O-GlcNAc modification of casein kinase 2 alpha alters the phosphoproteome
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Schwein, Paul Andrew
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Schwein, Paul Andrew. 2022. O-GlcNAc modification of casein kinase 2 alpha alters the phosphoproteome. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.Abstract
Post-translational modifications govern protein function and are paramount to manysignaling 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.
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