About the Project
Identification of substrates and regulators of protein kinases and phosphatases is pertinent to understanding their function in cells and in therapeutics. Conventional approaches used for identifying substrates exploit affinity-driven co-precipitation from cell extracts, but this approach has limitations: enzyme:substrate interactions are often transient and stimulus dependent; co-precipitation experiments from extracts overlook sub-cellular context; and potential substrates that precipitate in insoluble fractions during lysis are excluded. Recent phospho-proteomic technologies allow identification of global protein phosphorylation profiles upon manipulation of specific kinase and phosphatase activities but even these approaches cannot distinguish between direct and indirect changes in the phospho-proteome landscape. An innovative technology that potentially overcomes the limitations of the conventional co-precipitation techniques and streamlines the interpretation of phospho-proteomic data is desired.
Recently, the Sapkota lab has combined CRISPR/Cas9 genome editing (to introduce GFP tags on target proteins endogenously) and anti-GFP nanobodies (aGFP) tethered to VHL (the substrate adaptor of CUL2 E3 ubiquitin ligase) to achieve near-complete proteolysis of the endogenously GFP-tagged target proteins (Fulcher et al, 2016). This Affinity-directed PROtein Missile (AdPROM) approach can be adapted to identify protein interactors of potentially any endogenous protein, by replacing VHL with a recently engineered ascorbate peroxidase (APEX2) enzyme, which has been adapted to rapidly biotinylate proximal proteins and successfully used to identify proteins in different compartments of mitochondria in mammalian cells (Hung et al, 2015). Briefly, in the presence of biotin-phenol and hydrogen peroxide, APEX2 can rapidly (<1 ms) biotinylate proximal proteins within a 20 nM radius. Biotinylated proteins can be harvested in extreme denaturing lysis conditions and then enriched with streptavidin beads for identification by mass spectrometry. The key objectives of the project will be to: i. generate GFP and APEX2 knockins (and knockouts if feasible) at the endogenous loci of protein tyrosine phosphatases PTP1B & SHP2, and the poorly characterized protein kinase and phosphatase PFKFB3 by CRISPR/Cas9; ii. identify proximal biotinylated substrate proteins for the three targets using aGFP-APEX2 AdPROM and/or endogenously tagged APEX2 and compare interactors using conventional anti-GFP IPs by mass spectrometry; iii. validate associations and study the function of key interactors as either substrates or regulators; and iv. using wild type and knockout cells, establish global phospho-proteomic landscape and compare these with substrates/interactors identified above for streamlining bona fide substrates.
PTP1B and SHP2 are non-receptor tyrosine phosphatases. PTP1B is a critical regulator of cell proliferation, metabolism and survival, and is known to directly regulate insulin receptor and insulin receptor substrates as well as JAK-STAT (Grant et al, 2014) and plays an important role in tumorigenesis. SHP2 is implicated in Ras/MAPK signalling and thought to be an oncogene. Even though a few cellular substrates have been reported for PTB1B and SHP2, these substrates alone cannot fully explain the extensive physiological roles that these phosphatases are known to control. Finally, PFKFB3 is overexpressed in a variety of cancers and is highly phosphorylated in human cancer tissue. It is essential in Ras-dependent glycolysis and transformation and is implicated in a number of metabolic processes; however it is unclear if these are all directly regulated by PFKFB3 or its associated proteins.
The proposed project combines the expertise of the Sapkota lab in CRISPR/Cas9 genome editing, application of AdPROM technology and cutting-edge mass-spectrometry with the expertise of the Delibegovic lab in signalling and physiology associated with PTP1B, SHP2 and PFKB3. The student will initially exploit CRISPR/Cas9 technology to generate appropriate cell lines, develop and optimize APEX2 labelling and pulldown conditions, and then perform mass-spectrometry experiments for substrate identification in the Sapkota lab. The student will then draw on expertise and guidance from the Delibegovic lab to validate and prioritize substrates to undertake signalling and physiological studies. If successful, the technology can be applied to study the substrates and regulators of potentially any protein in cells.
References
References
Fulcher, L. J., Macartney, T., Bozatzi, P., Hornberger, A., Rojas-Fernandez, A., and Sapkota, G. P. (2016) An affinity-directed protein missile system for targeted proteolysis. Open Biol. DOI: 10.1098/rsob.160255
Hung, V., Udeshi, N. D., Lam, S. S., Loh, K. H., Cox, K. J., Pedram, K., Carr, S. A., and Ting, A. Y. (2016) Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456-475
Grant L, Shearer K, Czopek A, Lees E, Owen C, Agouni A, Martin-Granados C, Forrester JV, Wilson HM, Mody N, Delibegovic M. Myeloid-cell protein tyrosine phosphatase-1B deficiency in mice protects against high-fat diet and lipopolysaccharide induced inflammation, hyperinsulinemia and endotoxemia through an IL-10 STAT3 dependent mechanism. Diabetes 2014; 63: 456-470.