Engineered Manipulation of Signaling Networks: Control of Kinase Activation and Interactions Dissects Parallel Src Pathways

Abstract

Activation of a kinase in a specific protein complex or subcellular location determines what signaling pathway will be initiated. Current methods typically cannot activate a specific kinase and simultaneously cause it to interact with one specific downstream target. Here we provide such a tool, and use it to determine the role of individual Src-mediated signaling pathways in stimulation of different cell morphological changes. Focal adhesion kinase (FAK) and p130Cas can bind and activate Src kinase, propagating signals through parallel pathways. Localization of Src in the cytoplasm or at the plasma membrane also determines the function of Src. We therefore focused on activation of Src acting through FAK, p130Cas, or at the cytoplasm versus the plasma membrane. An engineered, “insertable” FKBP12 protein (iFKBP) was introduced into the catalytic domain of Src, while FKBP12-rapamycin binding domain (FRB) was attached to the specific downstream effector. This rendered the kinase inactive until rapamycin induced heterodimerization with FRB. Using this technology we restricted Src activation to the complex it formed with FRB-bearing downstream targets. Activation of Src, without targeting specific downstream molecules, led to cell spreading, reorganization of focal adhesions, and the production of filopodia and lamellipodial protrusions. Using the new approach (RapR-TAP), we showed that activation of Src specifically in complex with FAK led to focal adhesion rearrangement and only slow cell spreading, while activation in complex with p130Cas led to rapid spreading and filopodia formation, but no apparent effects on focal adhesions. Comparing Src activation in the cytosol versus at the plasma membrane showed that membrane localization is necessary to stabilize protrusions, whereas cytoplasmic Src drives rearrangement of focal adhesions. This demonstrates a broadly generalizable strategy to activate specific kinase-mediated signaling pathways in living cells.