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 alpha6beta4 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 alpha6beta4 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/alpha6beta4 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 alpha6beta4 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.

FIGURE 1. Expression of SSH and CIN proteins in human keratinocytes

Whole cell extracts of keratinocytes were processed for Western immunoblotting using antibodies against SSH1 (lane 1), SSH2 (lane 2), SSH3 (lane 3), or CIN (lane 4). Samples in lanes 1–3 were processed on 7.5% polyacrylamide gels, while the sample in lane 4 was processed on 15% polyacrylamide gel. Molecular weight markers are shown on the side of the blots.

FIGURE 2. Cofilin activity is regulated by SSH proteins and not CIN in human keratinocytes

A, extracts of human keratinocytes infected with adenovirus encoding phosphatase-dead V5-tagged SSH1CS, c-Myc-tagged SSH2CS, V5-tagged SSH3CS, or dominant inactive CIN (CIN D/N) were processed for Western immunoblotting and then probed with antibodies that recognize the V5 epitope (lanes 1 and 3), the c-Myc epitope (lane 2), or with an antibody against CIN (lane 4). Samples in lanes 1–3 were processed on 7.5% polyacrylamide gels, whereas the sample in lane 4 was processed on 15% polyacrylamide gel. Molecular weight markers are indicated on the sides of the probed blots. B, whole cells extracts of uninfected keratinocytes (Control) and keratinocytes infected with adenovirus encoding phosphatase-dead SSH1CS, SSH2CS, SSH3CS, or dominant inactive CIN (CIN D/N) were processed for SDS-PAGE/immunoblotting. Blots were probed first with antibodies against phosphorylated cofilin (pSer3) and then with antibodies against total cofilin. The percentage phosphorylation (mean ± S.E. for three independent trials) was calculated relative to total cofilin levels in each of three studies, and the quantification is shown.

FIGURE 3. Inactivation of SSH proteins changes the organization of the actin cytoskeleton in human keratinocytes

Uninfected human keratinocytes (Control) and keratinocytes infected with adenovirus encoding dominant inactive CIN (CIN D/N) or phosphatase-dead SSH proteins (SSH1CS, SSH2CS, and SSH3CS) were plated onto glass coverslips, allowed to adhere for 18 –24 h, and then were fixed and stained with rhodamine-phalloidin (left panels). The cell preparations were viewed by confocal laser scanning microscopy. In the right panels, the number of lamellipodia/cell were counted for each of the cell populations, and the graphs depict the results from 50 cells in each of at least three independent experiments (± S.E.). Representative images are shown in the left panels. Bars, 10 μm.

FIGURE 4. Motility assays of keratinocytes on uncoated glass

Human keratinocytes (Control) (n = 104), keratinocytes infected with control adenovirus (Viral control) (n = 32), or keratinocytes infected with adenovirus encoding dominant inactive CIN (CIN D/N) (n = 162), phosphatase-dead SSH1CS (n = 201), SSH2CS (n = 101), or SSH3CS (n = 74) as indicated were replated onto glass-bottomed dishes and allowed to adhere for 18 –24 h. The cells were then viewed by phase-contrast microscopy with images being captured every 2 min for 2 h. In each panel of six, images at 24-min intervals are presented. The graph depicts the percentage of cells exhibiting either migration along trails or motility in circles.

FIGURE 5. Analyses of laminin-332 matrix assembly by keratinocytes

Keratinocytes were infected with control adenovirus (Viral control) or adeno-virus encoding dominant inactive CIN D/N, or phosphatase-dead SSH1CS, SSH2CS, or SSH3CS and replated onto glass coverslips 24 h later. At 24 h after plating, cells were prepared for confocal immunofluorescence microscopy using antibody GB3 against the γ2 subunit of laminin-332. Left panels, the micrographs show laminin-332 staining patterns overlaid onto the phase-contrast images of the cells. Right panels, the percentage of cells displaying laminin-332 patterns in trails, circles, or a diffuse pattern was counted, and the graphs depict analyses of a minimum of 50 cells in each of at least two independent experiments (± S.E.). Bars, 20 μm.

FIGURE 6. Motility assays of keratinocytes expressing phosphatase-dead SSH1CS on preassembled arrays of laminin-332

Keratinocytes were infected with adenovirus encoding SSH1CS, and at 48 h post-infection, the cells were plated onto the laminin-332-rich matrix deposited by uninfected keratinocytes. The cells were allowed to adhere for 2– 4 h, and their motility was analyzed over a period of 2 h. In panel A, phase-contrast images of the cells are shown at the indicated time points. B, the graph depicts the percentage of cells exhibiting either migration along trails or motility in circles (n = 41). C, cofilin phosphorylation was measured in whole cell extracts from uninfected keratinocytes or keratinocytes infected with adenovirus encoding SSH1CS, SSH2CS, or SSH3CS that were replated on preformed laminin-332 matrix. Blots were probed first with antibodies against phosphorylated cofilin (pSer3) and then with antibodies against total cofilin.

FIGURE 7. Binding of 14-3-3 proteins to SSH proteins in the presence of a Rac1 inhibitor

Extracts of keratinocytes expressing V5-tagged SSH1WT (A), c-Myc-tagged SSH2WT (B), or V5-tagged SSH3WT (C) treated with or without the Rac1 inhibitor NSC23766 were processed for immunoprecipitation using V5 or c-Myc antibodies. A portion of the precipitated proteins was subjected to immunoblotting using a pan-14-3-3 antibody and monoclonal antibodies against V5 or c-Myc as indicated. The levels of SSH proteins that co-precipitate with 14-3-3 proteins from extracts of the Rac1 inhibitor-treated cells relative to non-treated cells, following normalization to either V5- or c-Myc-tagged SSH precipitated proteins, were quantified. These values are indicated under the 14-3-3 blots. B, there is a nonspecific reactive species indicated by *. An arrow points to the immunoprecipitated 14-3-3 protein in panel B.

FIGURE 8. This model depicts the putative signaling pathway by which β4 integrin regulates the organizational state of laminin-332 in the extracellular matrix in motile versus stationary keratinocytes

Our data indicate that it does so through signaling via Rac1, SSH proteins, 14-3-3 proteins, cofilin, and the actin cytoskeleton.