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Structure-Function Analysis of Core STRIPAK Proteins

A SIGNALING COMPLEX IMPLICATED IN GOLGI POLARIZATION*
  • Michelle J. Kean
    Footnotes
    Affiliations
    Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada

    Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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  • Derek F. Ceccarelli
    Affiliations
    Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
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  • Marilyn Goudreault
    Affiliations
    Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
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  • Mario Sanches
    Affiliations
    Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
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  • Stephen Tate
    Affiliations
    AB-SCIEX, Concord, Ontario L4K 4V8, Canada
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  • Brett Larsen
    Affiliations
    Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
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  • Lucien C.D. Gibson
    Affiliations
    Molecular Pharmacology Group, Institute of Psychology and Neuroscience, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • W. Brent Derry
    Affiliations
    Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada
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  • Ian C. Scott
    Affiliations
    Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada
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  • Laurence Pelletier
    Footnotes
    Affiliations
    Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada

    Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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  • George S. Baillie
    Affiliations
    Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada
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  • Frank Sicheri
    Footnotes
    Affiliations
    Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada

    Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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  • Anne-Claude Gingras
    Correspondence
    A Lea Reichmann chair. To whom correspondence should be addressed: Samuel Lunenfeld Research Institute at Mount Sinai Hospital, 600 University Ave., Rm. 992, Toronto, ON M5G 1X5, Canada. Tel.: 416-586-5027; Fax: 416-586-8869
    Footnotes
    Affiliations
    Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada

    Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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  • Author Footnotes
    * This work was supported in part by Canadian Institutes of Health Research Grants MOP-36399 (to F. S.) and MOP-84314 (to A.-C. G.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables I–V, Figs. 1–7, and additional references.
    1 Supported by Canadian Institutes of Health Research through a Banting and Best Canada graduate scholarship.
    2 Canada Research chairs.
Open AccessPublished:May 11, 2011DOI:https://doi.org/10.1074/jbc.M110.214486
      Cerebral cavernous malformations (CCMs) are alterations in brain capillary architecture that can result in neurological deficits, seizures, or stroke. We recently demonstrated that CCM3, a protein mutated in familial CCMs, resides predominantly within the STRIPAK complex (striatin interacting phosphatase and kinase). Along with CCM3, STRIPAK contains the Ser/Thr phosphatase PP2A. The PP2A holoenzyme consists of a core catalytic subunit along with variable scaffolding and regulatory subunits. Within STRIPAK, striatin family members act as PP2A regulatory subunits. STRIPAK also contains all three members of a subfamily of Sterile 20 kinases called the GCKIII proteins (MST4, STK24, and STK25). Here, we report that striatins and CCM3 bridge the phosphatase and kinase components of STRIPAK and map the interacting regions on each protein. We show that striatins and CCM3 regulate the Golgi localization of MST4 in an opposite manner. Consistent with a previously described function for MST4 and CCM3 in Golgi positioning, depletion of CCM3 or striatins affects Golgi polarization, also in an opposite manner. We propose that STRIPAK regulates the balance between MST4 localization at the Golgi and in the cytosol to control Golgi positioning.

      Introduction

      PP2A
      The abbreviations used are: PP2A
      phosphoprotein phosphatase 2A
      CCM
      cerebral cavernous malformation
      FAT
      focal adhesion targeting
      eGFP
      enhanced GFP
      STRIPAK
      striatin-interacting phosphatase and kinase
      AP-MS
      affinity purification coupled to mass spectrometry
      GCKIII
      germinal center kinase III
      esiRNA
      endoribonuclease-prepared siRNA
      N-mut
      L44D,A47D,I66D,L67D
      C-mut(4A)
      K132A,K139A,K172A,K179A.
      is an essential serine threonine phosphatase involved in many aspects of cell function (
      • Virshup D.M.
      • Shenolikar S.
      , ). PP2A acquires substrate and subcellular localization specificity via association with various scaffolding and regulatory subunits to form a number of different holoenzymes, most of which are trimers. In previous studies using affinity purification coupled to mass spectrometry, a portion of PP2A was also found in a higher order complex that we termed STRIPAK (striatin interacting phosphatase and kinase) (
      • Glatter T.
      • Wepf A.
      • Aebersold R.
      • Gstaiger M.
      ,
      • Goudreault M.
      • D'Ambrosio L.M.
      • Kean M.J.
      • Mullin M.J.
      • Larsen B.G.
      • Sanchez A.
      • Chaudhry S.
      • Chen G.I.
      • Sicheri F.
      • Nesvizhskii A.I.
      • Aebersold R.
      • Raught B.
      • Gingras A.C.
      ). In addition to the catalytic subunit PP2Acat, its scaffolding subunit PP2AA and members of the striatin family of regulatory subunits (
      • Moreno C.S.
      • Park S.
      • Nelson K.
      • Ashby D.
      • Hubalek F.
      • Lane W.S.
      • Pallas D.C.
      ), the core STRIPAK complex contains the striatin interactor Mob3 (
      • Moreno C.S.
      • Lane W.S.
      • Pallas D.C.
      ), the uncharacterized protein STRIP1, members of the germinal center kinase III (GCKIII) group (STK24, STK25, and MST4; Ref.
      • Dan I.
      • Ong S.E.
      • Watanabe N.M.
      • Blagoev B.
      • Nielsen M.M.
      • Kajikawa E.
      • Kristiansen T.Z.
      • Mann M.
      • Pandey A.
      ), and the small molecular weight protein CCM3 (Fig. 1A). Additional proteins can associate with this core STRIPAK complex in a mutually exclusive manner (
      • Goudreault M.
      • D'Ambrosio L.M.
      • Kean M.J.
      • Mullin M.J.
      • Larsen B.G.
      • Sanchez A.
      • Chaudhry S.
      • Chen G.I.
      • Sicheri F.
      • Nesvizhskii A.I.
      • Aebersold R.
      • Raught B.
      • Gingras A.C.
      ).
      Figure thumbnail gr1
      FIGURE 1Striatin is a scaffolding subunit within STRIPAK. A, composition of core STRIPAK. Proteins, paralogous genes (human nomenclature), and function of proteins are listed. B, schematic of the constructs used in this study. Human STRN3 constructs are in green, mouse Strn constructs are in purple. The first and last amino acid in each construct are indicated. C, summary of AP-MS results for the association of core STRIPAK components with the STRN3 (green) and Strn (purple) deletion mutants (top row). Identified proteins (hits; bottom row) in red do not interact with Strn(91–780) or STRN3(220–713), whereas hits in yellow do. The thickness of each line is proportional to the number of spectral counts (total number of peptides) recovered for each of the proteins in the analysis of the striatin mutants, relative to the spectral counts for the same protein in the AP-MS of full-length Strn. Note that each node (and its associated edges) represents paralogous families, as defined in A. The complete mass spectrometry data used to make this figure are presented in .
      CCM3 is encoded by one of the three genes mutated in familial cerebral cavernous malformations (CCMs; Ref.
      • Guclu B.
      • Ozturk A.K.
      • Pricola K.L.
      • Bilguvar K.
      • Shin D.
      • O'Roak B.J.
      • Gunel M.
      ) and was identified previously as an interactor for the GCKIII proteins (
      • Ma X.
      • Zhao H.
      • Shan J.
      • Long F.
      • Chen Y.
      • Chen Y.
      • Zhang Y.
      • Han X.
      • Ma D.
      ,
      • Rual J.F.
      • Venkatesan K.
      • Hao T.
      • Hirozane-Kishikawa T.
      • Dricot A.
      • Li N.
      • Berriz G.F.
      • Gibbons F.D.
      • Dreze M.
      • Ayivi-Guedehoussou N.
      • Klitgord N.
      • Simon C.
      • Boxem M.
      • Milstein S.
      • Rosenberg J.
      • Goldberg D.S.
      • Zhang L.V.
      • Wong S.L.
      • Franklin G.
      • Li S.
      • Albala J.S.
      • Lim J.
      • Fraughton C.
      • Llamosas E.
      • Cevik S.
      • Bex C.
      • Lamesch P.
      • Sikorski R.S.
      • Vandenhaute J.
      • Zoghbi H.Y.
      • Smolyar A.
      • Bosak S.
      • Sequerra R.
      • Doucette-Stamm L.
      • Cusick M.E.
      • Hill D.E.
      • Roth F.P.
      • Vidal M.
      ). CCMs are vascular lesions of the brain characterized by enlarged capillaries that lack structural integrity and that form caverns that tend to bleed, leading to symptoms ranging from headaches and dizziness to severe strokes and death (reviewed in Ref.
      • Riant F.
      • Bergametti F.
      • Ayrignac X.
      • Boulday G.
      • Tournier-Lasserve E.
      ). Recent studies have implicated defective Rho signaling as one of the consequences of depletion (or overexpression) of the CCM1, CCM2, and CCM3 proteins (
      • Borikova A.L.
      • Dibble C.F.
      • Sciaky N.
      • Welch C.M.
      • Abell A.N.
      • Bencharit S.
      • Johnson G.L.
      ,
      • Whitehead K.J.
      • Chan A.C.
      • Navankasattusas S.
      • Koh W.
      • London N.R.
      • Ling J.
      • Mayo A.H.
      • Drakos S.G.
      • Jones C.A.
      • Zhu W.
      • Marchuk D.A.
      • Davis G.E.
      • Li D.Y.
      ,
      • Stockton R.A.
      • Shenkar R.
      • Awad I.A.
      • Ginsberg M.H.
      ). Further links between CCM3 and its kinase partners and cytoskeletal dynamics via the Golgi were also uncovered. The Ser/Thr kinases STK25 and MST4 were found to localize to the Golgi apparatus via an association with the Golgi resident protein GM130 (
      • Preisinger C.
      • Short B.
      • De Corte V.
      • Bruyneel E.
      • Haas A.
      • Kopajtich R.
      • Gettemans J.
      • Barr F.A.
      ). Mislocalization of these kinases results in defects in Golgi positioning and cell migration (
      • Preisinger C.
      • Short B.
      • De Corte V.
      • Bruyneel E.
      • Haas A.
      • Kopajtich R.
      • Gettemans J.
      • Barr F.A.
      ). Recently, CCM3 was shown to participate in this effect by stabilizing the GCKIII proteins to promote Golgi orientation and assembly and proper cell orientation (
      • Fidalgo M.
      • Fraile M.
      • Pires A.
      • Force T.
      • Pombo C.
      • Zalvide J.
      ).
      Here, we define the structural organization of the STRIPAK complex, identifying direct interactions and interacting regions within the complex. Specifically, we demonstrate that the striatins and CCM3 act as adapter molecules to bridge the kinase and phosphatase catalytic activities (an accompanying publication by Ceccarelli et al. characterizes interactions between the GCKIII proteins and CCM3;
      • Ceccarelli D.F.
      • Laister R.C.
      • Mulligan V.K.
      • Kean M.J.
      • Goudreault M.
      • Scott I.
      • Derry W.B.
      • Chakrabartty A.
      • Gingras A.C.
      • Sicheri F.
      ). We also report the surprising finding that CCM3 and striatins exhibit opposing functions on the targeting of MST4 to the Golgi and Golgi positioning.

      DISCUSSION

      We have described the molecular organization of the STRIPAK complex and assigned a role to the disease-related CCM3 protein as an adaptor that links the kinase and phosphatase subunits of STRIPAK. We have also described a means by which the functions of striatin and CCM3 oppose each other, through the regulation of MST4 interactions and localization as well as their effect on Golgi positioning after stimulation by wounding the cell monolayer. These data suggest that the interaction between CCM3 and STRIPAK via direct association with striatin may serve as a regulatory mechanism to control the function of the MST4 kinase. Importantly, these results also suggest that Golgi localization of MST4 may be detrimental to polarization. The Golgi apparatus has emerged as a critical hub for intracellular signaling (
      • Farhan H.
      • Rabouille C.
      ), and signaling is essential for Golgi polarization. For example, phosphorylation of the Golgi protein, GORASP1 (also known as GRASP65, a GM130 interaction partner), by the kinase ERK is required for Golgi reorientation (
      • Bisel B.
      • Wang Y.
      • Wei J.H.
      • Xiang Y.
      • Tang D.
      • Miron-Mendoza M.
      • Yoshimura S.
      • Nakamura N.
      • Seemann J.
      ). Interestingly, ERK activity has been shown to be modulated by CCM3 and MST4 (
      • Ma X.
      • Zhao H.
      • Shan J.
      • Long F.
      • Chen Y.
      • Chen Y.
      • Zhang Y.
      • Han X.
      • Ma D.
      ); whether or not GORASP1 phosphorylation is modulated in our system remains to be tested.
      A large body of evidence suggests important roles for polarized localization of the Golgi (
      • Yadav S.
      • Puri S.
      • Linstedt A.D.
      ). Cell migration requires polarized secretion at the leading edge for the regulated transport of vesicles, the delivery of adhesion molecules and cytoskeletal components, as well as the addition of new membranes. The polarized localization of the Golgi has also been intimately linked to the small proteins of the Rho-GTPase family (
      • Etienne-Manneville S.
      • Hall A.
      ,
      • Jaffe A.B.
      • Hall A.
      ). In light of the defects in Rho signaling following modulation of CCM1, CCM2, or CCM3 expression (
      • Borikova A.L.
      • Dibble C.F.
      • Sciaky N.
      • Welch C.M.
      • Abell A.N.
      • Bencharit S.
      • Johnson G.L.
      ,
      • Whitehead K.J.
      • Chan A.C.
      • Navankasattusas S.
      • Koh W.
      • London N.R.
      • Ling J.
      • Mayo A.H.
      • Drakos S.G.
      • Jones C.A.
      • Zhu W.
      • Marchuk D.A.
      • Davis G.E.
      • Li D.Y.
      ,
      • Stockton R.A.
      • Shenkar R.
      • Awad I.A.
      • Ginsberg M.H.
      ), it is tempting to postulate that CCM3, MST4, and perhaps STRIPAK may regulate Golgi polarization via regulation of Rho-GTPases. Whether striatins, CCM3, and MST4 play a role in all aspects of Golgi polarization, including cell migration, remains to be answered.
      Given that STRIPAK contains both kinase and phosphatase activities, our results suggest the existence of a molecular switch defined by the balance of phosphorylation and dephosphorylation at the Golgi. At this point, the target(s) for the MST4 kinase (or the PP2A phosphatase) in the Golgi polarization process are still unknown. Additionally, whether and how Golgi polarization may contribute to the vascular defects observed in CCM patients remains to be investigated.
      New roles for STRIPAK complex components are beginning to emerge, in large part through analysis of STRIPAK paralogs across species. It is noteworthy that a portion of the STRIPAK complex (lacking CCM3 and the GCKIII protein component) has been conserved throughout eukaryotic evolution. Ancestral roles for STRIPAK point to cytoskeletal and membrane dynamics functions. In Saccharomyces cerevisiae, Far8 (striatin), Far11 (STRIP1/2), Vps64/Far10 (orthologous to the alternate STRIPAK component SLMAP), along with Far3 and Far7 (for which no human orthologs are known) form a protein complex implicated in cell cycle arrest following pheromone treatment (
      • Kemp H.A.
      • Sprague Jr., G.F.
      ). Orthologs of these ancestral STRIPAK genes are required for proper vegetative membrane fusion in filamentous fungi (
      • Simonin A.R.
      • Rasmussen C.G.
      • Yang M.
      • Glass N.L.
      ,
      • Xiang Q.
      • Rasmussen C.
      • Glass N.L.
      ). The function of STRIPAK in mediating membrane fusion appears to have been conserved in mammals, as deregulation of SLMAP prevents myoblast fusion to myotubes (
      • Guzzo R.M.
      • Wigle J.
      • Salih M.
      • Moore E.D.
      • Tuana B.S.
      ). More recently, deletion of the orthologs of striatin (FAR8), STRIP1/2 (FAR11), or one of the PP2A catalytic subunits (PPG1) was demonstrated to suppress lethality and actin cytoskeleton disorganization caused by mutations of TORC2 (target of rapamycin complex 2) (
      • Baryshnikova A.
      • Costanzo M.
      • Kim Y.
      • Ding H.
      • Koh J.
      • Toufighi K.
      • Youn J.Y.
      • Ou J.
      • San Luis B.J.
      • Bandyopadhyay S.
      • Hibbs M.
      • Hess D.
      • Gingras A.C.
      • Bader G.D.
      • Troyanskaya O.G.
      • Brown G.W.
      • Andrews B.
      • Boone C.
      • Myers C.L.
      ). Interestingly, TORC2 controls actin cytoskeleton assembly across multiple species, in part via regulation of the Rho1 GTPases (
      • Jacinto E.
      • Loewith R.
      • Schmidt A.
      • Lin S.
      • Rüegg M.A.
      • Hall A.
      • Hall M.N.
      ,
      • Loewith R.
      • Jacinto E.
      • Wullschleger S.
      • Lorberg A.
      • Crespo J.L.
      • Bonenfant D.
      • Oppliger W.
      • Jenoe P.
      • Hall M.N.
      ,
      • Helliwell S.B.
      • Howald I.
      • Barbet N.
      • Hall M.N.
      ). CCM disease, CCM3, and MST4 are intimately linked to Rho signaling in human cells (
      • Borikova A.L.
      • Dibble C.F.
      • Sciaky N.
      • Welch C.M.
      • Abell A.N.
      • Bencharit S.
      • Johnson G.L.
      ,
      • Whitehead K.J.
      • Chan A.C.
      • Navankasattusas S.
      • Koh W.
      • London N.R.
      • Ling J.
      • Mayo A.H.
      • Drakos S.G.
      • Jones C.A.
      • Zhu W.
      • Marchuk D.A.
      • Davis G.E.
      • Li D.Y.
      ,
      • Stockton R.A.
      • Shenkar R.
      • Awad I.A.
      • Ginsberg M.H.
      ,
      • Zheng X.
      • Xu C.
      • Di Lorenzo A.
      • Kleaveland B.
      • Zou Z.
      • Seiler C.
      • Chen M.
      • Cheng L.
      • Xiao J.
      • He J.
      • Pack M.A.
      • Sessa W.C.
      • Kahn M.L.
      ), suggesting that this function of STRIPAK has been evolutionarily conserved. In addition to these roles in cytoskeleton and membrane dynamics, a surprising recent report implicated the Drosophila STRIPAK complex (including CCM3) in Hippo signaling (
      • Ribeiro P.S.
      • Josué F.
      • Wepf A.
      • Wehr M.C.
      • Rinner O.
      • Kelly G.
      • Tapon N.
      • Gstaiger M.
      ), indicating that STRIPAK may control multiple signaling pathways. The elucidation of the substrates of the kinase and phosphatase components of STRIPAK will be required for a full molecular understanding of STRIPAK function.
      Finally, although our data point to the STRIPAK complex as the major interactor for epitope-tagged or endogenous CCM3 protein in HEK293 cells (
      • Goudreault M.
      • D'Ambrosio L.M.
      • Kean M.J.
      • Mullin M.J.
      • Larsen B.G.
      • Sanchez A.
      • Chaudhry S.
      • Chen G.I.
      • Sicheri F.
      • Nesvizhskii A.I.
      • Aebersold R.
      • Raught B.
      • Gingras A.C.
      ), HeLa cells, C2C12 myoblasts, and myotubes and in bovine endothelial aortic cells (data not shown) CCM3 is also capable of interacting with CCM2 (
      • Hilder T.L.
      • Malone M.H.
      • Bencharit S.
      • Colicelli J.
      • Haystead T.A.
      • Johnson G.L.
      • Wu C.C.
      ) and paxillin (
      • Li X.
      • Zhang R.
      • Zhang H.
      • He Y.
      • Ji W.
      • Min W.
      • Boggon T.J.
      ). Because these interactions are apparently mediated via the same surface as the striatin binding site on CCM3, we propose here that they may be mutually exclusive. Further studies on CCM3 function in vascular disease and elsewhere will need to take these alternative protein assemblies into consideration.

      Acknowledgments

      We thank A. Fernandes and members of the Gingras, Pelletier, and Sicheri laboratories for discussion and technical help and J.-P. Lambert for comments on the manuscript.

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