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Protein Kinase PKN1 Represses Wnt/β-Catenin Signaling in Human Melanoma Cells*

  • Richard G. James
    Correspondence
    To whom correspondence may be addressed: Center for Immunity and Immunotherapies, Seattle Children's Research Inst., Seattle, WA 98115
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    Department of Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109
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  • Katherine A. Bosch
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    Department of Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109
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  • Rima M. Kulikauskas
    Footnotes
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Medicine, Division of Dermatology, University of Washington, Seattle, Washington 98109
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  • Peitzu T. Yang
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    Department of Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98109
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  • Nick C. Robin
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    Department of Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98109
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  • Rachel A. Toroni
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Medicine, Division of Dermatology, University of Washington, Seattle, Washington 98109
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  • Travis L. Biechele
    Footnotes
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    Department of Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98109
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  • Jason D. Berndt
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    Department of Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109
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  • Priska D. von Haller
    Affiliations
    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Genome Sciences, University of Washington, Seattle, Washington 98109

    Department of Proteomics Resource, University of Washington, Seattle, Washington 98109
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  • Jimmy K. Eng
    Affiliations
    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Genome Sciences, University of Washington, Seattle, Washington 98109

    Department of Proteomics Resource, University of Washington, Seattle, Washington 98109
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  • Alejandro Wolf-Yadlin
    Affiliations
    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Genome Sciences, University of Washington, Seattle, Washington 98109

    Department of Proteomics Resource, University of Washington, Seattle, Washington 98109
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  • Andy J. Chien
    Affiliations
    Department of Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Medicine, Division of Dermatology, University of Washington, Seattle, Washington 98109
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  • Randall T. Moon
    Correspondence
    An Investigator of the Howard Hughes Medical Institute. To whom correspondence may be addressed. Tel.: 206-543-1722; Fax: 206-543-1183;
    Affiliations
    Department of Pharmacology, University of Washington, Seattle, Washington 98109

    Department of Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109

    University of Washington School of Medicine, Seattle, Washington 98109

    Department of Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98109
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant K99/R00 1K99HL103768-01 from the NHLBI (to R. G. J.), Grant K08CA128565 from the NCI (to A. J. C.), a grant from the NCI (to R. M. K.), and Training Grant T32AR056969 from the NIAMS (to T. L. B.). This work was also supported by University of Washington's Proteomics Resource Grant UWPR 95794.
    This article contains supplemental Databases S1–S6.
    3 Present address: Seattle Genetics, Inc., Bothell, WA 98021.
    2 Supported by an administrative supplemental grant through the American Recovery and Relief Act.
Open AccessPublished:October 10, 2013DOI:https://doi.org/10.1074/jbc.M113.500314
      Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of β-catenin-responsive transcription (β-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/β-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.

      Introduction

      The 19 members of the Wnt family of secreted glycoproteins regulate diverse intracellular signal transduction cascades in vertebrates. One Wnt pathway, herein referred to as the Wnt/β-catenin pathway, is activated by the binding of Wnt ligand to Frizzled (FZD)
      The abbreviations used are: FZD
      Frizzled
      LRP
      low density lipoprotein receptor-related protein
      BAR
      β-catenin-activated reporter
      PKN1
      protein kinase N1
      pPKN1
      phosphorylated protein kinase N1
      BRAF
      v-raf murine sarcoma viral oncogene homolog B
      BRAFi
      BRAF inhibitor
      GSK3
      glycogen synthase kinase 3
      EB
      elution buffer
      ANOVA
      analysis of variance
      PARP
      poly(ADP-ribose) polymerase.
      serpentine receptors and to low density lipoprotein receptor-related protein (LRP) co-receptors (for reviews, see Refs.
      • Angers S.
      • Moon R.T.
      Proximal events in Wnt signal transduction.
      ,
      • Clevers H.
      • Nusse R.
      Wnt/β-catenin signaling and disease.
      ,
      • MacDonald B.T.
      • Tamai K.
      • He X.
      Wnt/β-catenin signaling: components, mechanisms, and diseases.
      ). Following receptor activation, a series of events result in stabilization of nuclear and cytosolic pools of the multifunctional adapter protein β-catenin (encoded by CTNNB1). β-Catenin then promotes transcriptional changes that can result in modulation of differentiation and/or cell proliferation (
      • Chien A.J.
      • Conrad W.H.
      • Moon R.T.
      A Wnt survival guide: from flies to human disease.
      ).
      β-Catenin signaling plays diverse roles in embryonic development and in adults as well as following acute injury or chronic disease. For example, β-catenin specifies premigratory neural crest to adopt a melanocyte fate during development (
      • Dorsky R.I.
      • Moon R.T.
      • Raible D.W.
      Control of neural crest cell fate by the Wnt signalling pathway.
      ). In melanoma, a cancer generally considered to arise from melanocytes, β-catenin levels decrease during the progression of the disease (
      • Kageshita T.
      • Hamby C.V.
      • Ishihara T.
      • Matsumoto K.
      • Saida T.
      • Ono T.
      Loss of β-catenin expression associated with disease progression in malignant melanoma.
      ). Higher expression of β-catenin in tumors at the time of diagnosis correlates with improved patient survival in melanoma (
      • Kageshita T.
      • Hamby C.V.
      • Ishihara T.
      • Matsumoto K.
      • Saida T.
      • Ono T.
      Loss of β-catenin expression associated with disease progression in malignant melanoma.
      ,
      • Bachmann I.M.
      • Straume O.
      • Puntervoll H.E.
      • Kalvenes M.B.
      • Akslen L.A.
      Importance of P-cadherin, β-catenin, and Wnt5a/frizzled for progression of melanocytic tumors and prognosis in cutaneous melanoma.
      ,
      • Gould Rothberg B.E.
      • Berger A.J.
      • Molinaro A.M.
      • Subtil A.
      • Krauthammer M.O.
      • Camp R.L.
      • Bradley W.R.
      • Ariyan S.
      • Kluger H.M.
      • Rimm D.L.
      Melanoma prognostic model using tissue microarrays and genetic algorithms.
      ,
      • Maelandsmo G.M.
      • Holm R.
      • Nesland J.M.
      • Fodstad Ø.
      • Flørenes V.A.
      Reduced β-catenin expression in the cytoplasm of advanced-stage superficial spreading malignant melanoma.
      ,
      • Meyer S.
      • Fuchs T.J.
      • Bosserhoff A.K.
      • Hofstädter F.
      • Pauer A.
      • Roth V.
      • Buhmann J.M.
      • Moll I.
      • Anagnostou N.
      • Brandner J.M.
      • Ikenberg K.
      • Moch H.
      • Landthaler M.
      • Vogt T.
      • Wild P.J.
      A seven-marker signature and clinical outcome in malignant melanoma: a large-scale tissue-microarray study with two independent patient cohorts.
      ), which is opposite of the correlation observed with colorectal cancer. Understanding the roles and mechanisms of β-catenin signaling may thus inform us about disease as well as development.
      Changes in the phosphorylation of proteins play a central regulatory role in Wnt/β-catenin signaling (
      • Angers S.
      • Moon R.T.
      Proximal events in Wnt signal transduction.
      ,
      • Clevers H.
      • Nusse R.
      Wnt/β-catenin signaling and disease.
      ,
      • MacDonald B.T.
      • Tamai K.
      • He X.
      Wnt/β-catenin signaling: components, mechanisms, and diseases.
      ). Specifically, in the absence of Wnt stimulus, β-catenin is constitutively degraded by the proteasome following its phosphorylation by the kinases casein kinase 1α (encoded by CSNK1A1) and glycogen synthase kinase 3 (GSK3) (
      • Kimelman D.
      • Xu W.
      β-Catenin destruction complex: insights and questions from a structural perspective.
      ). Conversely, following Wnt stimulation, phosphorylation of the cytoplasmic tail of the FZD co-receptor LRP5/6 is thought to be necessary for the subsequent stabilization of CTNNB1. Upon phosphorylation of LRP5/6, the cytosolic pools of the scaffolding protein AXIN1 and its associated kinases, CSNK1A1 and GSK3, bind LRP5/6 at the cytoplasmic side of the plasma membrane (
      • Davidson G.
      • Wu W.
      • Shen J.
      • Bilic J.
      • Fenger U.
      • Stannek P.
      • Glinka A.
      • Niehrs C.
      Casein kinase 1γ couples Wnt receptor activation to cytoplasmic signal transduction.
      ,
      • Bilic J.
      • Huang Y.L.
      • Davidson G.
      • Zimmermann T.
      • Cruciat C.M.
      • Bienz M.
      • Niehrs C.
      Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.
      ), thus allowing newly translated β-catenin to accumulate in the cytosol, translocate to the nucleus, and regulate the transcription of target genes. Therefore, screening for kinases that regulate the Wnt/β-catenin pathway in melanoma is warranted given the continual discovery of new tissue-specific points of regulation of this pathway by kinases (
      • Huang X.
      • McGann J.C.
      • Liu B.Y.
      • Hannoush R.N.
      • Lill J.R.
      • Pham V.
      • Newton K.
      • Kakunda M.
      • Liu J.
      • Yu C.
      • Hymowitz S.G.
      • Hongo J.A.
      • Wynshaw-Boris A.
      • Polakis P.
      • Harland R.M.
      • Dixit V.M.
      Phosphorylation of dishevelled by protein kinase RIPK4 regulates Wnt signaling.
      ,
      • Rosenbluh J.
      • Nijhawan D.
      • Cox A.G.
      • Li X.
      • Neal J.T.
      • Schafer E.J.
      • Zack T.I.
      • Wang X.
      • Tsherniak A.
      • Schinzel A.C.
      • Shao D.D.
      • Schumacher S.E.
      • Weir B.A.
      • Vazquez F.
      • Cowley G.S.
      • Root D.E.
      • Mesirov J.P.
      • Beroukhim R.
      • Kuo C.J.
      • Goessling W.
      • Hahn W.C.
      β-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.
      ).
      To advance the characterization of changes in protein phosphorylation upon stimulation of the Wnt/β-catenin pathway, in the present study, we defined the WNT3A-regulated phosphoproteome in a melanoma cell line. We then integrated our phosphoproteomics data with a siRNA screen targeting known and predicted kinases. This approach identified protein kinase N1 (encoded by PKN1) as a putative inhibitor of the Wnt/β-catenin pathway. To begin to address the mechanisms by which PKN1 might attenuate Wnt/β-catenin signaling, we demonstrated that PKN1 inhibits WNT3A-dependent phosphorylation of LRP6 and regulates its expression on the cell surface. Suggesting that PKN1 might be relevant to future therapies in melanoma, we found that depletion of PKN1 can sensitize melanoma cells to cell death initiated by the WNT3A ligand.

      DISCUSSION

      Data from high throughput siRNA screens (
      • James R.G.
      • Biechele T.L.
      • Conrad W.H.
      • Camp N.D.
      • Fass D.M.
      • Major M.B.
      • Sommer K.
      • Yi X.
      • Roberts B.S.
      • Cleary M.A.
      • Arthur W.T.
      • MacCoss M.
      • Rawlings D.J.
      • Haggarty S.J.
      • Moon R.T.
      Bruton's tyrosine kinase revealed as a negative regulator of Wnt-β-catenin signaling.
      ,
      • Major M.B.
      • Roberts B.S.
      • Berndt J.D.
      • Marine S.
      • Anastas J.
      • Chung N.
      • Ferrer M.
      • Yi X.
      • Stoick-Cooper C.L.
      • von Haller P.D.
      • Kategaya L.
      • Chien A.
      • Angers S.
      • MacCoss M.
      • Cleary M.A.
      • Arthur W.T.
      • Moon R.T.
      New regulators of Wnt/β-catenin signaling revealed by integrative molecular screening.
      ,
      • Miller B.W.
      • Lau G.
      • Grouios C.
      • Mollica E.
      • Barrios-Rodiles M.
      • Liu Y.
      • Datti A.
      • Morris Q.
      • Wrana J.L.
      • Attisano L.
      Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway.
      ) and affinity purification-mass spectrometry experiments (
      • James R.G.
      • Biechele T.L.
      • Conrad W.H.
      • Camp N.D.
      • Fass D.M.
      • Major M.B.
      • Sommer K.
      • Yi X.
      • Roberts B.S.
      • Cleary M.A.
      • Arthur W.T.
      • MacCoss M.
      • Rawlings D.J.
      • Haggarty S.J.
      • Moon R.T.
      Bruton's tyrosine kinase revealed as a negative regulator of Wnt-β-catenin signaling.
      ,
      • Major M.B.
      • Roberts B.S.
      • Berndt J.D.
      • Marine S.
      • Anastas J.
      • Chung N.
      • Ferrer M.
      • Yi X.
      • Stoick-Cooper C.L.
      • von Haller P.D.
      • Kategaya L.
      • Chien A.
      • Angers S.
      • MacCoss M.
      • Cleary M.A.
      • Arthur W.T.
      • Moon R.T.
      New regulators of Wnt/β-catenin signaling revealed by integrative molecular screening.
      ,
      • Angers S.
      • Thorpe C.J.
      • Biechele T.L.
      • Goldenberg S.J.
      • Zheng N.
      • MacCoss M.J.
      • Moon R.T.
      The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-β-catenin pathway by targeting Dishevelled for degradation.
      ,
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ,
      • Hilger M.
      • Mann M.
      Triple SILAC to determine stimulus specific interactions in the Wnt pathway.
      ) reveal complex regulation of the Wnt/β-catenin pathway by kinases and phosphatases. One observation that we made in this study is that phosphorylation changes catalyzed by stimulation of melanoma cells with the WNT3A ligand are distinct from those caused by inhibition of GSK3. We found that the subset of phosphopeptides that are decreased in abundance following GSK3 inhibition that contain a GSK3 consensus site (SpXXXSp) are largely unaffected by stimulus with the WNT3A ligand. These findings contrast with the model presented by Taelman et al. (
      • Taelman V.F.
      • Dobrowolski R.
      • Plouhinec J.L.
      • Fuentealba L.C.
      • Vorwald P.P.
      • Gumper I.
      • Sabatini D.D.
      • De Robertis E.M.
      Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes.
      ) that proposes that the WNT3A ligand promotes trafficking of the majority of GSK3 to multivesicular bodies, thus sequestering GSK3 and preventing it from regulating cytosolic proteins. Instead, our data suggest that the WNT3A stimulus does not generally regulate proteins phosphorylated by GSK3 and are consistent with earlier studies that demonstrate that Wnt ligands can inhibit the ability of GSK3 proteins to phosphorylate CTNNB1 but not necessarily other substrates (
      • Ding V.W.
      • Chen R.H.
      • McCormick F.
      Differential regulation of glycogen synthase kinase 3β by insulin and Wnt signaling.
      ). One possible explanation for the contrast with the earlier study is that the majority of GSK3 substrates may migrate with and remain accessible to GSK3 in multivesicular bodies upon cellular stimulation with WNT3A. Regardless, our data suggest that inhibiting GSK3 is not synonymous with activating the Wnt/β-catenin pathway, which is an important consideration when using GSK3 inhibition as a proxy for activation of β-catenin by Wnt ligands.
      We found that WNT3A promotes phosphorylation changes in several proteins that have been previously reported to regulate RAS activation and phosphatidylinositol 3-kinase signaling (MTOR, RICTOR, PIK3CA, and RPS6KA4). Previous work has demonstrated that forced expression of a non-degradable phosphorylation mutant of CTNNB1 can increase the metastatic potential of melanoma cells harboring activating mutations in the NRAS/phosphatidylinositol 3-kinase pathways (
      • Damsky W.E.
      • Curley D.P.
      • Santhanakrishnan M.
      • Rosenbaum L.E.
      • Platt J.T.
      • Gould Rothberg B.E.
      • Taketo M.M.
      • Dankort D.
      • Rimm D.L.
      • McMahon M.
      • Bosenberg M.
      β-Catenin signaling controls metastasis in Braf-activated Pten-deficient melanomas.
      ,
      • Delmas V.
      • Beermann F.
      • Martinozzi S.
      • Carreira S.
      • Ackermann J.
      • Kumasaka M.
      • Denat L.
      • Goodall J.
      • Luciani F.
      • Viros A.
      • Demirkan N.
      • Bastian B.C.
      • Goding C.R.
      • Larue L.
      β-Catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development.
      ). Because many of the WNT3A-dependent changes in phosphorylation that we observed occurred within 60 min of stimulus, our data suggest that the WNT3A ligand may also regulate progrowth pathways (e.g. mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling) in melanoma cells that are possibly independent of CTNNB1.
      An interesting finding in these studies is that PKN1 depletion increases LRP6 cell surface expression and phosphorylation at serine 1490, a site associated with active Wnt/β-catenin signaling. Similarly, additional studies have shown that manipulations that increase surface expression of FZD (
      • Terabayashi T.
      • Funato Y.
      • Fukuda M.
      • Miki H.
      A coated vesicle-associated kinase of 104 kDa (CVAK104) induces lysosomal degradation of frizzled 5 (Fzd5).
      ,
      • Mukai A.
      • Yamamoto-Hino M.
      • Awano W.
      • Watanabe W.
      • Komada M.
      • Goto S.
      Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt.
      ) or LRP6 (
      • Carmon K.S.
      • Lin Q.
      • Gong X.
      • Thomas A.
      • Liu Q.
      LGR5 interacts and cointernalizes with Wnt receptors to modulate Wnt/β-catenin signaling.
      ,
      • Jiang Y.
      • He X.
      • Howe P.H.
      Disabled-2 (Dab2) inhibits Wnt/β-catenin signalling by binding LRP6 and promoting its internalization through clathrin.
      ) also increase expression of downstream markers of Wnt/β-catenin signaling. Collectively, these findings are inconsistent with the hypothesis that internalization of LRP6 is necessary for downstream Wnt/β-catenin signal transduction.
      Two models have been proposed to explain how Wnt ligands inhibit GSK3, enabling stabilization of CTNNB1 (
      • Metcalfe C.
      • Bienz M.
      Inhibition of GSK3 by Wnt signalling—two contrasting models.
      ). The first is based on the idea that the WNT3A stimulus causes recruitment of GSK3 to the cell membrane where it is subsequently internalized into multivesicular bodies (
      • Taelman V.F.
      • Dobrowolski R.
      • Plouhinec J.L.
      • Fuentealba L.C.
      • Vorwald P.P.
      • Gumper I.
      • Sabatini D.D.
      • De Robertis E.M.
      Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes.
      ), thus allowing cytosolic CTNNB1 to accumulate. In contrast, a second model proposes that phosphorylated LRP6 binds GSK3 and inhibits its catalytic activity (
      • Cselenyi C.S.
      • Jernigan K.K.
      • Tahinci E.
      • Thorne C.A.
      • Lee L.A.
      • Lee E.
      LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of β-catenin.
      ,
      • Mi K.
      • Dolan P.J.
      • Johnson G.V.
      The low density lipoprotein receptor-related protein 6 interacts with glycogen synthase kinase 3 and attenuates activity.
      ,
      • Piao S.
      • Lee S.H.
      • Kim H.
      • Yum S.
      • Stamos J.L.
      • Xu Y.
      • Lee S.J.
      • Lee J.
      • Oh S.
      • Han J.K.
      • Park B.J.
      • Weis W.I.
      • Ha N.C.
      Direct inhibition of GSK3beta by the phosphorylated cytoplasmic domain of LRP6 in Wnt/β-catenin signaling.
      ,
      • Wu G.
      • Huang H.
      • Garcia Abreu J.
      • He X.
      Inhibition of GSK3 phosphorylation of β-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6.
      ). Our data showing that PKN1 depletion increases LRP6 internalization while simultaneously decreasing GSK3-dependent phosphorylation of CTNNB1 are more consistent with the latter model.
      PKN1 is an atypical PKC kinase that acts as a Rho effector in several contexts and can regulate AKT (
      • Balendran A.
      • Casamayor A.
      • Deak M.
      • Paterson A.
      • Gaffney P.
      • Currie R.
      • Downes C.P.
      • Alessi D.R.
      PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2.
      ,
      • Koh H.
      • Lee K.H.
      • Kim D.
      • Kim S.
      • Kim J.W.
      • Chung J.
      Inhibition of Akt and its anti-apoptotic activities by tumor necrosis factor-induced protein kinase C-related kinase 2 (PRK2) cleavage.
      ,
      • Wick M.J.
      • Dong L.Q.
      • Riojas R.A.
      • Ramos F.J.
      • Liu F.
      Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1.
      ,
      • Yasui T.
      • Sakakibara-Yada K.
      • Nishimura T.
      • Morita K.
      • Tada S.
      • Mosialos G.
      • Kieff E.
      • Kikutani H.
      Protein kinase N1, a cell inhibitor of Akt kinase, has a central role in quality control of germinal center formation.
      ) and MAPK8 (also known as Jnk) (
      • Lu Y.
      • Settleman J.
      The Drosophila Pkn protein kinase is a Rho/Rac effector target required for dorsal closure during embryogenesis.
      ) signaling (for a review, see Ref.
      • Mukai H.
      The structure and function of PKN, a protein kinase having a catalytic domain homologous to that of PKC.
      ). PKN proteins localize to the endoplasmic reticulum and the endosome (
      • Mellor H.
      • Flynn P.
      • Nobes C.D.
      • Hall A.
      • Parker P.J.
      PRK1 is targeted to endosomes by the small GTPase, RhoB.
      ,
      • Gampel A.
      • Parker P.J.
      • Mellor H.
      Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.
      ,
      • Torbett N.E.
      • Casamassima A.
      • Parker P.J.
      Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3-phosphoinositide-dependent kinase 1.
      ) and are known to regulate vesicular traffic (
      • Manser C.
      • Stevenson A.
      • Banner S.
      • Davies J.
      • Tudor E.L.
      • Ono Y.
      • Leigh P.N.
      • McLoughlin D.M.
      • Shaw C.E.
      • Miller C.C.
      Deregulation of PKN1 activity disrupts neurofilament organisation and axonal transport.
      ). A protein array-based screen for substrates of the PKN1 kinase determined that it can phosphorylate several receptor proteins including EPHA5, EGF receptor, RET, and GRK4 (
      • Collazos A.
      • Michael N.
      • Whelan R.D.
      • Kelly G.
      • Mellor H.
      • Pang L.C.
      • Totty N.
      • Parker P.J.
      Site recognition and substrate screens for PKN family proteins.
      ). Our finding that PKN1 is required for WNT3A-dependent internalization of LRP6 is consistent with data showing that PKN1 promotes RHOB-dependent endocytosis of the EGF receptor (
      • Gampel A.
      • Parker P.J.
      • Mellor H.
      Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.
      ). Also consistent with these findings, we observed that PKN1 co-purifies with several proteins known to regulate vesicle trafficking. Based on these findings, we hypothesize that PKN proteins might directly regulate surface expression of several receptor proteins including LRP6. However, because PKN1 is known to regulate the AKT (
      • Yasui T.
      • Sakakibara-Yada K.
      • Nishimura T.
      • Morita K.
      • Tada S.
      • Mosialos G.
      • Kieff E.
      • Kikutani H.
      Protein kinase N1, a cell inhibitor of Akt kinase, has a central role in quality control of germinal center formation.
      ) and mitogen-activated protein kinase pathways (
      • Lu Y.
      • Settleman J.
      The Drosophila Pkn protein kinase is a Rho/Rac effector target required for dorsal closure during embryogenesis.
      ,
      • Sun W.
      • Vincent S.
      • Settleman J.
      • Johnson G.L.
      MEK kinase 2 binds and activates protein kinase C-related kinase 2. Bifurcation of kinase regulatory pathways at the level of an MAPK kinase kinase.
      ,
      • Marinissen M.J.
      • Chiariello M.
      • Gutkind J.S.
      Regulation of gene expression by the small GTPase Rho through the ERK6 (p38γ) MAP kinase pathway.
      ), its effects on WNT3A-dependent signaling could be mediated indirectly via its effects on those pathways. Further research to clarify how PKN1 regulates receptor dynamics may greatly enhance our understanding of the mechanisms governing LRP6 cell surface expression.
      Finally, our data showing that depletion of PKN1 increases WNT3A-dependent apoptosis in melanoma cells bolster previous data that PKN1 may be relevant to cancer biology: 1) the depletion of PKN1 also promotes programmed cell death in models of multiple myeloma (
      • Tiedemann R.E.
      • Zhu Y.X.
      • Schmidt J.
      • Yin H.
      • Shi C.X.
      • Que Q.
      • Basu G.
      • Azorsa D.
      • Perkins L.M.
      • Braggio E.
      • Fonseca R.
      • Bergsagel P.L.
      • Mousses S.
      • Stewart A.K.
      Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6.
      ), 2) PKN1 is overexpressed in prostate tumors (
      • Metzger E.
      • Müller J.M.
      • Ferrari S.
      • Buettner R.
      • Schüle R.
      A novel inducible transactivation domain in the androgen receptor: implications for PRK in prostate cancer.
      ) and in certain cohorts of malignant melanoma (Fig. 2B), and 3) PKN1 is a downstream effector of PDPK1, which is activated during phosphatidylinositol 3-kinase signaling (
      • Torbett N.E.
      • Casamassima A.
      • Parker P.J.
      Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3-phosphoinositide-dependent kinase 1.
      ). Finally, our demonstration that depletion of PKN1 increases the number of cells undergoing programmed cell death upon treatment with BRAFi could contribute to improvements in therapies.

      Acknowledgments

      We thank C. Yee (Fred Hutchinson Research Institute, Seattle, WA) for cell lines utilized in this study; J. Anastas for help with experiments not included in the final version; and J. Villén (University of Washington, Seattle, WA), M. MacCoss (University of Washington, Seattle, WA), and J. Annis (Quellos High Throughput Screening Core, Seattle, WA) for helpful discussions and technical suggestions.

      REFERENCES

        • Angers S.
        • Moon R.T.
        Proximal events in Wnt signal transduction.
        Nat. Rev. Mol. Cell Biol. 2009; 10: 468-477
        • Clevers H.
        • Nusse R.
        Wnt/β-catenin signaling and disease.
        Cell. 2012; 149: 1192-1205
        • MacDonald B.T.
        • Tamai K.
        • He X.
        Wnt/β-catenin signaling: components, mechanisms, and diseases.
        Dev. Cell. 2009; 17: 9-26
        • Chien A.J.
        • Conrad W.H.
        • Moon R.T.
        A Wnt survival guide: from flies to human disease.
        J. Invest. Dermatol. 2009; 129: 1614-1627
        • Dorsky R.I.
        • Moon R.T.
        • Raible D.W.
        Control of neural crest cell fate by the Wnt signalling pathway.
        Nature. 1998; 396: 370-373
        • Kageshita T.
        • Hamby C.V.
        • Ishihara T.
        • Matsumoto K.
        • Saida T.
        • Ono T.
        Loss of β-catenin expression associated with disease progression in malignant melanoma.
        Br. J. Dermatol. 2001; 145: 210-216
        • Bachmann I.M.
        • Straume O.
        • Puntervoll H.E.
        • Kalvenes M.B.
        • Akslen L.A.
        Importance of P-cadherin, β-catenin, and Wnt5a/frizzled for progression of melanocytic tumors and prognosis in cutaneous melanoma.
        Clin. Cancer Res. 2005; 11: 8606-8614
        • Gould Rothberg B.E.
        • Berger A.J.
        • Molinaro A.M.
        • Subtil A.
        • Krauthammer M.O.
        • Camp R.L.
        • Bradley W.R.
        • Ariyan S.
        • Kluger H.M.
        • Rimm D.L.
        Melanoma prognostic model using tissue microarrays and genetic algorithms.
        J. Clin. Oncol. 2009; 27: 5772-5780
        • Maelandsmo G.M.
        • Holm R.
        • Nesland J.M.
        • Fodstad Ø.
        • Flørenes V.A.
        Reduced β-catenin expression in the cytoplasm of advanced-stage superficial spreading malignant melanoma.
        Clin. Cancer Res. 2003; 9: 3383-3388
        • Meyer S.
        • Fuchs T.J.
        • Bosserhoff A.K.
        • Hofstädter F.
        • Pauer A.
        • Roth V.
        • Buhmann J.M.
        • Moll I.
        • Anagnostou N.
        • Brandner J.M.
        • Ikenberg K.
        • Moch H.
        • Landthaler M.
        • Vogt T.
        • Wild P.J.
        A seven-marker signature and clinical outcome in malignant melanoma: a large-scale tissue-microarray study with two independent patient cohorts.
        PLoS One. 2012; 7: e38222
        • Kimelman D.
        • Xu W.
        β-Catenin destruction complex: insights and questions from a structural perspective.
        Oncogene. 2006; 25: 7482-7491
        • Davidson G.
        • Wu W.
        • Shen J.
        • Bilic J.
        • Fenger U.
        • Stannek P.
        • Glinka A.
        • Niehrs C.
        Casein kinase 1γ couples Wnt receptor activation to cytoplasmic signal transduction.
        Nature. 2005; 438: 867-872
        • Bilic J.
        • Huang Y.L.
        • Davidson G.
        • Zimmermann T.
        • Cruciat C.M.
        • Bienz M.
        • Niehrs C.
        Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.
        Science. 2007; 316: 1619-1622
        • Huang X.
        • McGann J.C.
        • Liu B.Y.
        • Hannoush R.N.
        • Lill J.R.
        • Pham V.
        • Newton K.
        • Kakunda M.
        • Liu J.
        • Yu C.
        • Hymowitz S.G.
        • Hongo J.A.
        • Wynshaw-Boris A.
        • Polakis P.
        • Harland R.M.
        • Dixit V.M.
        Phosphorylation of dishevelled by protein kinase RIPK4 regulates Wnt signaling.
        Science. 2013; 339: 1441-1445
        • Rosenbluh J.
        • Nijhawan D.
        • Cox A.G.
        • Li X.
        • Neal J.T.
        • Schafer E.J.
        • Zack T.I.
        • Wang X.
        • Tsherniak A.
        • Schinzel A.C.
        • Shao D.D.
        • Schumacher S.E.
        • Weir B.A.
        • Vazquez F.
        • Cowley G.S.
        • Root D.E.
        • Mesirov J.P.
        • Beroukhim R.
        • Kuo C.J.
        • Goessling W.
        • Hahn W.C.
        β-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.
        Cell. 2012; 151: 1457-1473
        • Ong S.E.
        • Blagoev B.
        • Kratchmarova I.
        • Kristensen D.B.
        • Steen H.
        • Pandey A.
        • Mann M.
        Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
        Mol. Cell. Proteomics. 2002; 1: 376-386
        • James R.G.
        • Biechele T.L.
        • Conrad W.H.
        • Camp N.D.
        • Fass D.M.
        • Major M.B.
        • Sommer K.
        • Yi X.
        • Roberts B.S.
        • Cleary M.A.
        • Arthur W.T.
        • MacCoss M.
        • Rawlings D.J.
        • Haggarty S.J.
        • Moon R.T.
        Bruton's tyrosine kinase revealed as a negative regulator of Wnt-β-catenin signaling.
        Sci. Signal. 2009; 2: ra25
        • Villén J.
        • Gygi S.P.
        The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.
        Nat. Protoc. 2008; 3: 1630-1638
        • Yates 3rd, J.R.
        • Eng J.K.
        • McCormack A.L.
        • Schieltz D.
        Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.
        Anal. Chem. 1995; 67: 1426-1436
        • Keller A.
        • Eng J.
        • Zhang N.
        • Li X.J.
        • Aebersold R.
        A uniform proteomics MS/MS analysis platform utilizing open XML file formats.
        Mol. Syst. Biol. 2005; 1: 2005.0017
        • Han D.K.
        • Eng J.
        • Zhou H.
        • Aebersold R.
        Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.
        Nat. Biotechnol. 2001; 19: 946-951
        • Major M.B.
        • Roberts B.S.
        • Berndt J.D.
        • Marine S.
        • Anastas J.
        • Chung N.
        • Ferrer M.
        • Yi X.
        • Stoick-Cooper C.L.
        • von Haller P.D.
        • Kategaya L.
        • Chien A.
        • Angers S.
        • MacCoss M.
        • Cleary M.A.
        • Arthur W.T.
        • Moon R.T.
        New regulators of Wnt/β-catenin signaling revealed by integrative molecular screening.
        Sci. Signal. 2008; 1: ra12
        • Szklarczyk D.
        • Franceschini A.
        • Kuhn M.
        • Simonovic M.
        • Roth A.
        • Minguez P.
        • Doerks T.
        • Stark M.
        • Muller J.
        • Bork P.
        • Jensen L.J.
        • von Mering C.
        The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored.
        Nucleic Acids Res. 2011; 39: D561-D568
        • Stark C.
        • Breitkreutz B.J.
        • Chatr-Aryamontri A.
        • Boucher L.
        • Oughtred R.
        • Livstone M.S.
        • Nixon J.
        • Van Auken K.
        • Wang X.
        • Shi X.
        • Reguly T.
        • Rust J.M.
        • Winter A.
        • Dolinski K.
        • Tyers M.
        The BioGRID interaction database: 2011 update.
        Nucleic Acids Res. 2011; 39: D698-D704
        • Keshava Prasad T.S.
        • Goel R.
        • Kandasamy K.
        • Keerthikumar S.
        • Kumar S.
        • Mathivanan S.
        • Telikicherla D.
        • Raju R.
        • Shafreen B.
        • Venugopal A.
        • Balakrishnan L.
        • Marimuthu A.
        • Banerjee S.
        • Somanathan D.S.
        • Sebastian A.
        • Rani S.
        • Ray S.
        • Harrys Kishore C.J.
        • Kanth S.
        • Ahmed M.
        • Kashyap M.K.
        • Mohmood R.
        • Ramachandra Y.L.
        • Krishna V.
        • Rahiman B.A.
        • Mohan S.
        • Ranganathan P.
        • Ramabadran S.
        • Chaerkady R.
        • Pandey A.
        Human Protein Reference Database—2009 update.
        Nucleic Acids Res. 2009; 37: D767-D772
        • Shannon P.
        • Markiel A.
        • Ozier O.
        • Baliga N.S.
        • Wang J.T.
        • Ramage D.
        • Amin N.
        • Schwikowski B.
        • Ideker T.
        Cytoscape: a software environment for integrated models of biomolecular interaction networks.
        Genome Res. 2003; 13: 2498-2504
        • Biechele T.L.
        • Moon R.T.
        Assaying β-catenin/TCF transcription with β-catenin/TCF transcription-based reporter constructs.
        Methods Mol. Biol. 2008; 468: 99-110
        • James R.G.
        • Davidson K.C.
        • Bosch K.A.
        • Biechele T.L.
        • Robin N.C.
        • Taylor R.J.
        • Major M.B.
        • Camp N.D.
        • Fowler K.
        • Martins T.J.
        • Moon R.T.
        WIKI4, a novel inhibitor of tankyrase and Wnt/β-catenin signaling.
        PLoS One. 2012; 7: e50457
        • Angers S.
        • Thorpe C.J.
        • Biechele T.L.
        • Goldenberg S.J.
        • Zheng N.
        • MacCoss M.J.
        • Moon R.T.
        The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-β-catenin pathway by targeting Dishevelled for degradation.
        Nat. Cell Biol. 2006; 8: 348-357
        • Biechele T.L.
        • Kulikauskas R.M.
        • Toroni R.A.
        • Lucero O.M.
        • Swift R.D.
        • James R.G.
        • Robin N.C.
        • Dawson D.W.
        • Moon R.T.
        • Chien A.J.
        Wnt/β-catenin signaling and AXIN1 regulate apoptosis triggered by inhibition of the mutant kinase BRAFV600E in human melanoma.
        Sci. Signal. 2012; 5: ra3
        • Major M.B.
        • Camp N.D.
        • Berndt J.D.
        • Yi X.
        • Goldenberg S.J.
        • Hubbert C.
        • Biechele T.L.
        • Gingras A.C.
        • Zheng N.
        • Maccoss M.J.
        • Angers S.
        • Moon R.T.
        Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
        Science. 2007; 316: 1043-1046
        • Mammen A.L.
        • Huganir R.L.
        • O'Brien R.J.
        Redistribution and stabilization of cell surface glutamate receptors during synapse formation.
        J. Neurosci. 1997; 17: 7351-7358
        • Hoek K.S.
        • Schlegel N.C.
        • Brafford P.
        • Sucker A.
        • Ugurel S.
        • Kumar R.
        • Weber B.L.
        • Nathanson K.L.
        • Phillips D.J.
        • Herlyn M.
        • Schadendorf D.
        • Dummer R.
        Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature.
        Pigment Cell Res. 2006; 19: 290-302
        • Hoek K.S.
        DNA microarray analyses of melanoma gene expression: a decade in the mines.
        Pigment Cell Res. 2007; 20: 466-484
        • Liu G.
        • Bafico A.
        • Harris V.K.
        • Aaronson S.A.
        A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor.
        Mol. Cell. Biol. 2003; 23: 5825-5835
        • Miller B.W.
        • Lau G.
        • Grouios C.
        • Mollica E.
        • Barrios-Rodiles M.
        • Liu Y.
        • Datti A.
        • Morris Q.
        • Wrana J.L.
        • Attisano L.
        Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway.
        Mol. Syst. Biol. 2009; 5: 315
        • Hilger M.
        • Mann M.
        Triple SILAC to determine stimulus specific interactions in the Wnt pathway.
        J. Proteome Res. 2012; 11: 982-994
        • Taelman V.F.
        • Dobrowolski R.
        • Plouhinec J.L.
        • Fuentealba L.C.
        • Vorwald P.P.
        • Gumper I.
        • Sabatini D.D.
        • De Robertis E.M.
        Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes.
        Cell. 2010; 143: 1136-1148
        • Ding V.W.
        • Chen R.H.
        • McCormick F.
        Differential regulation of glycogen synthase kinase 3β by insulin and Wnt signaling.
        J. Biol. Chem. 2000; 275: 32475-32481
        • Damsky W.E.
        • Curley D.P.
        • Santhanakrishnan M.
        • Rosenbaum L.E.
        • Platt J.T.
        • Gould Rothberg B.E.
        • Taketo M.M.
        • Dankort D.
        • Rimm D.L.
        • McMahon M.
        • Bosenberg M.
        β-Catenin signaling controls metastasis in Braf-activated Pten-deficient melanomas.
        Cancer Cell. 2011; 20: 741-754
        • Delmas V.
        • Beermann F.
        • Martinozzi S.
        • Carreira S.
        • Ackermann J.
        • Kumasaka M.
        • Denat L.
        • Goodall J.
        • Luciani F.
        • Viros A.
        • Demirkan N.
        • Bastian B.C.
        • Goding C.R.
        • Larue L.
        β-Catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development.
        Genes Dev. 2007; 21: 2923-2935
        • Terabayashi T.
        • Funato Y.
        • Fukuda M.
        • Miki H.
        A coated vesicle-associated kinase of 104 kDa (CVAK104) induces lysosomal degradation of frizzled 5 (Fzd5).
        J. Biol. Chem. 2009; 284: 26716-26724
        • Mukai A.
        • Yamamoto-Hino M.
        • Awano W.
        • Watanabe W.
        • Komada M.
        • Goto S.
        Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt.
        EMBO J. 2010; 29: 2114-2125
        • Carmon K.S.
        • Lin Q.
        • Gong X.
        • Thomas A.
        • Liu Q.
        LGR5 interacts and cointernalizes with Wnt receptors to modulate Wnt/β-catenin signaling.
        Mol. Cell. Biol. 2012; 32: 2054-2064
        • Jiang Y.
        • He X.
        • Howe P.H.
        Disabled-2 (Dab2) inhibits Wnt/β-catenin signalling by binding LRP6 and promoting its internalization through clathrin.
        EMBO J. 2012; 31: 2336-2349
        • Metcalfe C.
        • Bienz M.
        Inhibition of GSK3 by Wnt signalling—two contrasting models.
        J. Cell Sci. 2011; 124: 3537-3544
        • Cselenyi C.S.
        • Jernigan K.K.
        • Tahinci E.
        • Thorne C.A.
        • Lee L.A.
        • Lee E.
        LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of β-catenin.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 8032-8037
        • Mi K.
        • Dolan P.J.
        • Johnson G.V.
        The low density lipoprotein receptor-related protein 6 interacts with glycogen synthase kinase 3 and attenuates activity.
        J. Biol. Chem. 2006; 281: 4787-4794
        • Piao S.
        • Lee S.H.
        • Kim H.
        • Yum S.
        • Stamos J.L.
        • Xu Y.
        • Lee S.J.
        • Lee J.
        • Oh S.
        • Han J.K.
        • Park B.J.
        • Weis W.I.
        • Ha N.C.
        Direct inhibition of GSK3beta by the phosphorylated cytoplasmic domain of LRP6 in Wnt/β-catenin signaling.
        PLoS One. 2008; 3: e4046
        • Wu G.
        • Huang H.
        • Garcia Abreu J.
        • He X.
        Inhibition of GSK3 phosphorylation of β-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6.
        PLoS One. 2009; 4: e4926
        • Balendran A.
        • Casamayor A.
        • Deak M.
        • Paterson A.
        • Gaffney P.
        • Currie R.
        • Downes C.P.
        • Alessi D.R.
        PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2.
        Curr. Biol. 1999; 9: 393-404
        • Koh H.
        • Lee K.H.
        • Kim D.
        • Kim S.
        • Kim J.W.
        • Chung J.
        Inhibition of Akt and its anti-apoptotic activities by tumor necrosis factor-induced protein kinase C-related kinase 2 (PRK2) cleavage.
        J. Biol. Chem. 2000; 275: 34451-34458
        • Wick M.J.
        • Dong L.Q.
        • Riojas R.A.
        • Ramos F.J.
        • Liu F.
        Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1.
        J. Biol. Chem. 2000; 275: 40400-40406
        • Yasui T.
        • Sakakibara-Yada K.
        • Nishimura T.
        • Morita K.
        • Tada S.
        • Mosialos G.
        • Kieff E.
        • Kikutani H.
        Protein kinase N1, a cell inhibitor of Akt kinase, has a central role in quality control of germinal center formation.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 21022-21027
        • Lu Y.
        • Settleman J.
        The Drosophila Pkn protein kinase is a Rho/Rac effector target required for dorsal closure during embryogenesis.
        Genes Dev. 1999; 13: 1168-1180
        • Mukai H.
        The structure and function of PKN, a protein kinase having a catalytic domain homologous to that of PKC.
        J. Biochem. 2003; 133: 17-27
        • Mellor H.
        • Flynn P.
        • Nobes C.D.
        • Hall A.
        • Parker P.J.
        PRK1 is targeted to endosomes by the small GTPase, RhoB.
        J. Biol. Chem. 1998; 273: 4811-4814
        • Gampel A.
        • Parker P.J.
        • Mellor H.
        Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.
        Curr. Biol. 1999; 9: 955-958
        • Torbett N.E.
        • Casamassima A.
        • Parker P.J.
        Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3-phosphoinositide-dependent kinase 1.
        J. Biol. Chem. 2003; 278: 32344-32351
        • Manser C.
        • Stevenson A.
        • Banner S.
        • Davies J.
        • Tudor E.L.
        • Ono Y.
        • Leigh P.N.
        • McLoughlin D.M.
        • Shaw C.E.
        • Miller C.C.
        Deregulation of PKN1 activity disrupts neurofilament organisation and axonal transport.
        FEBS Lett. 2008; 582: 2303-2308
        • Collazos A.
        • Michael N.
        • Whelan R.D.
        • Kelly G.
        • Mellor H.
        • Pang L.C.
        • Totty N.
        • Parker P.J.
        Site recognition and substrate screens for PKN family proteins.
        Biochem. J. 2011; 438: 535-543
        • Sun W.
        • Vincent S.
        • Settleman J.
        • Johnson G.L.
        MEK kinase 2 binds and activates protein kinase C-related kinase 2. Bifurcation of kinase regulatory pathways at the level of an MAPK kinase kinase.
        J. Biol. Chem. 2000; 275: 24421-24428
        • Marinissen M.J.
        • Chiariello M.
        • Gutkind J.S.
        Regulation of gene expression by the small GTPase Rho through the ERK6 (p38γ) MAP kinase pathway.
        Genes Dev. 2001; 15: 535-553
        • Tiedemann R.E.
        • Zhu Y.X.
        • Schmidt J.
        • Yin H.
        • Shi C.X.
        • Que Q.
        • Basu G.
        • Azorsa D.
        • Perkins L.M.
        • Braggio E.
        • Fonseca R.
        • Bergsagel P.L.
        • Mousses S.
        • Stewart A.K.
        Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6.
        Blood. 2010; 115: 1594-1604
        • Metzger E.
        • Müller J.M.
        • Ferrari S.
        • Buettner R.
        • Schüle R.
        A novel inducible transactivation domain in the androgen receptor: implications for PRK in prostate cancer.
        EMBO J. 2003; 22: 270-280