High Stoichiometry Phosphorylation of Talin at T144/T150 or S446 Produces Contrasting Effects on Calpain-mediated Talin Cleavage and Cell Migration.

Focal adhesions are large multi-protein complexes that serve as the linkage between extracellular matrix (ECM) and actin cytoskeleton and control the network of signaling cascades underlying cell migration. Talin plays a key role in focal adhesion turnover, and calpain-mediated proteolysis of talin is central to focal adhesion disassembly, but its regulation is not well elucidated. Here we demonstrate that talin phosphorylation at three high stoichiometry sites on its head domain, T144 and T150, or S446, have contrasting effects on calpain-mediated cleavage of talin and cell migration by using site-directed mutagenesis to inhibit phosphorylation. Expression of talin stimulated calpain-mediated cleavage of talin and accelerated focal adhesion disassembly, whereas expression of talin fully inhibited talin cleavage by calpain, preventing focal adhesion disassembly. A large decrease in phospho-threonine or phospho-serine levels was seen with talin or talin respectively, while more active ERK was present in talin than in talin. Cell adhesion and transwell assays using uniformly expressing cells showed that expression of talin or talin have opposing effects on cell adhesion and migration. These findings define and highlight the integral role of site-specific high stoichiometry phosphorylation of talin in regulating calpain-mediated cleavage of talin and focal adhesion disassembly, thus controlling adhesion stability, cell adhesion and ultimately, cell migration.