University of Dundee

BBSRC EASTBIO: Innovative methodologies for the identification of substrates and regulators of protein kinases and phosphatases. Supervisors: Dr G Sapkota; Dr M Delibegovic

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: i. enzyme:substrate interactions are often transient and stimulus dependent; ii. co-precipitation experiments from extracts overlook the sub-cellular context; and iii. 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 substrates. An innovative technology that potentially overcomes these limitations and streamlines the interpretation of phospho-proteomic data is desirable.

Recently, the Sapkota lab has pioneered an Affinity-directed PROtein Missile (AdPROM) system that can target endogenous proteins for functional modulation, such as degradation (1). The system exploits highly selective polypeptide binders (e.g. nanobodies or monobodies) of target proteins tethered to E3 ligase components (e.g. VHL for CUL2 E3 Ligase) for target protein degradation. AdPROM approach can be adapted to identify protein interactors of potentially any endogenous protein by replacing the E3 component 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 (2). 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-and aSHP2-APEX2 AdPROMs 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

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 (3) and plays an important role in tumorigenesis. SHP2 is implicated in Ras/MAPK signalling and thought to be an oncogene. Finally, PFKFB3 is overexpressed in a variety of cancers and is highly phosphorylated in human cancer tissue, however it is unclear if PFKFB3 directly regulates these.

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.

References

1. Fulcher, L. et al (2016) An affinity-directed protein missile system for targeted proteolysis. Open Biol. DOI: 10.1098/rsob.160255.

2. Hung, V., et al (2016) Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456-475.

3. Grant L, et al (2104) 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.

Eligibility