Formation of Kv2.1‐FAK complex as a mechanism of FAK activation, cell polarization and enhanced motility

Abstract

Focal adhesion kinase (FAK) plays key roles in cell adhesion and migration. We now report that the delayed rectifier Kv2.1 potassium channel, through its LD-like motif in N-terminus, may interact with FAK and enhance phosphorylation of FAK397 and FAK576/577. Overlapping distribution of Kv2.1 and FAK was observed on soma and proximal dendrites of cortical neurons. FAK expression promotes a polarized membrane distribution of the Kv2.1 channel. In Kv2.1-transfected CHO cells, formation of the Kv2.1-FAK complex was stimulated by fibronectin/integrin and inhibited by the K+ channel blocker tetraethylammonium (TEA). FAK phosphorylation was minimized by shRNA knockdown of the Kv2.1 channel, point mutations of the N-terminus, and TEA, respectively. Cell migration morphology was altered by Kv2.1 knockdown or TEA, hindering cell migration activity. In wound healing tests in vitro and a traumatic injury animal model, Kv2.1 expression and co-localization of Kv2.1 and FAK significantly enhanced directional cell migration and wound closure. It is suggested that the Kv2.1 channel may function as a promoting signal for FAK activation and cell motility.

Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation

“…Consistently, K ϩ channel blockers such as tetraethylammonium (TEA) and 4-aminopyridine inhibit cell migration (17,39,42,49). Our investigation has shown that the Kv2.1 K ϩ channel can act as an "adhesion protein," form a complex with FAK, and promote the activation of FAK in both neuronal and nonneuronal cells (51). The interaction of Kv2.1 with FAK enhances FAK 576/577 and FAK 397 phosphorylation, significantly increases cell adhesion and directional migration in wound healing processes both in vitro and in animal models (51).…”

“…Our investigation has shown that the Kv2.1 K ϩ channel can act as an "adhesion protein," form a complex with FAK, and promote the activation of FAK in both neuronal and nonneuronal cells (51). The interaction of Kv2.1 with FAK enhances FAK 576/577 and FAK 397 phosphorylation, significantly increases cell adhesion and directional migration in wound healing processes both in vitro and in animal models (51). Taken together, these data suggest that the Kv2.1 protein is a potent promoter for cell migration via increasing activation of FAK.…”

“…Protein concentration of each sample was determined using the bicinchoninic acid assay (Sigma, St. Louis, MO). Immunoprecipitation was performed as previously described (51). Briefly, cell lysates were incubated with specific antibodies overnight at 4°C, followed by incubation with protein G (Upstate, Charlottesville, VA) for 2 h. The immune complexes were washed three times with modified RIPA buffer without sodium deoxylcholate and SDS.…”

“…We showed in a previous investigation an important role of the potassium Kv2.1 channel in regulating cell adhesion and migration (51). Since hypoxia is one of the major regulatory signals affecting K ϩ channel expression (27), we analyzed protein expression levels of some important K ϩ channels in BMSCs by Western blot analysis.…”

“…We have shown in a recent investigation that interaction between Kv2.1 and FAK promoted FAK phosphorylation that increased FAK activation and cell adhesion/ migration (51). To examine whether hypoxic preconditioning increased Kv2.1-FAK interactions in BMSCs, immunoprecipitated protein complexes from N-BMSCs and HP-BMSCs were analyzed.…”