Writing and Erasing O-GlcNAc on Casein Kinase 2 Alpha Alters the Phosphoproteome.

O-GlcNAc is an essential carbohydrate modification that intersects with phosphorylation signaling pathways via crosstalk on protein substrates or by direct modification of the kinases that write the phosphate modification. Casein kinase 2 alpha (CK2α), the catalytic subunit of the ubiquitously expressed and constitutively active kinase CK2, is modified by O-GlcNAc, but the effect of this modification on the phosphoproteome in cells is unknown. Here, we apply complementary targeted O-GlcNAc editors, nanobody-OGT and -splitOGA, to selectively write and erase O-GlcNAc from a tagged CK2α to measure the effects on the phosphoproteome in cells. These tools effectively and selectively edit the Ser347 glycosite on CK2α. Using quantitative phosphoproteomics, we report 51 phosphoproteins whose enrichment changes as a function of editing O-GlcNAc on CK2α, including HDAC1, HDAC2, ENSA, SMARCAD1, and PABPN1. Specific phosphosites on HDAC1 Ser393 and HDAC2 Ser394, both reported CK2 substrates, are significantly enhanced by O-GlcNAcylation of CK2α. These data will propel future studies on the crosstalk between O-GlcNAc and phosphorylation.

The O-GlcNAc Modification on Kinases.

O-Linked N-acetyl glucosamine (O-GlcNAc) is a protein modification found on thousands of nuclear, cytosolic, and mitochondrial proteins. Many O-GlcNAc sites occur in proximity to protein sites that are likewise modified by phosphorylation. While several studies have uncovered crosstalk between these two signaling modifications on individual proteins and pathways, an understanding of the role of O-GlcNAc in regulating kinases, the enzymes that install the phosphate modification, is still emerging. Here we review recent methods to profile the O-GlcNAc modification on a global scale that have revealed more than 100 kinases are modified by O-GlcNAc and highlight existing studies about regulation of these kinases by O-GlcNAc. Continuing efforts to profile the O-GlcNAc proteome and understand the role of O-GlcNAc on kinases will reveal new mechanisms of regulation and potential avenues for manipulation of the signaling mechanisms at the intersection of O-GlcNAc and phosphorylation.