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 (CK2), 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 CK2 to measure the effects on the phosphoproteome in cells. These tools effectively and selectively edit the S347 glycosite on CK2. Using quantitative phosphoproteomics, we report 51 proteins whose enrichment changes as a function of editing O-GlcNAc on CK2, including HDAC1, HDAC2, ENSA, SMARCAD1, and PABPN1. Specific phosphosites on HDAC1 S393 and HDAC2 S394, both reported CK2 substrates, are significantly enhanced by O-GlcNAcylation of CK2. These data will propel future studies on the crosstalk between O-GlcNAc and phosphorylation.