We have used a c-Src-GFP fusion protein to address the spatial control of Src activation and the nature of Src-associated intracellular structures during stimulus-induced transit to the membrane. Src is activated during transit, particularly in RhoB-containing cytoplasmic endosomes associated with the perinuclear recycling compartment. Knocking out RhoB or expressing a dominant-interfering Rab11 mutant suppresses both catalytic activation of Src and translocation of active kinase to peripheral membrane structures. In addition, the Src- and RhoB-containing endosomes harbor proteins involved in actin polymerization and filament assembly, for example Scar1, and newly polymerized actin can associate with these endosomes in a Src-dependent manner. This implies that Src may regulate an endosome-associated actin nucleation activity. In keeping with this, Src controls the actin dependence of RhoB endosome movement toward the plasma membrane. This work identifies RhoB as a component of "outside-in" signaling pathways that coordinate Src activation with translocation to transmembrane receptors.

Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src-LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity.

A fundamental question in cell biology concerns how cells respond to their environment by polarizing after sensing directional cues. This requires the differential localization of protein complexes in cells, and it is important to identify and understand how these complexes function. Here we describe a novel "direction-sensing" pathway that links the integrin effector focal adhesion kinase (FAK), the molecular scaffold protein RACK1, and activity of one of its client proteins, PDE4D5, a cAMP-degrading phosphodiesterase. The complex is recruited to nascent adhesions and promotes cell polarity. We identify FAK FERM domain residues whose mutation impairs RACK1 binding. When re-expressed in cancer cells in which endogenous fak is deleted by Cre-lox-mediated recombination, the RACK1-binding-impaired FAK mutant protein does not support formation of nascent actin adhesion structures as cells spread. These cancer cells, like FAK-deficient cells, cannot undergo directional responses, including wound-induced polarization or chemotactic invasion into three-dimensional matrix gels. We show that RACK1 serves as the molecular bridge linking FAK to the recruitment of PDE4D5. FAK/RACK1/PDE4D5 is a novel 'direction-sensing' complex that acts to recruit specific components of the cAMP second-messenger system to nascent integrin adhesions and to the leading edge of polarizing cells.