Temporal and Spatial Control of Shp2 Phosphatase Activity through Engineered Allosteric Control

Cell signaling is inherently dynamic with temporally and spatially distinct functions contributing to physiological and pathological processes. As such, sophisticated tools capable of identifying transient or localized signaling events downstream of specific enzymes is essential to understand function on a physiological time scale. We report a broadly applicable chemogenetic protein engineering method for allosteric regulation of Protein Tyrosine Phosphatases as well enhanced tuning of optogenetic tools for allosteric regulation of proteins. Chemogenetic regulation of protein tyrosine phosphatases enable dissection of transient events and reconstruction of individual signaling pathways. With this approach we revealed parallel MAPK and ROCK II dependent pathways downstream of Shp2 mediating transient cell spreading and migration. Furthermore, we show that the N-SH2 domain of Shp2 regulates MAPK-independent, ROCK II-dependent cell migration. Engineered targeting of Shp2 activity to different protein complexes revealed that Shp2-FAK signaling induces cell spreading whereas Shp2-Gab1 or Shp2-Gab2 mediate cell migration. We identified specific transient morphodynamic processes induced by Shp2 and determined the role of individual signaling pathways downstream of Shp2 in regulating these events. The degree of spatial and temporal control of catalytic function can be enhanced through application of an optogenetic allosteric switch. This tool provides the combined advantages of tight spatiotemporal control, broad applicability, and modular tunability due to the design of the light regulated (LightR) switch. The LightR switch is composed of the two VVD domains joined by a flexible linker, modifications in the properties of the VVD domains as well as the flexible linker provide enhanced tuning capabilities for implementation of this regulatory domain. We demonstrated that combining a more rigid inter-VVD linker with lit state stabilizing mutations yielded a robustly activated tool which requires minimal blue light exposure. By contrast, the use of a contractile inter-VVD linkers enhances the activity of weakly activated LightR tools without influencing deactivation kinetics, as was demonstrated with a weakly activated but rapidly deactivated FastLightR-Src tool. Each of these modifications can be combined in a variety of ways to suit the needs of the enzyme and experiment. Both optogenetic and chemogenetic approaches enable broad application in the study of spatiotemporal dependent cell signaling.