Abstract
BackgroundEfficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.Principal FindingsUsing FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.ConclusionsOur results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement.
Highlights
Cell migration plays important roles during development and contributes to pathological processes such as tumor invasion and metastasis [1]
This involves a cycle of cortactin binding to focal adhesion kinase (FAK), cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by focal adhesion (FA) turnover and cell movement
As FAK2/2 mouse embryo fibroblasts (MEFs) stably reconstituted with GFPFAK exhibit motility properties identical to normal MEFs [41], comparisons were made with FAK2/2 MEFs stably-reconstituted with green fluorescent protein (GFP)-FAK WT or GFP-FAK constructs containing mutations disrupting kinase activity (R454), proline-rich region 2 (PRR2), or PRR3 (Fig. 1A and B)
Summary
Cell migration plays important roles during development and contributes to pathological processes such as tumor invasion and metastasis [1]. Cell movement is initiated by events including the formation of leading-edge membrane protrusions and integrinassociated focal adhesions (FAs) [2]. Leading edge cell projections are stabilized by FA formation and the severing of f-actin linkages can trigger FA turnover [4]. Various intracellular proteins act to regulate FA assembly and disassembly as this is an important control point for cell movement. One of these proteins is actin binding adaptor protein cortactin [5,6]. Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. FAK and cortactin interact, but the cellular role of this complex remains unclear
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