Talin High Stoichiometry Phosphorylation By PKC Or PKA Reversely Controls Calpain-mediated Cleavage Of Talin And Cell Migration

Focal adhesions are large multi-protein complexes, serving as the linkage between extracellular matrix(ECM) and actin cytoskeleton, and 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 the regulation is not well elucidated. Here we demonstrate that talin phosphorylation at three high stoichiometry sites of its head domain by PKC or PKA reversely controls calpain-mediated cleavage of talin and cell migration. Blockages of high stoichiometry phosphorylation by site-directed mutations lead to great decrease of phospho-threonine or phospho-serine level, and increase of PKC or PKA activity in talinT144A+T150A or talinS446A respectively. PKC activates ERK through MAPK cascade that eventually activates calpain,but PKA can not fully inhibit ERK activation, but the activation of calpain can be counteracted by PKA. Expression of talinT144A+TI50A stimulated calpain-mediated cleavage of talin, thus stimulating focal adhesion disassembly and cell migration, whereas expression of talinS446A fully inhibited talin cleavage by calpain, thus preventing focal adhesion disassembly and cell migration. These findings define and highlight the great role of talin site-specific high stoichiometry phosphorylation by PKC or PKA in regulating calpain-mediated cleavage of talin and focal adhesion disassembly, thus controlling adhesion stability, cell adhesion and cell migration ultimately.In this paper,the innovations are the following:Firstly,all cell lines chosen through BD FACSAriaIII flow cytometry sorter under488nm wavelength are finally completely uniform EGFP Positive cell group.Secondly, it is the first time to expound the mechanism of calpain cleavage together with mechanism of high stoichiometry phosphorylation.Thirdly,experimentaI results not only completely conform to the assumptions,but also can make new connection with MAPK signal channels.It is a new breakhrough in the foundation research.