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Regulation of Catalytic and Non-catalytic Functions of the Drosophila Ste20 Kinase Slik by Activation Segment Phosphorylation*

  • Vincent Panneton
    Affiliations
    Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada

    Molecular Biology Program, Université de Montréal, Montreal, Quebec H3T 3J7, Canada
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  • Apurba Nath
    Affiliations
    Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada

    Molecular Biology Program, Université de Montréal, Montreal, Quebec H3T 3J7, Canada
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  • Fadi Sader
    Footnotes
    Affiliations
    Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada
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  • Nathalie Delaunay
    Affiliations
    Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada
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  • Ariane Pelletier
    Affiliations
    Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada
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  • Dominic Maier
    Affiliations
    Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada

    Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Karen Oh
    Affiliations
    Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada
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  • David R. Hipfner
    Correspondence
    To whom correspondence should be addressed: Institut de Recherches Cliniques de Montréal, 110, Ave. des Pins Ouest, Montreal, QC, Canada. Tel.: 514-987-5508; Fax: 514-987-5991.
    Affiliations
    Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada

    Molecular Biology Program, Université de Montréal, Montreal, Quebec H3T 3J7, Canada

    Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada

    Department of Medicine, Université de Montréal, Montreal, Quebec H3C 3J7, Canada
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  • Author Footnotes
    * This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant (to D. H.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Present address: Départment de stomatologie, Faculté de médecine dentaire, Université de Montréal, Montreal, QC, H3C 3J7, Canada.
Open AccessPublished:July 13, 2015DOI:https://doi.org/10.1074/jbc.M115.645952
      Protein kinases carry out important functions in cells both by phosphorylating substrates and by means of regulated non-catalytic activities. Such non-catalytic functions have been ascribed to many kinases, including some members of the Ste20 family. The Drosophila Ste20 kinase Slik phosphorylates and activates Moesin in developing epithelial tissues to promote epithelial tissue integrity. It also functions non-catalytically to promote epithelial cell proliferation and tissue growth. We carried out a structure-function analysis to determine how these two distinct activities of Slik are controlled. We find that the conserved C-terminal coiled-coil domain of Slik, which is necessary and sufficient for apical localization of the kinase in epithelial cells, is not required for Moesin phosphorylation but is critical for the growth-promoting function of Slik. Slik is auto- and trans-phosphorylated in vivo. Phosphorylation of at least two of three conserved sites in the activation segment is required for both efficient catalytic activity and non-catalytic signaling. Slik function is thus dependent upon proper localization of the kinase via the C-terminal coiled-coil domain and activation via activation segment phosphorylation, which enhances both phosphorylation of substrates like Moesin and engagement of effectors of its non-catalytic growth-promoting activity.

      Introduction

      Protein kinases play key roles in most cellular processes through their well known catalytic function of reversibly phosphorylating their substrates. Catalytic activity-independent functions have been ascribed to an increasing number of these proteins, leading to the emerging view of protein kinases as molecular switches (analogous to small GTPases) rather than just enzymes (
      • Shaw A.S.
      • Kornev A.P.
      • Hu J.
      • Ahuja L.G.
      • Taylor S.S.
      Kinases and pseudokinases: lessons from RAF.
      ). The Sterile-20 (Ste20)
      The abbreviations used are: Ste20
      Sterile-20
      CCD
      C-terminal coiled-coiled repeat-containing domain
      NCD
      non-conserved central linker domain
      Slk
      Ste20-like kinase
      ERM
      ezrin/radixin/moesin
      dsRNA
      double-stranded RNA
      Moe.CT
      C-terminal actin-binding domain of Moesin
      DP
      disc proper
      PM
      peripodial membrane
      ptc
      patched
      ap
      apterous
      nub
      nubbin
      PI(4,5)P2
      phosphatidylinositol-4,5-bisphosphate
      EdU
      5-ethynyl-2′-deoxyuridine
      PAK
      p21-activated kinase
      GCK
      germinal center kinase
      Lok
      lymphocyte-oriented kinase.
      kinases are a large group of Ser/Thr kinases with 28 members in humans divided into 2 distinct families (PAK- and GCK-like) and 10 subfamilies. Outside of the catalytic domain, which shows homology to the yeast kinase Ste20p, the 10 subfamilies show little sequence similarity to one another. Despite their structural diversity, many Ste20 kinases appear to regulate a few common cellular functions including cell proliferation and survival, cytoskeletal dynamics, and ion transport (
      • Delpire E.
      The mammalian family of sterile 20p-like protein kinases.
      ). Some of the Ste20 kinases are well known proteins with clearly defined physiological functions, e.g. the PAK subfamilies in cytoskeletal regulation (
      • Kumar R.
      • Gururaj A.E.
      • Barnes C.J.
      p21-activated kinases in cancer.
      ), the GCK-II subfamily kinases Mst1/Mst2/Hippo in tissue growth and tumor suppression (
      • Avruch J.
      • Zhou D.
      • Fitamant J.
      • Bardeesy N.
      • Mou F.
      • Barrufet L.R.
      Protein kinases of the Hippo pathway: regulation and substrates.
      ), and the GCK-VI subfamily kinases Osr1 and Spak in regulating ion channels (
      • Gagnon K.B.
      • Delpire E.
      Molecular physiology of SPAK and OSR1: two Ste20-related protein kinases regulating ion transport.
      ). Many others are less well characterized. Although most of the identified functions of these kinases have been attributed to substrate phosphorylation, catalytic activity-independent functions have been proposed for some (
      • Rauch J.
      • Volinsky N.
      • Romano D.
      • Kolch W.
      The secret life of kinases: functions beyond catalysis.
      ).
      The GCK-V subfamily is composed of two kinases in mammals, Slk and Lok/Stk10. These kinases are characterized by an N-terminal Ste20-like kinase domain and a C-terminal coiled-coiled repeat-containing domain (CCD) connected by a non-conserved central linker domain (NCD) of variable length. Slk has been implicated in the regulation of a variety of cellular processes, including cell cycle progression (
      • O'Reilly P.G.
      • Wagner S.
      • Franks D.J.
      • Cailliau K.
      • Browaeys E.
      • Dissous C.
      • Sabourin L.A.
      The Ste20-like kinase SLK is required for cell cycle progression through G2.
      ), apoptosis (
      • Hao W.
      • Takano T.
      • Guillemette J.
      • Papillon J.
      • Ren G.
      • Cybulsky A.V.
      Induction of apoptosis by the Ste20-like kinase SLK, a germinal center kinase that activates apoptosis signal-regulating kinase and p38.
      ,
      • Sabourin L.A.
      • Rudnicki M.A.
      Induction of apoptosis by SLK, a Ste20-related kinase.
      ), and cell migration (
      • Burakov A.V.
      • Zhapparova O.N.
      • Kovalenko O.V.
      • Zinovkina L.A.
      • Potekhina E.S.
      • Shanina N.A.
      • Weiss D.G.
      • Kuznetsov S.A.
      • Nadezhdina E.S.
      Ste20-related protein kinase LOSK (SLK) controls microtubule radial array in interphase.
      ,
      • Quizi J.L.
      • Baron K.
      • Al-Zahrani K.N.
      • O'Reilly P.
      • Sriram R.K.
      • Conway J.
      • Laurin A.A.
      • Sabourin L.A.
      SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration.
      • Wagner S.
      • Flood T.A.
      • O'Reilly P.
      • Hume K.
      • Sabourin L.A.
      Association of the Ste20-like kinase (SLK) with the microtubule: role in Rac1-mediated regulation of actin dynamics during cell adhesion and spreading.
      ). The one fundamental function of these kinases that is evolutionarily conserved is the regulation of ezrin/radixin/moesin (ERM) family proteins. ERM proteins are important regulators of the cell cortex, acting as cross-linkers to connect the actin cytoskeleton to diverse transmembrane proteins at the plasma membrane. Their ability to do so requires phosphorylation of a highly conserved Thr residue near the C terminus, which disrupts autoinhibitory interactions between the N- and C-terminal domains (
      • Fehon R.G.
      • McClatchey A.I.
      • Bretscher A.
      Organizing the cell cortex: the role of ERM proteins.
      ). In Drosophila, phosphorylation at the critical residue (and thus activation) of Moesin was negligible in epithelial cells mutant for Slik, the single GCK-V orthologue in flies (
      • Hipfner D.R.
      • Keller N.
      • Cohen S.M.
      Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth.
      ). Recently, depletion of Slk and/or Lok in mammalian cells was shown to strongly reduce ERM protein phosphorylation (
      • Machicoane M.
      • de Frutos C.A.
      • Fink J.
      • Rocancourt M.
      • Lombardi Y.
      • Garel S.
      • Piel M.
      • Echard A.
      SLK-dependent activation of ERMs controls LGN-NuMA localization and spindle orientation.
      ,
      • Viswanatha R.
      • Ohouo P.Y.
      • Smolka M.B.
      • Bretscher A.
      Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells.
      ). Mutating or depleting these GCK-V kinases in Drosophila or mammalian cells produces cellular and tissue phenotypes similar to those caused by mutating or depleting the ERM proteins themselves, including impaired epithelial tissue integrity (
      • Hipfner D.R.
      • Keller N.
      • Cohen S.M.
      Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth.
      ,
      • Speck O.
      • Hughes S.C.
      • Noren N.K.
      • Kulikauskas R.M.
      • Fehon R.G.
      Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity.
      ), disrupted organization of apical microvilli (
      • Hipfner D.R.
      • Keller N.
      • Cohen S.M.
      Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth.
      ,
      • Karagiosis S.A.
      • Ready D.F.
      Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis.
      ), reduced cortical stiffness (
      • Carreno S.
      • Kouranti I.
      • Glusman E.S.
      • Fuller M.T.
      • Echard A.
      • Payre F.
      Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells.
      ,
      • Kunda P.
      • Pelling A.E.
      • Liu T.
      • Baum B.
      Moesin controls cortical rigidity, cell rounding, and spindle morphogenesis during mitosis.
      ), and misorientation of the mitotic spindle and cytokinesis defects (
      • Machicoane M.
      • de Frutos C.A.
      • Fink J.
      • Rocancourt M.
      • Lombardi Y.
      • Garel S.
      • Piel M.
      • Echard A.
      SLK-dependent activation of ERMs controls LGN-NuMA localization and spindle orientation.
      ,
      • Carreno S.
      • Kouranti I.
      • Glusman E.S.
      • Fuller M.T.
      • Echard A.
      • Payre F.
      Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells.
      ,
      • Kunda P.
      • Pelling A.E.
      • Liu T.
      • Baum B.
      Moesin controls cortical rigidity, cell rounding, and spindle morphogenesis during mitosis.
      ). Taken together, these studies strongly highlight the importance of GCK-V kinase function in ERM regulation to control cell structure and epithelial organization and their potential involvement in pathological conditions where these are affected.
      Drosophila slik mutants have an additional developmental phenotype that is separable from Moesin regulation. The mutant animals grow slowly, requiring approximately three times as long to reach full size in the larval stage before subsequently dying (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ). Overexpression of Slik in wing imaginal discs (the epithelial precursors to the adult wing) has the opposite effect, increasing cell proliferation rates and causing overgrowth of the wing (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ). Thus, as with other Ste20 kinases, including Hippo/Mst and Tao1 (
      • Harvey K.F.
      • Pfleger C.M.
      • Hariharan I.K.
      The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis.
      • Pantalacci S.
      • Tapon N.
      • Léopold P.
      The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila.
      ,
      • Udan R.S.
      • Kango-Singh M.
      • Nolo R.
      • Tao C.
      • Halder G.
      Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway.
      ,
      • Wu S.
      • Huang J.
      • Dong J.
      • Pan D.
      hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts.
      ,
      • Poon C.L.
      • Lin J.I.
      • Zhang X.
      • Harvey K.F.
      The sterile 20-like kinase Tao-1 controls tissue growth by regulating the Salvador-Warts-Hippo pathway.
      • Boggiano J.C.
      • Vanderzalm P.J.
      • Fehon R.G.
      Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway.
      ), Slik regulates tissue growth. There are two unusual features of Slik-driven growth. First, Slik expression had nonautonomous effects, with not only Slik-expressing cells but also surrounding cells displaying the proliferative response (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ). Second, a point mutant form of the kinase that is expected to impair catalytic activity also induced the proliferative response (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ). This suggests that the effect does not require catalytic activity, in line with the catalytic activity-independent allosteric functions of a number of kinases and pseudokinases (
      • Shaw A.S.
      • Kornev A.P.
      • Hu J.
      • Ahuja L.G.
      • Taylor S.S.
      Kinases and pseudokinases: lessons from RAF.
      ).
      Because of its involvement in both processes, Slik is well positioned to serve as one of the mechanisms for coordinating epithelial cell organization with epithelial tissue growth (
      • Tepass U.
      The apical polarity protein network in Drosophila epithelial cells: regulation of polarity, junctions, morphogenesis, cell growth, and survival.
      ). To understand how these distinct activities of Slik may be regulated, we undertook a structure-function analysis of this kinase. Our results confirm that Slik kinase activity is not required for its ability to promote proliferation and point to both apical localization via the CCD and phosphorylation as key mechanisms regulating both the epithelial integrity (catalytic) and growth-promoting (non-catalytic) functions of Slik.

      Results

      The imaginal discs in Drosophila are a series of epithelial sacs that give rise to the adult appendages and body wall. Initially formed from invaginations of up to 50 cells in the embryonic epidermis, the discs grow rapidly during the 4 days of larval life to reach up to ∼50,000 cells. The discs are divided into two continuous but morphologically distinct epithelial monolayers with their apical domains apposed and enclosing a central lumen. On one side of the lumen is the disc proper (DP) composed of pseudostratified columnar epithelial cells that will make most of the adult structures. DAPI staining of nuclei reveals the tight packing of DP cells (Fig. 1A). On the other side is the peripodial membrane (PM), made up of large flattened squamous cells with distinctly spaced nuclei (Fig. 1A). This difference in nuclear spacing makes it easy to distinguish the two layers both in XY (Fig. 1A) and XZ (Fig. 1B) confocal optical sections. EdU incorporation assays reveal that DP cells are highly proliferative, whereas PM cells are not (Fig. 1C).
      Transgenic expression of wild-type Slik in DP cells using any one of several different GAL4 drivers (e.g. patched (ptc)-GAL4, apterous (ap)-GAL4, nubbin-GAL4) has a striking non-autonomous effect on the PM cells (Fig. 1, D and G, and not shown) (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ). For example, in discs where ptc-GAL4 was used to drive expression of Slik in a central stripe of DP cells (marked by co-expression of GFP) (Fig. 1D), a large and abnormal cluster of densely packed PM cells appeared in a position directly overlying the Slik-expressing DP cells, as if these PM cells were responding to a signal from the Slik-expressing cells by proliferating. EdU incorporation assays confirmed that the PM cells within these clusters were rapidly proliferating (Fig. 1E), unlike normal PM cells (Fig. 1C). In Z-sections it was clear that this cluster of proliferating cells was indeed in the PM and distinct from the DP and that the GAL4 driver was not active in these cells (based on expression of GFP) (Fig. 1F). Slik expression using an independent GAL4 driver, ap-GAL4, which drives expression throughout the dorsal compartment of the DP (marked by co-expression of GFP), led to the appearance of a similar dense cluster of overproliferating PM cells overlying the dorsal compartment (Fig. 1, G–I).
      As we previously reported (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ), the same non-autonomous effect was observed when expressing a form of Slik (Slikkd) with the critical Asp residue involved in binding the catalytic magnesium ion (Asp176) mutated to Asn, which is expected to disrupt catalytic activity (Fig. 1, J–L). To confirm that this pro-proliferative effect is independent of Slik kinase activity, we tested the catalytic activity of Slikkd in cells and in vitro using Moesin phosphorylation as a readout (
      • Hipfner D.R.
      • Keller N.
      • Cohen S.M.
      Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth.
      ). Treatment of S2 cells with a dsRNA targeting the Slik 5′-untranslated region (UTR) efficiently depleted Slik protein and led to a strong reduction of Moesin Thr556 phosphorylation in the cells (detected using a phosphospecific antiserum) (Fig. 2A). Transfection of a wild-type slik transgene lacking the 5′-UTR into slik 5′-UTR dsRNA-treated cells restored Moesin phosphorylation, whereas the comparable slikkd mutant did not (Fig. 2A). In in vitro kinase assays using [γ-32P]ATP and kinase immunoprecipitated from transfected S2 cells, wild-type Slik phosphorylated a truncated form of Moesin consisting of the C-terminal actin-binding domain (Moe.CT) (Fig. 2B). Phosphorylation of Moe.CT was abolished by mutation of the critical regulatory Thr (Thr556 in Moesin) to Ala, confirming that Slik specifically phosphorylates this residue (Fig. 2B). In the same assay, Slikkd did not show any activity (Fig. 2B). We conclude that Slikkd does indeed lack catalytic activity.
      Catalytically inactive mutant forms of some kinases can dimerize with and activate wild-type kinases; for example, catalytically inactive BRAF can activate CRAF in heterodimers (
      • Heidorn S.J.
      • Milagre C.
      • Whittaker S.
      • Nourry A.
      • Niculescu-Duvas I.
      • Dhomen N.
      • Hussain J.
      • Reis-Filho J.S.
      • Springer C.J.
      • Pritchard C.
      • Marais R.
      Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
      ). To rule out the possibility that Slikkd promotes proliferation by activating the wild-type kinase through dimerization, we expressed it in wing discs depleted of endogenous Slik. ptc-GAL4-driven expression of a slik 5′-UTR dsRNA transgene efficiently depleted endogenous Slik (Fig. 2C) and did not affect PM cell proliferation (Fig. 2D). In this background, reintroduction of Slikkd had a similar effect as wild-type Slik in promoting non-autonomous proliferation of PM cells (Fig. 2, E and F). Taken together these results confirm that the ability of Slik to drive proliferation does not require catalytic activity.

      Slik Specifically Stimulates Non-autonomous Proliferation

      Slik expression has pleiotropic effects in discs, accelerating cell proliferation rates and tissue growth while also increasing apoptosis (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ). Altering these processes can have compensatory non-autonomous effects on tissue growth (
      • Johnston L.A.
      Competitive interactions between cells: death, growth, and geography.
      ,
      • Ryoo H.D.
      • Gorenc T.
      • Steller H.
      Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways.
      ). To see if the non-autonomous pro-proliferative effects of Slik could be an indirect consequence of its effect on growth and cell survival, we tested whether expression of other genes that affect these processes in DP cells could have a similar effect on PM morphology. Genes whose expression accelerates primarily cell growth, such as the phosphatidylinositol 3-kinase catalytic subunit Dp110 (
      • Leevers S.J.
      • Weinkove D.
      • MacDougall L.K.
      • Hafen E.
      • Waterfield M.D.
      The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth.
      ), Myc (
      • Johnston L.A.
      • Prober D.A.
      • Edgar B.A.
      • Eisenman R.N.
      • Gallant P.
      Drosophila myc regulates cellular growth during development.
      ), and activated Ras85D (
      • Prober D.A.
      • Edgar B.A.
      Ras1 promotes cellular growth in the Drosophila wing.
      ) had strong cell autonomous effects in DP cells (as evidenced by an obvious increase in space between nuclei within the expression domain) but did not lead to the appearance of a cluster of PM cell nuclei as did Slik (Fig. 3, A–E). CyclinD and Cdk4, which together promote cell growth in Drosophila (
      • Datar S.A.
      • Jacobs H.W.
      • de la Cruz A.F.
      • Lehner C.F.
      • Edgar B.A.
      The Drosophila cyclin D-Cdk4 complex promotes cellular growth.
      ), also did not noticeably alter PM morphology (Fig. 3F). Accelerating cell division rates by co-expressing the cell cycle regulator E2F and its co-factor DP (
      • Du W.
      • Xie J.E.
      • Dyson N.
      Ectopic expression of dE2F and dDP induces cell proliferation and death in the Drosophila eye.
      ) led to the expected autonomous reduction in DP cell size (and hence decreased nuclear spacing) without affecting PM morphology (Fig. 3G). Manipulations that coordinately accelerate cell proliferation and tissue growth, such as expression of the miRNA bantam (
      • Hipfner D.R.
      • Weigmann K.
      • Cohen S.M.
      The bantam gene regulates Drosophila growth.
      ) or expression of a dominant negative form of the tumor suppressor Fat (FatΔicd) (
      • Matakatsu H.
      • Blair S.S.
      Separating the adhesive and signaling functions of the Fat and Dachsous protocadherins.
      ), induced robust overgrowth in adult wings (data not shown) but did not noticeably alter PM morphology (Fig. 3, H–I). Finally, induction of apoptosis by expressing either the tumor necrosis factor ligand Eiger (
      • Igaki T.
      • Kanda H.
      • Yamamoto-Goto Y.
      • Kanuka H.
      • Kuranaga E.
      • Aigaki T.
      • Miura M.
      Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway.
      ) or the pro-apoptotic protein Hid (
      • Grether M.E.
      • Abrams J.M.
      • Agapite J.
      • White K.
      • Steller H.
      The head involution defective gene of Drosophila melanogaster functions in programmed cell death.
      ), which had obvious effects on DP morphology (Eiger) and DP cell survival (Hid), did not noticeably alter PM morphology (Fig. 3, J–L). The fact that each of these regulators produced robust cell autonomous effects in DP cells without affecting PM morphology strongly suggests that the non-autonomous effect of Slik on cell proliferation is the result of Slik signaling rather that an indirect consequence of altered proliferation or apoptosis.

      Slik-driven Tissue Growth, but Not Moesin Phosphorylation, Depends on Proper Localization

      In immunostainings, Slik is diffusely localized throughout imaginal disc cells and is concentrated apically, where the majority of P-Moe staining is observed (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ). To see which of the three domains of Slik (kinase, NCD, or CCD) (Fig. 4A) might mediate this localization, we generated and expressed transgenes encoding the domains either individually or in various combinations. A Myc-tagged full-length form of Slik recapitulated the normal localization pattern (Fig. 4, B and C). We previously reported that expression of just the kinase domain (Slikkin) caused a redistribution of P-Moe away from the apical domain in wing disc cells (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ), implying that localization of the kinase was affected in the absence of the linker and/or CCD. In fact, Slikkin accumulated primarily in the nucleus of imaginal disc cells, as did a form of Slik lacking just the CCD (SlikΔCCD) (Fig. 4, D and E). Conversely, the CCD of Slik was sufficient for apical localization (Fig. 4F). The comparable domain of mammalian Lok mediates apical microvilli localization of the kinase in cultured cells, suggesting it is a conserved localization domain (
      • Viswanatha R.
      • Ohouo P.Y.
      • Smolka M.B.
      • Bretscher A.
      Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells.
      ).
      Domains outside of the kinase domain of some protein kinases are responsible for their non-catalytic activities (
      • Rauch J.
      • Volinsky N.
      • Romano D.
      • Kolch W.
      The secret life of kinases: functions beyond catalysis.
      ). For example, the regulatory domain of c-Raf alone is sufficient to mediate c-Raf-dependent inhibition of Mst2 (
      • O'Neill E.
      • Rushworth L.
      • Baccarini M.
      • Kolch W.
      Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1.
      ). Therefore, we hypothesized that the CCD might not only localize Slik in cells but also directly mediate its growth-promoting activity. To test this, we examined the requirement for the CCD in Slik function. The CCD was dispensable for Moesin phosphorylation in S2 cells (Fig. 4G), consistent with the in vivo effects of Slikkin expression on P-Moe re-distribution referred to above (
      • Hipfner D.R.
      • Cohen S.M.
      The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.
      ). In contrast, removal of the CCD abrogated the effect of Slik on proliferation (Fig. 4, H–J). Although required, the CCD alone was not sufficient to stimulate proliferation (Fig. 4K). Together these results imply that multiple regions of the protein are required for non-catalytic signaling. We conclude that the CCD is important for localizing Slik within cells, restricting Moesin phosphorylation to the appropriate apical site and positioning the protein to activate pro-proliferative signaling.
      Moesin activation occurs at sites of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) accumulation, as its phosphorylation is dependent upon prior binding to PI(4,5)P2 (
      • Fievet B.T.
      • Gautreau A.
      • Roy C.
      • Del Maestro L.
      • Mangeat P.
      • Louvard D.
      • Arpin M.
      Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin.
      ). To see if this might also be involved in membrane recruitment of Slik, we tested the ability of the CCD to bind to phosphoinositides. In pulldown assays of cell lysates from S2 cells expressing SlikCCD, we did not detect interaction of the CCD with any phosphoinositides (Fig. 4L). Pulldown of the PI(4,5)P2 binding PH domain of PLC-δ from S2 cell lysates using PI(4,5)P2-coupled beads confirmed that the assay was working (Fig. 4L). We conclude that apical localization of Slik likely does not involve direct interaction of the CCD with membrane phosphoinositides.

      Regulation of Slik Catalytic Activity by Phosphorylation

      Both in wing discs and in S2 cells, Slik is a phosphoprotein, as evidenced by an increase in mobility in SDS-PAGE gels after treatment of lysates with λ-phosphatase (Fig. 5A). At least some of this is likely attributable to autophosphorylation, as Slik is capable of phosphorylating itself in in vitro kinase assays (Fig. 5B). To see if Slik may also be a substrate of other kinases, we compared the phosphorylation status of transfected Slik and Slikkd in S2 cells depleted of endogenous Slik. As expected, transfected Slik shifted in response to λ-phosphatase treatment (Fig. 5C). Interestingly, we observed a very similar shift of Slikkd in response to λ-phosphatase treatment (Fig. 5C). This suggests that Slik is also phosphorylated and potentially regulated by other kinases.
      Figure thumbnail gr5
      FIGURE 5Slik is a phosphoprotein in vivo. Western blot of lysates from S2 cells or third-instar wing discs treated without (−) or with (+) λ-phosphatase, probed with anti-Slik antibody, reveals that Slik is a phosphoprotein in cells and in tissues. B, in vitro kinase assay using Slik or Slikkd immunoprecipitated from S2 cells. 32P-Labeling indicates that wild-type Slik but not Slikkd is capable of autophosphorylation. C, Western blot of lysates from S2 cells treated without (−) or with (+) dsRNA targeting the 5′-UTR of slik and transfected with empty vector (−) or transgenes encoding Slik or Slikkd. Depletion of endogenous Slik was efficient. Despite being unable to autophosphorylate, Slikkd shows a similar phosphorylation shift as wild-type Slik after λ-phosphatase treatment, suggesting that Slik is phosphorylated by other kinases. D, summary of phosphorylation site mapping by LC-MS/MS. Sites in yellow were identified in Slik isolated from S2 cells, sites in blue were identified in Slik from wing discs, and sites in green were identified in both. Sites identified more than five times in the Phosphosite database for mammalian Slk are indicated below.
      We used a LC-MS/MS approach to map phosphorylated residues in Slik. We carried out this analysis on Slik protein isolated from transfected S2 cells and on endogenous Slik isolated from third instar larvae. All 19 of the phosphosites we identified (Table 1) mapped to either the kinase domain or NCD, with 15 of them observed in both S2 cells and embryos (Fig. 5D). Two clusters of phosphorylation sites, one in the activation segment (Thr186, Thr192, Thr196) and one just beyond the C-terminal end of the kinase domain (Ser340, Ser342, Ser345, and Ser354), stood out, with both corresponding to conserved phosphorylation sites that have been observed in LC-MS/MS analyses of the mammalian orthologues Slk and Lok (according to the Phosphosite Plus database (
      • Hornbeck P.V.
      • Kornhauser J.M.
      • Tkachev S.
      • Zhang B.
      • Skrzypek E.
      • Murray B.
      • Latham V.
      • Sullivan M.
      PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse.
      )).
      TABLE 1Summary of Slik phosphopeptides identified by LC-MS/MS
      PhosphositePeptide sequencem/zChargeErrorMascot score
      ppm
      Thr31LYNNIKMDpTDPAEFWEMVGELGDGAFGK1076.47790+3343
      Thr186, Thr192NKHpTMQKHDpTFIGTPYWMAPELVLCETFR936.41748+4531
      Thr192, Thr196NKHTMQKHDpTFIGpTPYWMAPELVLCETFR936.416500+4429
      Ser340NSALQLDLDDDpSASLQSQDIDKLPGTPTSILR1169.568600+3<184
      Ser342NSALQLDLDDDSApSLQSQDIDKLPGTPTSILR1169.56555+3−185
      Ser342, Ser345NSALQLDLDDDSApSLQpSQDIDKLPGTPTSILR1196.22485+3148
      Thr354NSALQLDLDDDSASLQSQDIDKLPGpTPTSILR1169.56824+3<187
      Thr392ATpTPDRPNHTKDDNAEAAAQQPPHTK579.067020+5<125
      Thr437VPAPAPPSSQQTPPPQVQQPPpTPPAQPTAAVLQK1177.604860+3−130
      Ser482GKAPPPPpSPLGLANAKPAASDSQTSPK666.838620+4−249
      Ser499PAASDSQTpSPK584.748050+2141
      Ser640STHLHESpSTDDDFPSLDDSLGDATTPPHK801.343260+4164
      Thr641STHLHESSpTDDDFPSLDDSLGDATTPPHK801.343510+4166
      Ser647, Ser651STHLHESSTDDDFPpSLDDpSLGDATTPPHK821.338260+4562
      Ser781pSPDSSLSENGSVR707.796810+2172
      Ser781, Ser784FKQEKRpSPDpSSLSENGSVR771.008970+3<137
      Ser791SPDSSLSENGpSVR707.795590+2<182
      The activation segment is a common site of regulatory phosphorylation in most eukaryotic protein kinases. This extended polypeptide region, situated between the highly conserved Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE) tripeptide motifs, wraps across the surface of the large lobe of the kinase domain. In its unphosphorylated state, the activation segment is highly dynamic and disordered. Phosphorylation at one or more residues in this region stabilizes the kinase in its active conformation, with the so-called contiguous “hydrophobic spine” (regulatory or R-spine) at the kinase domain core, composed of residues from both the small (N-) and large (C-) lobes, properly aligned. This is generally required for efficient catalysis (for review, see Refs.
      • Taylor S.S.
      • Keshwani M.M.
      • Steichen J.M.
      • Kornev A.P.
      Evolution of the eukaryotic protein kinases as dynamic molecular switches.
      and
      • Nolen B.
      • Taylor S.
      • Ghosh G.
      Regulation of protein kinases; controlling activity through activation segment conformation.
      ). Ste20 kinases are typical in this respect, with activation segment phosphorylation being crucial for the activity of several (
      • Praskova M.
      • Khoklatchev A.
      • Ortiz-Vega S.
      • Avruch J.
      Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras.
      • Arnold R.
      • Patzak I.M.
      • Neuhaus B.
      • Vancauwenbergh S.
      • Veillette A.
      • Van Lint J.
      • Kiefer F.
      Activation of hematopoietic progenitor kinase 1 involves relocation, autophosphorylation, and transphosphorylation by protein kinase D1.
      ,
      • King C.C.
      • Gardiner E.M.
      • Zenke F.T.
      • Bohl B.P.
      • Newton A.C.
      • Hemmings B.A.
      • Bokoch G.M.
      p21-activated kinase (PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1 (PDK1).
      • Vitari A.C.
      • Deak M.
      • Morrice N.A.
      • Alessi D.R.
      The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases.
      ).
      Phosphorylation at three Ser/Thr residues in the activation segments of Slk and Lok has been observed numerous times (≥48 occurrences at each site for the two proteins combined) in independent LC-MS/MS analyses (
      • Hornbeck P.V.
      • Kornhauser J.M.
      • Tkachev S.
      • Zhang B.
      • Skrzypek E.
      • Murray B.
      • Latham V.
      • Sullivan M.
      PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse.
      ). Phosphorylation at two of these sites, Thr183 and Ser189 of human Slk (corresponding to Thr186 and Thr192 in Slik), was observed in a crystal structure of the kinase domain of this protein (
      • Pike A.C.
      • Rellos P.
      • Niesen F.H.
      • Turnbull A.
      • Oliver A.W.
      • Parker S.A.
      • Turk B.E.
      • Pearl L.H.
      • Knapp S.
      Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites.
      ), and mutational analysis indicated that both phospho-residues are important for activation of its catalytic activity toward an exogenous substrate (
      • Luhovy A.Y.
      • Jaberi A.
      • Papillon J.
      • Guillemette J.
      • Cybulsky A.V.
      Regulation of the Ste20-like kinase, SLK: involvement of activation segment phosphorylation.
      ). Mutation of the third site, Thr193 (corresponding to Thr196 in Slik), impaired catalytic activity of the isolated kinase domain of Slk (
      • Luhovy A.Y.
      • Jaberi A.
      • Papillon J.
      • Guillemette J.
      • Cybulsky A.V.
      Regulation of the Ste20-like kinase, SLK: involvement of activation segment phosphorylation.
      ). Consistent with this, we found that mutating both Thr186 and Thr192 in Slik to Ala (SlikT186A/T192A) strongly impaired its ability to rescue Moesin phosphorylation in endogenous Slik-depleted cells (Fig. 6A). Phosphorylation at either site was sufficient for at least some activity, as both single mutants either fully (SlikT186A) or partially (SlikT192A) rescued Moesin phosphorylation (Fig. 6A). We observed the same trend in in vitro kinase assays (Fig. 6B). In contrast, mutation of the single site Thr196 to Ala (SlikT196A) strongly impaired catalytic activity both in cells and in vitro (Fig. 6A and B).
      To carry out similar tests in vivo, we generated transgenic flies for expressing the wild-type and mutant forms of Slik. All transgenes were recombined into the same site in the genome using the φC31-based integration system (
      • Bischof J.
      • Maeda R.K.
      • Hediger M.
      • Karch F.
      • Basler K.
      An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.
      ) to ensure equal mRNA expression. We used nub-GAL4 to drive expression of the slik 5′-UTR RNAi transgene, which efficiently depleted endogenous Slik protein and caused a strong reduction of Moesin phosphorylation specifically throughout the wing pouch of developing wing discs, as compared with controls (Fig. 6, C and D). Co-expression of wild-type Slik increased Moesin phosphorylation to well above endogenous levels (Fig. 6E). Co-expression of either SlikT186A/T192A or SlikT196A partially rescued Moesin phosphorylation but only to about endogenous levels (Fig. 6, F and G), confirming that both mutants have impaired activity. Taken together, our results suggest that phosphorylation of Slik at Thr196 and either Thr186 or Thr192 is required for efficient phosphorylation of its substrates. We were unable to detect any difference in the subcellular distribution of the activation segment mutants versus wild-type Slik (Fig. 6, E–G, insets), suggesting that apical localization occurs independently of activation.

      The Non-catalytic Function of Slik in Growth Control Is Dependent upon Kinase Activation

      Because Slik can promote tissue growth independently of substrate phosphorylation, it remained an open question whether this activity is regulated in any way. To test whether activation loop phosphorylation could play a role in this regard, we used the rescue assay to assess the ability of activation loop mutants of Slik to drive non-autonomous proliferation. Expression of wild-type Slik or SlikD176N with ptc-GAL4 caused fully penetrant lethality, with many animals dying at pupal stage. In contrast, SlikT186A/T192A and SlikT196A expression was much better tolerated, with some or all animals surviving to adult stage, respectively (data not shown). Interestingly, whereas expression of wild-type Slik or SlikD176N in endogenous Slik-depleted DP cells triggered the expected non-autonomous proliferation effect (Fig. 2, E and F), SlikT186A/T192A did so much more weakly and SlikT196A not at all (Fig. 7, A and B). Thus, although it does not require catalytic activity per se, the ability of Slik to induce non-autonomous proliferation in discs does require that the kinase be “activated” by activation segment phosphorylation, providing a level of regulation.
      Slik has been linked to regulation of the tumor suppressor protein Mer/NF2 which, like Moesin, is a FERM domain-containing protein. Based on work in both flies and mammals, Mer/NF-2 mediates its growth-suppressive effects by activating the Hippo (Hpo)/Mst pathway (
      • Hamaratoglu F.
      • Willecke M.
      • Kango-Singh M.
      • Nolo R.
      • Hyun E.
      • Tao C.
      • Jafar-Nejad H.
      • Halder G.
      The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis.
      • Zhang N.
      • Bai H.
      • David K.K.
      • Dong J.
      • Zheng Y.
      • Cai J.
      • Giovannini M.
      • Liu P.
      • Anders R.A.
      • Pan D.
      The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals.
      ,
      • Yu J.
      • Zheng Y.
      • Dong J.
      • Klusza S.
      • Deng W.M.
      • Pan D.
      Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded.
      • Yin F.
      • Yu J.
      • Zheng Y.
      • Chen Q.
      • Zhang N.
      • Pan D.
      Spatial organization of Hippo signaling at the plasma membrane mediated by the tumor suppressor Merlin/NF2.
      ). Like Moesin, Mer/NF2 exists in active and inactive conformations, with the transition from active to inactive states driven by phosphorylation at a critical residue (Ser518 in human NF2) (
      • Rong R.
      • Surace E.I.
      • Haipek C.A.
      • Gutmann D.H.
      • Ye K.
      Serine 518 phosphorylation modulates merlin intramolecular association and binding to critical effectors important for NF2 growth suppression.
      ). Manipulating Slik activity altered Mer phosphorylation, which led to the suggestion that Slik promotes growth by phosphorylating and inhibiting Mer (
      • Hughes S.C.
      • Fehon R.G.
      Phosphorylation and activity of the tumor suppressor Merlin and the ERM protein Moesin are coordinately regulated by the Slik kinase.
      ). This is hard to reconcile with the observation that Slik promotes tissue growth independently of its catalytic activity. Furthermore, the residue in Mer/NF2 corresponding to the critical Thr in Moesin that is phosphorylated by Slik (Thr576 in human NF2) does not appear to be involved in regulating its activity (
      • Surace E.I.
      • Haipek C.A.
      • Gutmann D.H.
      Effect of merlin phosphorylation on neurofibromatosis 2 (NF2) gene function.
      ).
      To see if inhibition of Mer by Slik could be sufficient to explain its effects on growth, we compared the phenotypic effects of expressing Slik or inhibiting Mer in discs. ap-GAL4-driven expression of Slik in dorsal DP cells induced robust non-autonomous proliferation of PM cells directly overlying the DP dorsal compartment (Fig. 8A). In contrast, under the same conditions neither expression of a dominant negative form of Mer (MerΔBB) (
      • LaJeunesse D.R.
      • McCartney B.M.
      • Fehon R.G.
      Structural analysis of Drosophila merlin reveals functional domains important for growth control and subcellular localization.
      ) nor mer dsRNA had nonautonomous effects on PM cell proliferation (Fig. 8, B and C), although both induced robust overgrowth of the dorsal wing surface that caused the wings to curve downward (Fig. 8, D and E, and not shown). Taken together, our results suggest that Mer is not the main target of Slik in regulating tissue growth.

      Discussion

      The Drosophila Ste20 kinase Slik is one of a growing number of kinases with both catalytic activity-dependent and -independent functions (
      • Rauch J.
      • Volinsky N.
      • Romano D.
      • Kolch W.
      The secret life of kinases: functions beyond catalysis.
      ). The non-catalytic functions of kinases are mediated in different ways, including scaffolding of protein complexes, allosteric regulation of other proteins, and competition for binding partners. We were interested to know whether the regulatory mechanisms that control Slik catalytic activity, which is required for phosphorylation of the substrate Moesin and thus epithelial tissue integrity in vivo, also control its non-catalytic growth-promoting function. Our results indicate that activation segment phosphorylation, which typically stabilizes kinases in their active conformation, activates the catalytic activity of Slik and is also important for its non-catalytic function. In contrast, localization of the kinase via its conserved CCD is not required for catalytic activity but is essential for Slik to drive cell proliferation.
      Analysis of the crystal structures of many protein kinase domains in the phosphorylated active and non-phosphorylated inactive states has revealed how activation segment phosphorylation affects kinase conformation and thus catalytic activity (
      • Taylor S.S.
      • Keshwani M.M.
      • Steichen J.M.
      • Kornev A.P.
      Evolution of the eukaryotic protein kinases as dynamic molecular switches.
      ,
      • Nolen B.
      • Taylor S.
      • Ghosh G.
      Regulation of protein kinases; controlling activity through activation segment conformation.
      ). In the structures of protein kinase A (PKA) and others, the primary activation segment phosphate group (on Thr196 in PKA) stabilizes the active conformation by making electrostatic contacts with several conserved motifs in the kinase core. Some kinases also contain a secondary phosphorylation site, whose main function is less clear. Of the three activation segment residues we identified, Thr192 in Slik aligns most closely with Thr196 in PKA and is conserved (Ser or Thr) in all Ste20 kinases. In most of these proteins, this residue is the primary regulatory phosphorylation site in the activation loop (
      • Delpire E.
      The mammalian family of sterile 20p-like protein kinases.
      ). For some, this residue is autophosphorylated (e.g. Pak2 (
      • Walter B.N.
      • Huang Z.
      • Jakobi R.
      • Tuazon P.T.
      • Alnemri E.S.
      • Litwack G.
      • Traugh J.A.
      Cleavage and activation of p21-activated protein kinase γ-PAK by CPP32 (caspase 3): effects of autophosphorylation on activity.
      ), hMink (
      • Lim J.
      • Lennard A.
      • Sheppard P.W.
      • Kellie S.
      Identification of residues which regulate activity of the STE20-related kinase hMINK.
      ), Slk (
      • Pike A.C.
      • Rellos P.
      • Niesen F.H.
      • Turnbull A.
      • Oliver A.W.
      • Parker S.A.
      • Turk B.E.
      • Pearl L.H.
      • Knapp S.
      Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites.
      )). For others, such as Osr1 and Spak (
      • Vitari A.C.
      • Deak M.
      • Morrice N.A.
      • Alessi D.R.
      The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases.
      ), Hippo (
      • Boggiano J.C.
      • Vanderzalm P.J.
      • Fehon R.G.
      Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway.
      ), Pak1 (
      • King C.C.
      • Gardiner E.M.
      • Zenke F.T.
      • Bohl B.P.
      • Newton A.C.
      • Hemmings B.A.
      • Bokoch G.M.
      p21-activated kinase (PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1 (PDK1).
      ), and Hpk1 (
      • Arnold R.
      • Patzak I.M.
      • Neuhaus B.
      • Vancauwenbergh S.
      • Veillette A.
      • Van Lint J.
      • Kiefer F.
      Activation of hematopoietic progenitor kinase 1 involves relocation, autophosphorylation, and transphosphorylation by protein kinase D1.
      ), the site can also be trans-phosphorylated, providing a point of intersection with other signaling pathways. The residue corresponding to Thr186 is less well conserved, being present in Slk, Lok, and Hippo/Mst kinases but not in the PAK kinases or Osr1 and Spak. In those kinases where it is present, this site appears to function as a secondary phosphorylation site (
      • Delpire E.
      The mammalian family of sterile 20p-like protein kinases.
      ,
      • Nolen B.
      • Taylor S.
      • Ghosh G.
      Regulation of protein kinases; controlling activity through activation segment conformation.
      ). Our results with Slik fit this model, as mutation of Thr192 alone had more of an effect on catalytic activity than Thr186. Phosphorylation of at least one of these residues is important for efficient catalysis, as the double mutant had low activity. Both are likely autophosphorylated, as phosphate groups were observed at the equivalent sites in bacterially expressed recombinant Slk and Lok kinase domains (
      • Pike A.C.
      • Rellos P.
      • Niesen F.H.
      • Turnbull A.
      • Oliver A.W.
      • Parker S.A.
      • Turk B.E.
      • Pearl L.H.
      • Knapp S.
      Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites.
      ). However, our genetic analyses suggest that at least one of these sites is likely also trans-phosphorylated (see below).
      In addition to these two well defined sites, Slik activity was strongly impaired by mutation of Thr196. This residue is situated at a hinge point near the start of the “p + 1” loop region of the activation segment, so-called because several residues in this region make contact with the p + 1 residue in the substrate. The equivalent residue in other kinases also makes contact with conserved residues in the catalytic loop (
      • Nolen B.
      • Taylor S.
      • Ghosh G.
      Regulation of protein kinases; controlling activity through activation segment conformation.
      ), meaning that modification of this residue could influence both the active structure and interaction with substrates. This Thr residue is invariant in all Ste20 kinases (
      • Delpire E.
      The mammalian family of sterile 20p-like protein kinases.
      ), and phosphorylation at this site appears to be fairly common (based on analysis of the Phosphosite MS/MS database (
      • Hornbeck P.V.
      • Kornhauser J.M.
      • Tkachev S.
      • Zhang B.
      • Skrzypek E.
      • Murray B.
      • Latham V.
      • Sullivan M.
      PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse.
      )), especially for certain kinases (e.g. Slk and Lok, Spak and Osr1, and Ysk/Stk25). In several Ste20 kinases, including Slk, Spak, and Mst1, Ala substitution at this site impairs substrate phosphorylation (
      • Luhovy A.Y.
      • Jaberi A.
      • Papillon J.
      • Guillemette J.
      • Cybulsky A.V.
      Regulation of the Ste20-like kinase, SLK: involvement of activation segment phosphorylation.
      ,
      • Gagnon K.B.
      • England R.
      • Delpire E.
      Characterization of SPAK and OSR1, regulatory kinases of the Na-K-2Cl cotransporter.
      ). Thus the role of phosphorylation at this site in regulating kinase function is conserved both between at least some Ste20 kinase family members and between species. The mechanism remains to be determined.
      Although there are few cases where the involvement of activation segment phosphorylation in regulating catalytic activity-independent activities of kinases has been assessed, there is evidence that it can either be required (as in activation of topo-IIα and Parp-1 by ERKs (
      • Shapiro P.S.
      • Whalen A.M.
      • Tolwinski N.S.
      • Wilsbacher J.
      • Froelich-Ammon S.J.
      • Garcia M.
      • Osheroff N.
      • Ahn N.G.
      Extracellular signal-regulated kinase activates topoisomerase IIalpha through a mechanism independent of phosphorylation.
      ,
      • Cohen-Armon M.
      • Visochek L.
      • Rozensal D.
      • Kalal A.
      • Geistrikh I.
      • Klein R.
      • Bendetz-Nezer S.
      • Yao Z.
      • Seger R.
      DNA-independent PARP-1 activation by phosphorylated ERK2 increases Elk1 activity: a link to histone acetylation.
      )) or can cause a switch from catalytic activity-independent to -dependent signaling (for yeast Kss1 (
      • Madhani H.D.
      • Styles C.A.
      • Fink G.R.
      MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation.
      )). We found that the catalytic activity-independent growth promoting function of Slik shows a similar dependence on activation loop phosphorylation as catalytic activity; Thr196 and either Thr186 or Thr192 need to be phosphorylated for an efficient proliferative response. Although Slik can autophosphorylate, it is likely also trans-phosphorylated by one or more other kinases, as the wild-type and catalytically inactive proteins were phosphorylated to a similar extent in endogenous Slik-depleted cells. Interestingly, the catalytically inactive Slikkd mutant protein cannot autophosphorylate but is capable of promoting cell proliferation even when expressed in cells depleted of endogenous Slik. This suggests that the activation segment of Slik can be phosphorylated by another kinase to activate non-catalytic pro-proliferative signaling.
      We identified 16 phosphorylation sites outside of the activation segment of Slik in our MS/MS analyses. The majority of these were in the non-conserved central domain, and their relevance is unclear. One cluster of sites just beyond the C-terminal limit of the kinase domain (Ser340, Ser342, Ser345, and Ser354) appears to be conserved in spacing, if not in actual primary sequence, with phosphorylation sites in mammalian Slk and Lok. Interestingly, two of the corresponding sites in mammalian Slk (Ser347 and Ser348) mediate negative regulation by casein kinase II in response to v-Src activity (
      • Chaar Z.
      • O'reilly P.
      • Gelman I.
      • Sabourin L.A.
      v-Src-dependent down-regulation of the Ste20-like kinase SLK by casein kinase II.
      ), suggesting the possibility that these may be conserved trans-regulatory phosphorylation sites.
      Consistent with studies of mammalian Slk (
      • Viswanatha R.
      • Ohouo P.Y.
      • Smolka M.B.
      • Bretscher A.
      Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells.
      ), we found that the coiled-coil-containing CCD of Slik mediates apical localization of the protein in epithelial cells of the imaginal disc. Although a model involving membrane recruitment of Slik by PI(4,5)P2 would have fit well with ERM protein biology, we did not detect any interaction of the CCD with phosphoinositol lipids. Interestingly, the CCD also appears to be involved in regulated membrane recruitment of Slk in non-polarized mouse embryonic fibroblasts through interactions with the LIM domain transcription cofactors Ldb1 and Ldb2 (
      • Storbeck C.J.
      • Wagner S.
      • O'Reilly P.
      • McKay M.
      • Parks R.J.
      • Westphal H.
      • Sabourin L.A.
      The Ldb1 and Ldb2 transcriptional cofactors interact with the Ste20-like kinase SLK and regulate cell migration.
      ). Although we found that it determines where in cells Moesin gets activated, the CCD is not required for phosphorylation per se. In fact, previous work suggests that the Slk CCD acts as an autoinhibitory domain (
      • Storbeck C.J.
      • Wagner S.
      • O'Reilly P.
      • McKay M.
      • Parks R.J.
      • Westphal H.
      • Sabourin L.A.
      The Ldb1 and Ldb2 transcriptional cofactors interact with the Ste20-like kinase SLK and regulate cell migration.
      ), something that we have not tested. In contrast, the CCD is absolutely required for activation of pro-proliferative signaling. Although non-catalytic activities of many kinases are mediated by parts of the protein outside of the kinase domain, the CCD alone is not capable of stimulating proliferation. Our data are more consistent with a model in which localization via the CCD instead brings Slik into the vicinity of other proteins required for signaling, and activation loop phosphorylation places Slik in a conformation that enables it to interact with these proteins to initiate the signal.
      We do not know how Slik promotes cell proliferation in the wing disc. Although Slik in some way influences the phosphorylation of Mer, the importance of that for the Slik-driven growth phenotypes is not clear for two reasons. First, Slik-driven growth does not require catalytic activity. Second, although it causes the expected tissue overgrowth response, direct inhibition of Mer activity in wing discs does not induce non-autonomous proliferation, which is a characteristic effect of Slik. Thus, although Mer regulation may contribute to the growth effects of Slik, it does not appear to be the main target. The direct target of Slik in growth signaling remains to be identified.

      Author Contributions

      V. P. was involved in the design of and performed and analyzed experiments shown in Figs. 1, 2, 5, 6, and 7. A. N. and N. D. designed, performed, and analyzed experiments shown in Fig. 8. F. S. performed and analyzed experiments shown in Figs. 4G, 5A, and 5D. A. P. performed and analyzed the experiment shown in Fig. 2B. D. M. was involved in the design of experiments in FIGURE 2, FIGURE 5. K. O. performed and analyzed experiments shown in Fig. 4L. D. R. H. conceived of the study, was involved in the design of all experiments, and performed and analyzed experiments shown in Figs. 1, 3, 4, and 6.

      Acknowledgments

      We thank Denis Faubert of the Institut de Recherches Cliniques de Montréal (IRCM) Proteomics Core Facility for the LC-MS/MS analysis of Slik phosphorylation and Helen McNeill, Bruce Edgar, Sally Leevers, Rick Fehon, Christian Lehner, and Jean-Francois Côté for generously providing Drosophila stocks and other reagents.

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