Cell signaling through O-GlcNAc reader proteins

Project Summary/Abstract
O-linked b-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification (PTM) in
mammals, decorating thousands of nuclear and cytoplasmic proteins. O-GlcNAc cycling is an essential regulator
of myriad aspects of cell physiology and is dysregulated in numerous human diseases, such as cancer, diabetes
and neurodegeneration. Despite this broad pathophysiological significance, major aspects of O-GlcNAc signaling
remain incompletely understood, including the biochemical mechanisms through which O-GlcNAc transduces
information. At the start of our project, several studies had shown that O-GlcNAcylation mediates protein-protein
interactions (PPIs) in processes as diverse as chromatin remodeling, deubiquitination and nuclear envelope
assembly. These results suggested that O-GlcNAc might signal through conserved modes of PPI. However, little
was known about either the structure or function of these intracellular glycoprotein-protein complexes.
We hypothesized that mammalian “reader” proteins might exist and transduce O-GlcNAc signals. In the
first project period, we devised and deployed a method to identify proteins that bind specifically and directly to O-
GlcNAcylated peptides and proteins in vitro and in human cells. These include the 14-3-3 family, which are well-
known to bind to phosphoproteins. Our results indicate that 14-3-3 proteins may be signal integrators, mediating
the extensively documented crosstalk between O-GlcNAc and O-phosphate, with broad implications for
intracellular signaling. In addition, we identified and characterized O-GlcNAc-mediated PPIs among nuclear import
receptors, intermediate filament (IF) cytoskeleton proteins and ubiquitin E3 ligase complex components. Building
on these results, we will study a focused set of diverse model O-GlcNAc-binding proteins in the next project period.
This strategy will generate new insight into the functions of individual O-GlcNAc-mediated PPIs in particular cell
biological processes, while also providing a comparative perspective across reader proteins, allowing us to discern
general themes of O-GlcNAc-mediated PPIs at the biophysical, biochemical and physiological levels.
The long-term objective of our work is to understand the principles, mechanisms and functions of O-
GlcNAc-mediated PPIs in mammals. In the next project period, we will advance this goal through three Specific
Aims. In Aim 1, we will define the biochemical scope and phenotypic effects of O-GlcNAc binding by 14-3-3
proteins. In Aim 2, we will determine the role of O-GlcNAc/nuclear transport receptor interactions in inter-organelle
trafficking. In Aim 3, we will dissect the functional importance of O-GlcNAc-mediated interactions in two model IF
proteins. We believe our proposed work will significantly advance the field of intracellular signaling both by
elucidating the mechanisms and functions of individual O-GlcNAc-mediated PPIs and by revealing conserved
biochemical principles of O-GlcNAc recognition across reader proteins.

Grant Number: 5R01GM118847-09
NIH Institute/Center: NIH
Principal Investigator: MICHAEL BOYCE