The authors first showed that CDK5 phosphorylates the talin head domain at Ser425 both in vitro and in vivo. Because talin is known to regulate focal adhesion dynamics, they next analysed the turnover of two talin mutants — non-phosphorylatable talinS425A and phosphomimetic talinS425D — fused to enhanced green fluorescent protein (EGFP) at protrusions in Chinese hamster ovary (CHO) cells. Focal adhesion disassembly was increased in the presence of EGFP–talinS425A and decreased in the presence of EGFP–talinS425D. Furthermore, the reduction in talin phosphorylation prevented the stabilization of protrusions, whereas EGFP–talinS425D expression resulted in more persistent protrusions, excessively firm adhesion of cells to the substrate, and impaired cell migration. This effect was not cell type-specific, as phosphorylation at talin Ser425 was also confirmed to be important for the migration of neuronal cells.
But how does CDK5-mediated talin phosphorylation regulate focal adhesion disassembly? The authors focused on the role of SMURF1, which targets proteins for proteasomal degradation and localizes to focal adhesions. SMURF1 binds tightly to the head domain of talin but not to full-length talin both in vitro and in vivo, and this interaction is affected by CDK5 activity. In vitro phosphorylation inhibited the binding of the talin head domain to SMURF1, whereas mutation of Ser425 to prevent phosphorylation increased talin binding to SMURF1. This suggests that the phosphorylation of talin by CDK5 inhibits its interaction with SMURF1. Levels of the talin head domain also decreased when co-transfected with SMURF1 into CHO cells, but degradation was inhibited on treatment with a proteasome inhibitor, suggesting that SMURF1-mediated ubiquitylation leads to degradation of the head domain. The authors confirmed that ubiquitylation of talin is mediated by SMURF1, and that this is inhibited following talin phosphorylation by CDK5 in vivo.
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