Abstract
Talin is a large (∼2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs), where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell.
Highlights
Cell migration involves a complex cycle of inter-related events initiated by extracellular cues that establish cell polarity and membrane protrusion at the leading edge driven by actin polymerisation [1]
Cell-extracellular matrix interactions are typically mediated by members of the integrin family of transmembrane ab-heterodimers, and both ‘‘inside-out’’ and ‘‘outside-in’’ signalling [6] can trigger the formation of multi-protein complexes on the cytoplasmic face of integrins that are important in cell adhesion and migration [7,8,9]
We have identified a mutation (E2492G) that suppresses cleavage at this site in vitro, and show that it leads to an increase in the steady state levels of talin1 in HEK293 cells
Summary
Cell migration involves a complex cycle of inter-related events initiated by extracellular cues that establish cell polarity and membrane protrusion at the leading edge driven by actin polymerisation [1]. This is followed by the assembly of small highly dynamic nascent adhesions, a fraction of which mature into larger more stable structures, the subsequent translocation of the cell body, and the detachment of the trailing edge [2]. One of the key proteins required for the assembly of cell-matrix adhesions is the adaptor protein talin [10,11], which can bind both integrins and F-actin, and can switch integrins from a low to high affinity state [12,13]. Talin is required to form the slip bond between fibronectin/integrin complexes and the actomyosin contractile apparatus within the cell [18]
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