O-GlcNAc modification of casein kinase 2 alpha alters the phosphoproteome

Abstract

Post-translational modifications govern protein function and are paramount to many signaling networks in cells. How different post-translational modifications intersect to result in complex signaling outcomes represents a fascinating field of study. O-GlcNAc is an essential carbohydrate post-translational modification that intersects with phosphorylation signaling pathways in two major ways – via crosstalk on protein substrates, or by direct modification of the kinases that write the phosphate modification. Disparate cellular pathways are tuned to the nutritional state of the cell through the O-GlcNAc modification by the highly promiscuous enzyme pair that regulates it. In Chapter 1, I discuss the history of the O-GlcNAc modification, methods to manipulate it, and how O-GlcNAc intersects with phosphorylation and with kinases. In Chapter 2, I discuss the role of O-GlcNAc in T cell activation, and efforts to identify the glycosite of tyrosine kinase Zap-70 using site-directed mutagenesis, mass spectrometry, and techniques based in Western blotting to visualize O-GlcNAc stoichiometry. In Chapter 3, I discuss another OGlcNAcylated kinase, casein kinase 2 alpha, the catalytic subunit of the ubiquitously expressed and constitutively active kinase CK2. Here, complementary targeted O-GlcNAc editors, nanobody-OGT and -splitOGA, are applied to selectively write and erase O-GlcNAc from a tagged CK2a. These tools effectively and selectively edit the S347 glycosite on CK2a. Using quantitative phosphoproteomics, we report 51 proteins whose enrichment changes as a function of editing OGlcNAc on CK2a, 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 CK2a. Chapter 4 contains a discussion on how these data will propel future studies on the crosstalk between O-GlcNAc and phosphorylation.