Identification and Functional Study of Novel Substrates of GSK-3 Revealed by Quantitative Phosphoproteome Analysis

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase involved in diverse biological functions including glycogen metabolism, protein synthesis, apoptosis, cell cycle, motility, differentiation, embryogenesis and oncogenesis. Dyregulation of GSK-3 has been known to closely associate with some human diseases such as type II diabetes, Alzheimer’s disease and cancer, rendering this kinase as a promising target for therapeutic drug development. Our current understanding of how GSK-3 can execute its biological functions is mainly based on the identification of bona fide substrates of GSK-3 in vivo; GSK-3 can phosphorylate its substrate on specific serine/threonine residue(s), by which the function/stability (for protein substrate) or activity (for enzyme substrate) of substrate protein can be altered in cells. Using traditional biochemical and molecular biology approaches, approximately 90 substrates of GSK-3 have been identified since the discovery of GSK-3 in 1980. It is generally believed that there are still many unknown substrates of GSK-3 waiting for discovery, and that discovery of novel substrates of GSK-3 should provide key clues for exploring the new biological function(s) of GSK-3. To systemically identify novel substrates of GSK-3, we have established a sensitive quantitative phosphoproteomics analysis technology to profile the alterations of proteome and phosphoproteome of HEK293 cells when the cellular GSK-3 activity was selectively inhibited by a pharmacological inhibitor SB216763, allowing us to quantify ~5200 proteins and ~3700/3400 phosphopeptides/phosphosites. Form the two data sets, we can for the first time to uncover dozens of proteins as potential novel substrates of GSK-3. Meanwhile, we have verified one protein, neural precursor cell expressed, developmentally down-regulated gene 4-like (NEDD4L), as a novel substrate of GSK-3 which is phosphorylated at S475 in HEK293 cells. NEDD4L is a newly discovered E3 ligase involved in the ubiquitination of several ion channels, amino acid transporters, and membrane receptors and associated with Liddle’s syndrome (an inherited form of hypertension) and cystic fibrosis-like lung disease. Based on these preliminary but convincing data, this 4-year proposal plans to (1) perform in-depth analysis and verification of the two proteome and phosphoproteome data sets to evaluate the effects of pharmacological inhibition of GSK-3 on the overall signal networks in human cells; (2) investigate the biological meaning of GSK-3-mediated S475 phosphorylation of NEDD4L, including the effects on E3 ligase activity, subcellular location and interactome of NEDD4L, as well as the NEDD4L-mediated substrate ubiquitination/degradation and cell growth control; (3) verify additional 5-10 novel potential GSK-3 substrates (at both protein level and phosphorylation level) predicted from the bioinformatics analysis of substrate specificity of GSK-3. The results obtained from this project are expected to provide a system view of the effect of GSK-3 activity on the signal networks in human cells and shed new light on the biological function(s) of GSK-3 via identification of its novel substrates.

Project ID:PC10401-0173
External Project ID:NSC102-2628-B182-013-MY3