title: High Stoichiometry Phosphorylation of Talin at T144/T150 or S446 Produces Contrasting Effects on Calpain-mediated Talin Cleavage and Cell Migration.
abstract: 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 talinT144A+T150Astimulated calpain-mediated cleavage of talin and accelerated focal adhesion disassembly, whereas expression of talinS446Afully inhibited talin cleavage by calpain, preventing focal adhesion disassembly. A large decrease in phospho-threonine or phospho-serine levels was seen with talinT144A+T150Aor talinS446Arespectively, while more active ERK was present in talinT144A+T150Athan in talinS446A. Cell adhesion and transwell assays using uniformly expressing cells showed that expression of talinT144A+T150Aor talinS446Ahave 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.
authors.affiliation: Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha 410081, China.
authors.forename: Youjun
authors.initials: Y
authors.lastname: Li
authors.affiliation: Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha 410081, China.
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Luo
authors.affiliation: Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha 410081, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha 410081, China.
authors.forename: Zhenyi
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha 410081, China.
authors.forename: Yizhou
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Laboratory of Medical Molecular and Immunological Diagnostics, College of Medicine, Hunan Normal University, Changsha 410013, China.
authors.forename: Rushi
authors.initials: R
authors.lastname: Liu
authors.affiliation: Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha 410081, China.
authors.forename: Xiangrong
authors.initials: X
authors.lastname: Guo
keywords: Calpain-mediated cleavage; Focal adhesion disassembly; High stoichiometry phosphorylation; Talin