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JNK Pathway-associated Phosphatase Dephosphorylates Focal Adhesion Kinase and Suppresses Cell Migration*

  • Ju-Pi Li
    Affiliations
    Immunology Research Center, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan
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  • Yu-Ning Fu
    Affiliations
    Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan
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  • Yi-Rong Chen
    Correspondence
    To whom correspondence may be addressed: 35 Keyan Rd., Zhunan, Miaoli County 35053, Taiwan. Tel.: 886-37-246166 (Ext. 35311); Fax: 886-37-586459;
    Affiliations
    Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan
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  • Tse-Hua Tan
    Correspondence
    To whom correspondence may be addressed: 35 Keyan Rd., Zhunan, Miaoli County 35053, Taiwan. Tel.: 886-37-246166 (Ext. 37600); Fax: 886-37-586642;
    Affiliations
    Immunology Research Center, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan

    Department of Immunology, Baylor College of Medicine, Houston, Texas 77030
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant 1R01-AI066895 (to T.-H. T.). This work was also supported by National Health Research Institutes, Taiwan, Grants MG-097-PP-03 (to Y.-R. C.) and 98A1-IMPP01-014 (to T.-H. T.) and Department of Health, Taiwan, Grant DOH97-TD-G-111-0037 (to Y.-R. C.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.
Open AccessPublished:December 14, 2009DOI:https://doi.org/10.1074/jbc.M109.060186
      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.

      Introduction

      Dual specificity phosphatases (DUSPs),
      The abbreviations used are: DUSP
      dual specificity phosphatase
      FAK
      focal adhesion kinase
      GFP
      green fluorescent protein
      GST
      glutathione S-transferase
      JNK
      c-Jun N-terminal kinase
      JKAP
      JNK pathway-associated phosphatase
      JKAP-CS
      JKAP mutant lacking catalytic activity
      MAPK
      mitogen-activated protein kinase
      PTP
      protein-tyrosine phosphatase
      TRITC
      tetramethylrhodamine isothiocyanate
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      TR
      tetracycline repressor
      sh
      short hairpin.
      including MAPK phosphatases, can dephosphorylate both tyrosine and serine/threonine residues (
      • Patterson K.I.
      • Brummer T.
      • O'Brien P.M.
      • Daly R.J.
      ). Although DUSPs display limited sequence identity to the classical protein-tyrosine phosphatases (PTPs), they use the same cysteine-based catalytic mechanism and share striking structural similarity with the PTPs. Most MAPK phosphatases consist of a conserved catalytic region and an extended regulatory region, the cdc25 homology domain (
      • Barford D.
      • Flint A.J.
      • Tonks N.K.
      ,
      • Neel B.G.
      • Tonks N.K.
      ). Some DUSPs lack this regulatory domain and, accordingly, have a low molecular weight; they have been classified as atypical DUSPs (
      • Alonso A.
      • Sasin J.
      • Bottini N.
      • Friedberg I.
      • Friedberg I.
      • Osterman A.
      • Godzik A.
      • Hunter T.
      • Dixon J.
      • Mustelin T.
      ). JNK pathway-associated phosphatase (JKAP), an atypical DUSP, was originally identified from a differential display analysis of genes that are preferentially expressed in murine hematopoietic stem cells (
      • Chen A.J.
      • Zhou G.
      • Juan T.
      • Colicos S.M.
      • Cannon J.P.
      • Cabriera-Hansen M.
      • Meyer C.F.
      • Jurecic R.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Fletcher F.
      • Tan T.H.
      • Belmont J.W.
      ). JKAP is also named as VHR-related MKPX (VHX) (
      • Alonso A.
      • Merlo J.J.
      • Na S.
      • Kholod N.
      • Jaroszewski L.
      • Kharitonenkov A.
      • Williams S.
      • Godzik A.
      • Posada J.D.
      • Mustelin T.
      ), JNK stimulatory phosphatase-1 (JSP-1) (
      • Shen Y.
      • Luche R.
      • Wei B.
      • Gordon M.L.
      • Diltz C.D.
      • Tonks N.K.
      ), and LMW-DSP2 (
      • Aoyama K.
      • Nagata M.
      • Oshima K.
      • Matsuda T.
      • Aoki N.
      ). This gene is now designated as DUSP22. JKAP has a canonical PTP signature motif, HCXXGXXR, at residues 87–94. JKAP is expressed in various types of tissues and cells (
      • Chen A.J.
      • Zhou G.
      • Juan T.
      • Colicos S.M.
      • Cannon J.P.
      • Cabriera-Hansen M.
      • Meyer C.F.
      • Jurecic R.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Fletcher F.
      • Tan T.H.
      • Belmont J.W.
      ), suggesting that JKAP may participate in essential biological processes. Previous studies have demonstrated that JKAP selectively activates JNK in human embryonic kidney 293T cells (
      • Chen A.J.
      • Zhou G.
      • Juan T.
      • Colicos S.M.
      • Cannon J.P.
      • Cabriera-Hansen M.
      • Meyer C.F.
      • Jurecic R.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Fletcher F.
      • Tan T.H.
      • Belmont J.W.
      ) and COS-1 cells (
      • Shen Y.
      • Luche R.
      • Wei B.
      • Gordon M.L.
      • Diltz C.D.
      • Tonks N.K.
      ). JKAP-deficient murine embryonic fibroblasts lack JNK activation in response to tumor necrosis factor α and transforming growth factor β (
      • Chen A.J.
      • Zhou G.
      • Juan T.
      • Colicos S.M.
      • Cannon J.P.
      • Cabriera-Hansen M.
      • Meyer C.F.
      • Jurecic R.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Fletcher F.
      • Tan T.H.
      • Belmont J.W.
      ). In contrast, JKAP was also shown to dephosphorylate and inactivate JNK and p38, but not ERK, in transfected COS-7 cells (
      • Aoyama K.
      • Nagata M.
      • Oshima K.
      • Matsuda T.
      • Aoki N.
      ). JKAP expression has been found to suppress T cell antigen receptor-induced ERK2 activation in Jurkat T cells (
      • Alonso A.
      • Merlo J.J.
      • Na S.
      • Kholod N.
      • Jaroszewski L.
      • Kharitonenkov A.
      • Williams S.
      • Godzik A.
      • Posada J.D.
      • Mustelin T.
      ). The effects of JKAP on the activation of MAPKs are controversial, implying JKAP may exert distinct properties dependent on cell type and tissue specificity. Additionally, JKAP also acts as a negative regulator through decreased phosphorylation of estrogen receptor-α and STAT3 in estrogen- and interleukin-6/leukemia inhibitory factor-mediated signaling pathways, respectively (
      • Sekine Y.
      • Ikeda O.
      • Hayakawa Y.
      • Tsuji S.
      • Imoto S.
      • Aoki N.
      • Sugiyama K.
      • Matsuda T.
      ,
      • Sekine Y.
      • Tsuji S.
      • Ikeda O.
      • Sato N.
      • Aoki N.
      • Aoyama K.
      • Sugiyama K.
      • Matsuda T.
      ). Thus, JKAP may have multiple physiological substrates and participate in various signaling cascades.
      The dynamic change of focal adhesions plays a central role in cell migration. Many proteins in focal adhesions, such as cytoskeletal proteins and signaling proteins, are regulated by phosphorylation (
      • Larsen M.
      • Tremblay M.L.
      • Yamada K.M.
      ). Although the roles of kinases in focal adhesions have been elucidated, the importance of phosphatases remains largely unknown. Focal adhesion kinase (FAK) is associated with the formation of focal contacts and is activated by tyrosine phosphorylation (
      • Parsons J.T.
      ). Phosphorylation on Tyr-397, the autophosphorylated residue in FAK, creates an Src-binding site (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      ). Phosphorylation of FAK by Src on Tyr-576 and Tyr-577 within the catalytic domain, in turn, promotes the optimal activation of FAK (
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      ). FAK could influence the cytoskeleton, structures of cell adhesion, and membrane protrusions to regulate cell motility (
      • Mitra S.K.
      • Hanson D.A.
      • Schlaepfer D.D.
      ). In this study, we show that FAK is a substrate of JKAP. Furthermore, JKAP co-localizes with actin filaments. By manipulating the expression and activity of JKAP, we have demonstrated a novel role of this phosphatase in coordination of cell motility through regulating FAK phosphorylation.

      DISCUSSION

      DUSPs were first found to be involved in the down-regulation of MAPK signaling cascades (reviewed in Refs.
      • Neel B.G.
      • Tonks N.K.
      , ). Recently, many newly identified DUSPs were shown to have little or no phosphatase activity against MAPKs and to have different substrate specificities and physiological roles from those of typical MAPK phosphatases (
      • Hood K.L.
      • Tobin J.F.
      • Yoon C.
      ,
      • Niwa R.
      • Nagata-Ohashi K.
      • Takeichi M.
      • Mizuno K.
      • Uemura T.
      ,
      • Wang J.Y.
      • Lin C.H.
      • Yang C.H.
      • Tan T.H.
      • Chen Y.R.
      ,
      • Wang J.Y.
      • Yang C.H.
      • Yeh C.L.
      • Lin C.H.
      • Chen Y.R.
      ). Here, we demonstrated that JKAP, an atypical DUSP, suppressed Src-induced FAK phosphorylation and reduced cell migration.
      The movement of cells is controlled by the dynamic assembly and disassembly of actin filaments underlying the plasma membrane (
      • Ridley A.J.
      ). Results presented here showed that JKAP co-localized with actin filaments in the lamellipodia of the leading edges of migratory cells, suggesting that JKAP could regulate cell motility. Furthermore, JKAP-expressing cells migrated significantly slower. Conversely, expression of JKAP-CS and knockdown of endogenous JKAP markedly enhanced growth factor-induced cell migration. These results further indicate that JKAP could modulate cell migration and that this function is dependent on phosphatase activity of JKAP.
      FAK was first identified as a tyrosine-phosphorylated, Src-associated protein in v-Src-transformed cells (
      • Kanner S.B.
      • Reynolds A.B.
      • Vines R.R.
      • Parsons J.T.
      ). Phosphorylation of FAK at Tyr-397 in response to integrin or growth factor signaling leads to the recruitment and activation of Src (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      ); subsequently, the activated Src phosphorylates other tyrosine residues of FAK to induce maximal kinase activity (
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      ). In addition to regulation by Src, phosphorylation of FAK may be regulated by tyrosine phosphatases, given that vanadate treatment of fibroblasts leads to sustained phosphorylation of FAK (
      • Maa M.C.
      • Leu T.H.
      ). Several PTPs have been implicated in regulation of FAK phosphorylation status and function. Both PTP1B and PTPD1 positively regulate FAK phosphorylation (
      • Arregui C.O.
      • Balsamo J.
      • Lilien J.
      ,
      • Carlucci A.
      • Gedressi C.
      • Lignitto L.
      • Nezi L.
      • Villa-Moruzzi E.
      • Avvedimento E.V.
      • Gottesman M.
      • Garbi C.
      • Feliciello A.
      ). Expression of PTP1B induces FAK phosphorylation by activating Src through dephosphorylation of Src at residue Tyr-527 (
      • Liang F.
      • Lee S.Y.
      • Liang J.
      • Lawrence D.S.
      • Zhang Z.Y.
      ). Conversely, PTP-SHP2 and PTP-PEST are negative regulators of FAK phosphorylation (
      • Yu D.H.
      • Qu C.K.
      • Henegariu O.
      • Lu X.
      • Feng G.S.
      ,
      • Angers-Loustau A.
      • Côté J.F.
      • Charest A.
      • Dowbenko D.
      • Spencer S.
      • Lasky L.A.
      • Tremblay M.L.
      ). Our study indicates that JKAP is another phosphatase negatively regulating FAK phosphorylation.
      Different from PTPs, DUSPs have shallow but broader catalytic pockets, which would allow them to dephosphorylate both tyrosine and serine/threonine residues. Interestingly, the structure around the catalytic site in several atypical DUSPs, including JKAP, VHR, and VHY, is identical to PTPs, although little sequence homology is shown (
      • Yuvaniyama J.
      • Denu J.M.
      • Dixon J.E.
      • Saper M.A.
      ,
      • Yoon T.S.
      • Jeong D.G.
      • Kim J.H.
      • Cho Y.H.
      • Son J.H.
      • Lee J.W.
      • Ryu S.E.
      • Kim S.J.
      ,
      • Yokota T.
      • Nara Y.
      • Kashima A.
      • Matsubara K.
      • Misawa S.
      • Kato R.
      • Sugio S.
      ). In fact, VHR has been shown to dephosphorylate the tyrosine residue of STAT5 in the context of interferon signaling (
      • Hoyt R.
      • Zhu W.
      • Cerignoli F.
      • Alonso A.
      • Mustelin T.
      • David M.
      ). In this study, we demonstrate that JKAP can dephosphorylate FAK at tyrosine residues. A previous study shows that JKAP also dephosphorylates serine 118 of estrogen receptor-α, which in turn attenuates estrogen-mediated signaling (
      • Sekine Y.
      • Ikeda O.
      • Hayakawa Y.
      • Tsuji S.
      • Imoto S.
      • Aoki N.
      • Sugiyama K.
      • Matsuda T.
      ). These studies suggest that atypical DUSPs can have various substrate specificities and efficiently exert dephosphorylation activity. It deserves further exploration how these atypical DUSPs, which lack apparent substrate-binding domain, exhibit selective activity toward their substrates.
      The results of previous reports (
      • Sekine Y.
      • Ikeda O.
      • Hayakawa Y.
      • Tsuji S.
      • Imoto S.
      • Aoki N.
      • Sugiyama K.
      • Matsuda T.
      ,
      • Sekine Y.
      • Tsuji S.
      • Ikeda O.
      • Sato N.
      • Aoki N.
      • Aoyama K.
      • Sugiyama K.
      • Matsuda T.
      ) and this study showed that JKAP can regulate cellular functions through targeting molecules other than MAPKs. Furthermore, in addition to FAK, JKAP may also regulate other proteins (e.g. paxillin) in focal adhesions directly or indirectly (Fig. 4). Further studies can elucidate the mechanisms by which JKAP regulates cell migration through other signaling proteins in coordination with the suppression of FAK phosphorylation.

      Acknowledgments

      We thank the Optical Biology Core, National Health Research Institute, for confocal microscopy assistance.

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