JNK pathway-associated phosphatase dephosphorylates focal adhesion kinase and suppresses cell migration

Abstract

JNK pathway-associated phosphatase (JKAP, also named DUSP22) is expressed in various tissues, indicating that JKAP may have an important biological function. We showed that JKAP localized in the actin filament-enriched region. Expression of JKAP reduced cell migration, whereas a JKAP mutant lacking catalytic activity promoted cell motility. JKAP efficiently removed tyrosine phosphorylation of several proteins. We have identified focal adhesion kinase (FAK) as a substrate of JKAP. Overexpression of JKAP, but not JKAP mutant lacking catalytic activity, decreased FAK phosphorylation at tyrosines 397, 576, and 577 in H1299 cells. Consistent with these results, decreasing JKAP expression by RNA interference promoted cell migration and Src-induced FAK phosphorylation. Taken together, this study identified a new role for JKAP in the modulation of FAK phosphorylation and cell motility.

FIGURE 1.

JKAP co-localizes with actin filaments and reduces cell migration. A, H1299 cells were stably transfected with GFP (upper panel) or GFP-tagged JKAP (lower panel, Field 1 and Field 2) and stained with TRITC-conjugated phalloidin for F-actin and 4′,6-diamidino-2-phenylindole for nucleus. B and C, H1299-JKAP_TR cells and H1299-JKAP-CS_TR cells were cultured with or without 2 μg/ml tetracycline (Tet) for 15 h. The protein levels and cell migration abilities were examined by Western blotting (left panel) or by Transwell assays (right panel), respectively. EGF, epidermal growth factor; exo, exogenous JKAP; endo, endogenous JKAP. Quantitative data were means ± S.E. of one representative result from three independent experiments. *, p < 0.05; **, p < 0.01, compared with the Tet− group.

FIGURE 2.

Expression of JKAP decreases cell motility. H1299-JKAP_TR cells were cultured with or without 2 μg/ml tetracycline (Tet) overnight before the gap-closing assay. A, after a gap was created, cells migrating into the gap region were monitored at 0, 15, and 24 h. Quantitative data were means ± S.E. of one representative result from three independent experiments. **, p < 0.01, compared with the Tet− group. B, migrating H1299-JKAP_TR cells that were cultured with 2 μg/ml tetracycline were fixed at 15 h (Field 1 and Field 2) and subjected to double immunostaining with anti-Myc antibody for JKAP-Myc and TRITC-conjugated phalloidin for F-actin. Magnifications of the insets are shown in the right panels.

FIGURE 3.

JKAP decreases FAK phosphorylation in vitro. A, tyrosine-phosphorylated proteins were immunoprecipitated (IP) from pervanadate (5 μm, 1 h)-treated cell extracts and subjected to JKAP phosphatase assay. B and C, cellular FAKs were pulled down with GST-Src-SH2 fusion protein (B) or immunoprecipitated with the anti-FAK antibody (C) as substrates and then left untreated or treated with GST-JKAP-WT or GST-JKAP-CS proteins to perform phosphatase reactions. Equal amounts of reaction mixtures were subjected to Western blot (WB) analyses using specific antibodies as indicated. Results shown are one representative result of three independent experiments.

FIGURE 4.

JKAP dephosphorylates FAK and decreases paxillin-rich focal adhesions. A, H1299-JKAP_TR cells and H1299-JKAP-CS_TR cells were transfected with Src and then incubated with 2 μg/ml tetracycline (Tet) for 6 and 12 h. The cell extracts were subjected to Western blotting using specific antibodies as indicated. Results shown are one representative result from three independent experiments. The relative band intensities on Western blot assay shown under blot were determined using a computing densitometer equipped with the Gel-Pro analyzer program, which were normalized by arbitrarily setting the densitometry of control to 1. B, bar graphs depicted the results from A (open bars, H1299-JKAP_TR; shaded bars, H1299-JKAP-CS_TR cells). Quantitative data were means ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01, compared with Src-only control. C, H1299-JKAP_TR cells were cultured with or without 2 μg/ml tetracycline for 12 h and subjected to double immunostaining with an anti-paxillin antibody (red) as well as an anti-Myc antibody (green). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Arrows indicate those cells with higher JKAP expression and less paxillin-rich focal adhesions.

FIGURE 5.

Knockdown of JKAP expression increases cell migration and FAK phosphorylation. A, levels of indicated proteins and cell migration abilities of H1299-pSUPER, H1299-shJKAP-1, and H1299-shJKAP-2 cells were examined by Western blotting (upper panel) or by Transwell assays (lower panel), respectively. Quantitative data were means ± S.E. of one representative result from three independent experiments. *, p < 0.05; **, p < 0.01, compared with H1299-pSUPER cells. B, H1299-pSUPER and H1299-shJKAP-2 cells were stably transfected with Src. The levels of indicated proteins in cell extracts were examined by Western blot analyses. Results shown are one representative of three independent experiments.