Aberrant signaling by protein tyrosine phosphatases (PTPases) has been identified for multiple cancers. However, dissection of PTPase-mediated signaling pathways that drive oncogenesis is limited by the capabilities of current tools. It remains difficult to manipulate PTPase activity with precise timing and specificity in living cells. Furthermore, targeted manipulation of PTPase activity only in selected protein complexes is currently impossible for the majority of biological studies. To overcome these limitations we propose to employ novel protein engineering strategy that will enable specific activation a PTPase by rapamycin or its non- immunosuppresive analogs. To demonstrate broad applicability of this method for different PTPases we propose to generate engineered PTPases Shp2, PTP1B and RPTP-?. To achieve stimulation of specific pathways downstream of a PTPase we will target activation of Shp2 to different protein complexes with known binding partners. The reagents used in this method are genetically encoded or membrane permeable, enabling ready application in many systems. This method will provide tight temporal and spatial control of PTPase activity. Tight temporal control of engineered Shp2 will allow us to determine signaling events at different time points following activation Shp2. Targeted activation of Shp2 in complexes with it binding partners Gab1 and PZR will allow us to identify signaling pathways specifically mediated by these signaling complexes. Using combination of a recently developed method for BirA fusion protein-based biotinylation (BioID) coupled with proteomics analysis we will determine changes in Shp2-associated protein interactions and accompanied changes in protein phosphorylation.
The proposed project is focused on the development of new tools for targeted manipulation and interrogation of specific signaling pathways mediated by protein tyrosine phosphatases, a class of enzymes often involved in development and progression of human cancers. In particular, the work will enable scientists to turn on phosphatases in specific complexes and with precise timing in living cells. These new approaches will allow scientists to identify biological processes critical for tumorigenesis.
