged regions show the O-GlcNAcylation and protein levels of a few of the kinases in detail.
Figure 3
Figure 3
Selected kinases are O-GlcNAcylated in vivo. Lysates from HEK293 cells transfected with empty HA plasmid, ERK5-HA, S6LK1-HA, PKC-ζ-Flag were imunoprecipitated for HA or Flag tags. When indicated, the cells were treated with NAG-thiazole overnight.
See this image and copyright information in PMC

References

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