The slingshot family of phosphatases mediates Rac1 regulation of cofilin phosphorylation, laminin-332 organization, and motility behavior of keratinocytes

The motility of keratinocytes is an essential component of wound closure and the development of epidermal tumors. In vitro, the specific motile behavior of keratinocytes is dictated by the assembly of laminin-332 tracks, a process that is dependent upon α6β4 integrin signaling to Rac1 and the actin-severing protein cofilin. Here we have analyzed how cofilin phosphorylation is regulated by phosphatases (slingshot (SSH) or chronophin (CIN)) downstream of signaling by α6β4 integrin/Rac1 in human keratinocytes. Keratinocytes express all members of the SSH family (SSH1, SSH2, and SSH3) and CIN. However, expression of phosphatase-dead versions of all three SSH proteins, but not dominant inactive CIN, results in phosphorylation/ inactivation of cofilin, changes in actin cytoskeleton organization, loss of cell polarity, and assembly of aberrant arrays of laminin-332 in human keratinocytes. SSH activity is regulated by 14-3-3 protein binding, and intriguingly, 14-3-3/α6β4 integrin protein interaction is required for keratinocyte migration. Wewondered whether 14-3-3 proteins function as regulators of Rac1-mediated keratinocyte migration patterns. In support of this hypothesis, inhibition of Rac1 results in an increase in 14-3-3 protein association with SSH. Thus, we propose a novel mechanism in which α6β4 integrin signaling via Rac1, 14-3-3 proteins, and SSH family members regulates cofilin activation, cell polarity, and matrix assembly, leading to specific epidermal cell migration behavior.