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Writing and erasing O-GlcNAc on casein kinase 2 alpha alters the phosphoproteome

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell, Cell Culture

SUBMITTER: Christina Woo

LAB HEAD: Christina May Woo

PROVIDER: PXD030466 | Pride | 2022-04-26

REPOSITORIES: Pride

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Project description:Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from DSS-induced murine colitis. While tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal Disease Activity Index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation Total RNAs from the colon of three control and three Hdac2 IEC-specific knockout mice were isolated with the Rneasy kit (Qiagen, Mississauga, ON, Canada).

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Writing and erasing O-GlcNAc on casein kinase 2 alpha alters the phosphoproteome

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