The Slingshot Family of Phosphatases Mediates Rac1 Regulation of Cofilin Phosphorylation, Laminin-332 Organization, and Motility Behavior of Keratinocytes

Abstract

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 ␣6␤4 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 ␣6␤4 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/␣6␤4 integrin protein interaction is required for keratinocyte migration. We wondered 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 ␣6␤4 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.

Fluorescently tagged laminin subunits facilitate analyses of the properties, assembly and processing of laminins in live and fixed lung epithelial cells and keratinocytes

“…5A,B). This is consistent with recent reports from our group that laminin-332 matrices are key to the regulation of epidermal motile behavior (Kligys et al, 2007;Sehgal et al, 2006).…”

“…Cells were maintained at 37°C in a cell culture, stage top incubator (Tokai Hit Co., Ltd, Shizuokaken, Japan) and then viewed on a Zeiss laser-scanning microscope (LSM) 510 confocal microscope (Zeiss Inc., Thornwood, NY). Images were taken at 2-5 min intervals over 2 h. In the case of fixed cell preparations, cells plated onto glass coverslips were processed for microscopical analyses as detailed elsewhere (Kligys et al, 2007;Sehgal et al, 2006). All preparations were viewed with a Zeiss laser-scanning microscope (LSM) 510 confocal microscope.…”

The NAV2 homolog Sickie regulates F-actin-mediated axonal growth inDrosophilamushroom body neurons via the non-canonical Rac-Cofilin pathway

“…Although several pieces of evidence have suggested the importance of the non-canonical pathway and predicted the existence of its mediator, whether this pathway finally converges upon the downstream Cofilin pathway and subsequent F-actin reorganization remains unclear (Kligys et al, 2007;Nagata-Ohashi et al, 2004;Ng and Luo, 2004). Moreover, many biochemical studies have assessed the regulation of Cofilin function and F-actin states using in vitro systems; the endogenous changes in F-actin and Cofilin phosphorylation appear not to have been analyzed simultaneously with an internal control in developing brain.…”

βArrestin1 Regulates the Guanine Nucleotide Exchange Factor RasGRF2 Expression and the Small GTPase Rac-mediated Formation of Membrane Protrusion and Cell Motility

“…Decitabine also increased rasgrf2 expression level in wild-type cells, implying additional factors are involved in the methylation of rasgrf2 gene promoter. It can be envisioned that activated Rac leads to activation of slingshot family phosphatase to dephosphorylate and activate cofilin, as has been reported for keratinocyte migration (39). It is also worth mentioning that Rac was reported to inactivate cofilin through LIM kinase-mediated phosphorylation (40,41).…”