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Fission Yeast Germinal Center (GC) Kinase Ppk11 Interacts with Pmo25 and Plays an Auxiliary Role in Concert with the Morphogenesis Orb6 Network (MOR) in Cell Morphogenesis*

  • Tetsuya Goshima
    Footnotes
    Affiliations
    From the Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan,
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  • Kazunori Kume
    Footnotes
    Affiliations
    From the Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan,
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  • Takayuki Koyano
    Affiliations
    From the Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan,
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  • Yoshikazu Ohya
    Affiliations
    the Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan, and
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  • Takashi Toda
    Affiliations
    the Laboratory of Cell Regulation, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
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  • Dai Hirata
    Correspondence
    To whom correspondence should be addressed: 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan. Tel.: 81-82-424-7764; Fax: 81-82-424-7045;
    Affiliations
    From the Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan,
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  • Author Footnotes
    * This work was supported by grants from the Ministry of Education, Science, and Culture of Japan (to K. K. and D. H.) and the Cancer Research UK (to T. T.).
    1 Both authors contributed equally to this work.
    2 Recipient of a JSPS fellowship (DC1).
Open AccessPublished:September 07, 2010DOI:https://doi.org/10.1074/jbc.M110.176867
      How cell morphology and the cell cycle are coordinately regulated is a fundamental subject in cell biology. In fission yeast, 2 germinal center kinases (GCKs), Sid1 and Nak1, play an essential role in septation/cytokinesis and cell separation/cell polarity control, respectively, as components of the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR). Here we show that a third GCK, Ppk11, is also required for efficient cell separation particularly, at a high temperature. Although Ppk11 is not essential for cell division, this kinase plays an auxiliary role in concert with MOR in cell morphogenesis. Ppk11 physically interacts with the MOR component Pmo25 and is localized to the septum, by which Ppk11 is crucial for Pmo25 targeting/accumulation to the septum. The conserved C-terminal WDF motif of Ppk11 is essential for both septum accumulation of Pmo25 and efficient cell separation. In contrast its kinase activity is required only for cell separation. Thus, both interaction of Ppk11 with Pmo25 and Ppk11 kinase activity are critical for efficient cell separation.

      Introduction

      Cell morphogenesis and the cell cycle are coordinately regulated. The fission yeast Schizosaccharomyces pombe is an excellent model system in which to study this coordinated regulation (for reviews, see Refs.
      • Hayles J.
      • Nurse P.
      ,
      • Chang F.
      • Martin S.G.
      ), because growth polarity dynamically changes at 3 stages during the cell cycle (
      • Mitchison J.M.
      ,
      • Marks J.
      • Hagan I.
      • Hyams J.S.
      ), i.e. the initiation of growth upon cell division, NETO (new end take off) (
      • Mitchison J.M.
      • Nurse P.
      ), and septum formation. Upon cell division, cortical F-actin moves from the new end, which is newly produced by division, to the old end, which existed in the previous cell cycle; and the cell growth is initiated from only the old end. Upon NETO at about 0.34 of the cell cycle, F-actin localization shifts from the old end to both ends, thereby changing growth polarity from monopolar to bipolar. This growth polarity is maintained during the following interphase. At the onset of mitosis, the cell growth ceases; and the cortical F-actin patches at both ends disappear, having been translocated to the medial ring, which corresponds to the following division site. The onset of cytokinesis is triggered by the septation initiation network (SIN),
      The abbreviations used are: SIN
      septation initiation network
      GCK
      germinal center kinase
      MBM
      MO25 binding motif
      KD
      kinase domain
      MOR
      morphogenesis Orb6 network.
      analogous to the budding yeast mitotic exit network (MEN), that comprises Spg1 GTPase and a downstream kinase cascade (Cdc7, Sid1, and Sid2; Ref.
      • Simanis V.
      ). SIN promotes actomyosin ring constriction and septum formation in the middle of the cell (
      • Simanis V.
      ).
      The Ste20 group of kinases has been implicated in various cellular events including the regulation of cell morphogenesis, cytoskeletal rearrangements, and apoptosis (
      • Daniels R.H.
      • Bokoch G.M.
      ,
      • Dan I.
      • Watanabe N.M.
      • Kusumi A.
      ). This group includes germinal center kinases (GCKs) and p21-activated kinases (PAKs). GCKs have an N-terminal kinase domain followed by less conserved C-terminal putative regulatory regions but lack the conserved G-protein binding sites possessed by PAKs. Genome sequencing shows that fission yeast cells have 3 GCKs, i.e. Sid1, Nak1, and Ppk11. Sid1 and Nak1 are essential for cell growth, and play a critical role in septation/cytokinesis and cell morphogenesis (cell separation and cell polarity control), respectively, as components of SIN and the morphogenesis Orb6 network (MOR) (
      • Simanis V.
      ,
      • Kanai M.
      • Kume K.
      • Miyahara K.
      • Sakai K.
      • Nakamura K.
      • Leonhard K.
      • Wiley D.J.
      • Verde F.
      • Toda T.
      • Hirata D.
      ). However, a function of Ppk11, that is non-essential for cell growth, remains unclear.
      Members of the MO25 family are evolutionally conserved proteins that are structurally related armadillo-repeat scaffold proteins (
      • Miyamoto H.
      • Matsushiro A.
      • Nozaki M.
      ,
      • Nozaki M.
      • Onishi Y.
      • Togashi S.
      • Miyamoto H.
      ). Accumulating evidence indicates that MO25 proteins are important for regulation of cell polarity and are also related functionally to GCK (
      • Nozaki M.
      • Onishi Y.
      • Togashi S.
      • Miyamoto H.
      ,
      • Karos M.
      • Fischer R.
      ,
      • Hergovich A.
      • Stegert M.R.
      • Schmitz D.
      • Hemmings B.A.
      ). In fission yeast, MO25/Pmo25 interacts with Nak1 and functions as an upstream component of the Drosophila Furry-like Mor2 and the NDR kinase Orb6 in the MOR pathway (
      • Kanai M.
      • Kume K.
      • Miyahara K.
      • Sakai K.
      • Nakamura K.
      • Leonhard K.
      • Wiley D.J.
      • Verde F.
      • Toda T.
      • Hirata D.
      ,
      • Mendoza M.
      • Redemann S.
      • Brunner D.
      ). The Pmo25 localization to the spindle pole bodies (SPBs) and the Nak1-Orb6 kinase activities in early interphase following cytokinesis are under the control of Cdc7-Sid1, indicating that Pmo25 plays a connecting role between SIN and MOR by interacting functionally with 2 GCKs, Sid1 and Nak1 (
      • Kanai M.
      • Kume K.
      • Miyahara K.
      • Sakai K.
      • Nakamura K.
      • Leonhard K.
      • Wiley D.J.
      • Verde F.
      • Toda T.
      • Hirata D.
      ,
      • Kume K.
      • Goshima T.
      • Miyahara K.
      • Toda T.
      • Hirata D.
      ). The budding yeast MO25/Hym1 is involved in the RAM (regulation of Ace2p activity and cellular morphogenesis) pathway that consists of the budding yeast homologs (Kic1, Tao3, and Cbk1) of fission yeast Nak1, Mor2, and Orb6 (
      • Nelson B.
      • Kurischoko C.
      • Horecka J.
      • Mody M.
      • Nair P.
      • Pratt L.
      • Zougman A.
      • McBroom L.D.
      • Hughes T.R.
      • Boone C.
      • Luca F.C.
      ). RAM controls not only cell separation via regulation of the Ace2 transcription factor but also polarized cell growth (
      • Nelson B.
      • Kurischoko C.
      • Horecka J.
      • Mody M.
      • Nair P.
      • Pratt L.
      • Zougman A.
      • McBroom L.D.
      • Hughes T.R.
      • Boone C.
      • Luca F.C.
      ). In Drosophila, Mo25 and the GCK Fray act in concert to regulate asymmetric division in embryonic neuroblasts (
      • Yamamoto Y.
      • Izumi Y.
      • Matsuzaki F.
      ). In vertebrates, MO25 functionally interacts with the GCK family pseudokinase STRAD (Ste-20-related adaptor) to activate serine/threonine kinase LKB1, which is implicated as a regulator of multiple biological processes, including the control of cell-cycle arrest and cell polarity control, and whose mutation causes the Peutz-Jeghers cancer syndrome (
      • Boudeau J.
      • Baas A.F.
      • Deak M.
      • Morrice N.A.
      • Kieloch A.
      • Schutkowski M.
      • Prescott A.R.
      • Clevers H.C.
      • Alessi D.R.
      ,
      • Baas A.F.
      • Smit L.
      • Clevers H.
      ,
      • Alessi D.R.
      • Sakamoto K.
      • Bayascas J.R.
      ). ATP and MO25 regulate the conformational state of STRAD and activation of LKB1 (
      • Zeqiraj E.
      • Filippi B.M.
      • Goldie S.
      • Navratilova I.
      • Boudeau J.
      • Deak M.
      • Alessi D.R.
      • van Aalten D.M.
      ). LKB1 activation by STRAD can establish complete polarity in single epithelial cells through the induction of brush borders at the apical domain (
      • Baas A.F.
      • Smit L.
      • Clevers H.
      ). Recently, it has been shown that human MO25 interacts with the GCK Mst4 and that the active MO25/STRAD/LKB1 complex induces the brush-border formation through Mst4 and the actin remodeler Ezrin (
      • ten Klooster J.P.
      • Jansen M.
      • Yuan J.
      • Oorschot V.
      • Begthel H.
      • Di Giacomo V.
      • Colland F.
      • de Koning J.
      • Maurice M.M.
      • Hornbeck P.
      • Clevers H.
      ).
      In this present study, we characterized the third GCK, Ppk11, in detail and obtained data indicating that Ppk11 interacted with Pmo25 and was important for efficient cell separation under high temperature conditions.

      DISCUSSION

      GCKs play pivotal roles in various aspects of cell signaling in eukaryotes, and normally constitute kinase cascades like MAPK pathways (
      • Daniels R.H.
      • Bokoch G.M.
      ,
      • Dan I.
      • Watanabe N.M.
      • Kusumi A.
      ). Fission yeast contains 3 GCKs and previous work including our laboratory shows that Sid1 and Nak1 are components of SIN and MOR, and play essential roles in septum formation/cytokinesis and cell morphogenesis, respectively (
      • Simanis V.
      ,
      • Kanai M.
      • Kume K.
      • Miyahara K.
      • Sakai K.
      • Nakamura K.
      • Leonhard K.
      • Wiley D.J.
      • Verde F.
      • Toda T.
      • Hirata D.
      ). In this study, we analyzed the role of the third GCK, Ppk11, in the cell morphogenesis. Ppk11 is not essential for cell growth but is important for efficient cell separation at the high temperature. Ppk11 is localized to the medial region after mitosis, more specifically probably to the septum, as it shows double-ring patterns upon cytokinesis. Localization of Ppk11 to the medial region requires a functional actomyosin ring, rather than SIN activation per se. Like other GCKs, Ppk11 does not exert its function on its own, but instead it acts at least in part with MOR, as this kinase physically interacts with Pmo25, one of the MOR components. Importantly Pmo25 accumulation/targeting to the medial region substantially depends on the Ppk11 protein. The absence of Ppk11 in MOR mutants exaggerates cell morphogenesis defects. These results lead us to conclude that Ppk11 plays an auxiliary but independent function in cell separation and cell polarity control coordinately with MOR. A model for functional interaction between Ppk11 and SIN/MOR in cell morphogenesis is depicted in Fig. 8.
      Figure thumbnail gr8
      FIGURE 8A model for functional interaction between Ppk11 and SIN/MOR in cell morphogenesis. Ppk11 has an N-terminal KD and the conserved C-terminal MBM. The kinase activity of Ppk11 is essential for efficient cell separation at high temperature (red line). The MBM of Ppk11 is crucial for Pmo25 targeting/accumulation to the septum. Septum localization of the proteins, Ppk11, Nak1, or Pmo25, is indicated by red, green, or yellow circle, respectively. Translocation of proteins from the mitotic spindle pole body (SPB) to the septum is also indicated by gray line.

      Role of Ppk11 in Cell Separation

      Ppk11 was important for efficient cell separation particularly at the high temperature. The Ppk11 kinase dead mutant cells, in which Pmo25 localized to the septum relatively normally, showed a defect in cell separation. These results suggest that Ppk11 protein plays a dual role in cell separation, one as a protein kinase, while the other acts as a structural platform for the Pmo25 accumulation/recruitment, which would contribute to the activation of other MOR component kinases such as Nak1 and Orb6, especially under the high temperature condition (Fig. 8, red dotted line). Consistent with this notion, the localization of Ppk11 to the septum precedes that of Pmo25. We envision that, by interacting with and accumulating Pmo25 to the medial region, Ppk11 is further activated at the site of cytokinesis, thereby in turn activating a downstream substrate(s)/kinase(s) for efficient cell separation. In Δppk11 cells, a certain amount of Pmo25 was still localized at the septum, indicating that Ppk11 per se is not essential for the localization of Pmo25 to the septum. Under a stringent condition such as the high temperature, a larger amount of Pmo25 protein at the septum, mediated by Ppk11, would be important for completion of cell separation through full activation of GCKs Nak1 and Ppk11. The Ppk11 protein level was not changed by the high temperature-shift (data not shown). It is possible that a target molecule(s) of Ppk11 might be induced under the high temperature condition.

      Functional Interactions among MO25 and 3 GCKs

      The partnership between MO25 and GCK appears to be evolutionally conserved among eukaryotes (
      • Nozaki M.
      • Onishi Y.
      • Togashi S.
      • Miyamoto H.
      ,
      • Karos M.
      • Fischer R.
      ,
      • Hergovich A.
      • Stegert M.R.
      • Schmitz D.
      • Hemmings B.A.
      ). We showed that in fission yeast Pmo25 physically/functionally interacts with 3 GCKs, i.e. Sid1, Nak1, and Ppk11 (
      • Kanai M.
      • Kume K.
      • Miyahara K.
      • Sakai K.
      • Nakamura K.
      • Leonhard K.
      • Wiley D.J.
      • Verde F.
      • Toda T.
      • Hirata D.
      ,
      • Kume K.
      • Goshima T.
      • Miyahara K.
      • Toda T.
      • Hirata D.
      ). In other organisms, it has been shown that MO25 interacts with just 1 GCK. Our result is thus the first evidence showing the interaction of MO25 with multiple GCKs. The biological significance of this Pmo25 network is still not fully understood. To clarify this point, it would be important for construction of the pmo25 mutant that lacks the ability to interact with GCKs or the double/triple GCK mutants that lack the ability to interact with Pmo25.

      Downstream Kinase(s) of Ppk11

      MO25 physically interacts with GCK and activates downstream kinases. Sid1 and Pmo25 are important for the activities of the downstream kinases Nak1 and Orb6. In the MOR pathway, Pmo25 and Nak1 activate the downstream NDRK Orb6. The identification of a putative downstream kinase of Ppk11, other than Orb6, would be important for understanding a role of Ppk11 in cell morphogenesis. As in general GCK activates NDRK, we searched for a putative downstream NDRK(s) whose deletion exhibits the defect in cell separation similar to that seen in Δppk11 cells. Our homology search indicates that fission yeast has 5 non-essential NDRKs (Orb6-homologous NDRK-like kinases) on the genome that might act together with Ppk11. However, so far we have not found any NDRKs whose deletion results in defective cell separation, at least a single deletion.
      T. Goshima and D. Hirata, unpublished results.
      It is possible that some of these NDRKs share functions in cell separation in a redundant fashion. Multiple deletions would be necessary to address this point further. GCK Nak1 physically interacts with NDRK Orb6. To identify the downstream kinase of Ppk11, it would be intriguing to search for a kinase(s) that interacts with Ppk11.

      Domain Analysis of GCK

      In the ppk11-WDF/AAA mutant cells the septum localization of Pmo25 decreased significantly to 41% compared with that in WT cells; however, the Ppk11-WDF/AAA mutant protein was localized normally to the septum. This result indicates that the WDF motif of Ppk11 is important for the interaction with Pmo25 but not for the septum localization of Ppk11. A septum-targeting sequence might exist on Ppk11. It would be interesting to identify such a sequence on Ppk11 for the septum localization. Two GCKs, Sid1 and Nak1, also possess a MO25 binding motif (MBM) in their C-terminal regulatory regions. The importance of MBM in these GCKs for their function should be clarified. Further domain analysis should provide us with valuable information on the biological significance of the MO25-GCK network.

      Acknowledgments

      We thank M. Balasubramanian for the generous gift of the kinase deletion set used and I. Hagan and V. Simanis for strains. We thank D. McCollum, F. Verde, M. Balasubramanian, E. Tsuchiya, and I. Yamashita for helpful discussions. We are grateful to all other members of Hirata's laboratory for help.

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