bioRxiv2021
Activation of receptor tyrosine kinases (RTKs) promotes the intracellular assembly of protein complexes formed through phosphotyrosine-SH2 domain linkages. These interactions are weak and reversible, and the dephosphorylation of uncomplexed phosphorylated proteins can be rapid enough to reduce the time and length scales of protein complex persistence distal from the receptor. This limitation may be overcome by cytosolic kinases that rephosphorylate constituent complex proteins as they diffuse. To develop quantitative understanding of the spatiotemporal regulation of these processes, we generated a reaction-diffusion model describing how membrane-bound epidermal growth factor receptor (EGFR) activates the SH2 domain-containing protein tyrosine phosphatase SHP2 by assembling SHP2 complexes with the adaptor GRB2-associated binder 1 (GAB1). Src family kinases (SFKs) were included as a diffusible EGFR-activated intermediary kinase for GAB1. Contrary to the textbook picture of EGFR-mediated SHP2 activation, the model predicts that GAB1-SHP2 complexes can remain intact distal from membrane-retained EGFR at [~]80% of their plasma membrane concentration over the entire intracellular length scale. Parameter sensitivity analysis identified SFK inactivation as one of the most important kinetic processes controlling GAB1-SHP2 spatial persistence, revealing the key role of repeated adaptor phosphorylation in maintaining SHP2 activity. A simple order of magnitude analysis of the controlling rate processes supports the predictions of the coupled differential equation model. Furthermore, proximity ligation assays and immunofluorescence microscopy in carcinoma cells demonstrate that GAB1-SHP2 complexes are increasingly cytoplasmic and distal from EGFR as time proceeds after acute EGFR activation, consistent with model predictions. These results provide quantitative insight into a mechanism that may be alternately tuned for different receptors and downstream effector complexes to enable receptor control of signaling at a distance. STATEMENT OF SIGNIFICANCEThe textbook understanding of receptor-mediated signaling involves the assembly of protein complexes at the receptor cytosolic tail and complex movement within the cell by protein trafficking. However, reaction-diffusion processes can also play an important role in distributing signaling proteins throughout the cell, potentially quite distal from signal-initiating receptors. Indeed, receptors can activate cytosolic kinases that may maintain the phosphorylation-dependent complexation of downstream effector complexes through many rounds of complex dissociation and protein dephosphorylation. Here, we develop quantitative understanding of this mode of signal regulation through development of a mechanistic computational model and demonstrate, with experimental validation, that membrane-bound receptors can regulate signaling through downstream effector complexes over the entire intracellular length scale.