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Epithelial Polarity: Dual Lkb1 Pathways Regulate Apical Microvilli
Developmental Cell
Previews Epithelial Polarity: Dual Lkb1 Pathways Regulate Apical Microvilli Anthony Bretscher1,* 1Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2009.03.012
The Lkb1/Strad/Mo25 complex can polarize single epithelial cells, leading to the formation of a brush border containing microvilli on the apical surface. In this issue of Developmental Cell, ten Klooster et al. show that the Lkb1/Strad/Mo25 complex mediates two separate pathways, and that both are required for assembly of apical microvilli. Cells have regulatory mechanisms for placing the appropriate proteins and lipids in specific domains at the cell surface to achieve a state known as cell polarity. A classical example is the polarized epithelial cell, with its microvilli-studded apical membrane—comprising the brush border—segregated by tight junctions from the distinct basolateral domain. The signaling pathways that polarize cells have been studied in a diversity of systems, including yeasts, nematodes, flies, and vertebrate systems, and many of these pathways have been conserved through evolution. For example, a search for factors important for the first asymmetric division in the nematode embryo led to the discovery of the PAR proteins, many of which are now known to be critical factors in the establishment of polarity in multiple systems. In epithelial cells, cues from adjacent cells and the extracellular matrix were generally believed to provide the polarity input. Thus the report that single epithelial cells could be induced to polarize spontaneously upon induction of the Lkb1 kinase, the human homolog of nematode PAR-4, was a fascinating finding (Baas et al., 2004). The authors showed that upon activation of Lkb1, single epithelial cells become polarized with a morphologically distinct apical brush border containing microvilli, and with apical proteins segregated from the basolateral membrane by rudimentary complexes containing tight junction markers. At the time, Lkb1 kinase was known to regulate cell proliferation and to be mutated in Peutz-Jeghers cancer syndrome, and to also have many other roles, including responding to low levels of energy metabolism by phosphorylating AMPK (Alessi et al., 2006), but its role in
cell polarity was novel. A paper in this issue of Developmental Cell (ten Klooster et al., 2009) begins to dissect the pathways from Lkb1 that set up this polarity. Active Lkb1 exists in a complex with the pseudokinase Strad and an adaptor protein, Mo25 (Baas et al., 2003; Boudeau et al., 2003). To test the role of Mo25, the authors knocked down expression of Mo25 in cells expressing Lkb1 and Strad. The cells were unable to polarize: no actin-rich brush borders were formed and apical and basolateral proteins were not segregated. A key feature of microvilli on polarized epithelial cells is the presence of the membrane-microfilament linking protein Ezrin (Bretscher et al., 2002). Ezrin is required for normal microvilli on these cells (Saotome et al., 2004), and its activity is regulated by phosphorylation of threonine 567. In single epithelial cells polarized by the expression of Lkb1/Strad/Mo25, phosphorylated Ezrin is enriched in apical microvilli, whereas in Mo25-depleted cells, Ezrin phosphorylation drops dramatically. Moreover, overexpression of a nonphosphorylatable mutant, Ezrin T567A, inhibits Lkb1/Strad/Mo25-induced actin polarization. This suggests that Ezrin phosphorylation is a critical aspect of the polarization process, but how is it linked to Lkb1 activation? Two strategies to identify the relevant kinase converged on the Ste20-related Mst4. First, the authors undertook a twohybrid screen with Mo25 as bait that yielded Strad, as expected, and Mst4. Second, they assayed a panel of 80 kinases to ask which can phosphorylate the appropriate Ezrin peptide. Along with kinases already known to phosphorylate Ezrin on threonine 567, they identified members of the Mst family of kinases. In
cells expressing Lkb1/Strad/Mo25, knockdown of Mst4 eliminated Ezrin phosphorylation and the polarized distribution of actin. These findings implicated Mst4 downstream of Lkb1/Strad/Mo25 in the polarization pathway. Mst4 is activated by autophosphorylation, but the level of this phosphorylation is independent of the presence of active Lkb1, so how does Lkb1/Strad/Mo25 regulate Mst4’s function? Mst4 is normally localized to the Golgi in fibroblasts through binding of GM130; however, the authors show that upon Lkb1/Strad/ Mo25 activation in epithelial cells, Mst4 becomes partially translocated to the apical domain, where it presumably phosphorylates Ezrin. What aspects of epithelial cell polarization does the Mst4 kinase control? As originally shown by Baas et al. (2004), active Lkb1 polarizes both the actin cytoskeleton to form microvilli containing the actin cross-linking protein villin, and the asymmetric distribution of membrane markers to define the apical and basolateral membranes. When Mst4 is depleted in the presence of active Lkb1/Strad/Mo25, membrane proteins are still polarized, but Ezrin is not phosphorylated and no villincontaining apical microvilli are assembled. In these Mst4-depleted cells, apical microvilli can be restored by expression of the phosphomimetic Ezrin T567D. Since expression of Ezrin T567D without active Lkb1/Strad/Mo25 cannot polarize the cells, full polarization depends on both Ezrin phosphorylation by Mst4 and on other aspects of the Lkb1/Strad/Mo25dependent pathway to polarize apical and basolateral membrane proteins. This pathway has been elucidated in cultured epithelial cells, but does it relate
Developmental Cell 16, April 21, 2009 ª2009 Elsevier Inc. 491
Developmental Cell
Previews to epithelial cell polarization in tissues? As ten Klooster et al. show, Mst4 is enriched in the apical aspect of epithelial cells that line the small intestine. However, while intestinal epithelial cells in mice lacking Ezrin have less ordered microvilli, microvilli are present and they still contain villin (Saotome et al., 2004). Thus, it seems likely that in the context of tissues, additional signaling pathways independent of Ezrin contribute to drive apical microvilli assembly or maintenance. The Lkb1/Strad/Mo25/Mst4 pathway seems to be widely conserved in regulating cell polarity. Nematodes contain homologs of these proteins (PAR-4/STRD-1/ MOP-25/GCK-1) and RNAi knockdown of gck-1 reduces cortical ruffling in the onecell embryo (Sonnichsen et al., 2005), a process that in vertebrates involves Ezrin-related proteins. Although yeasts do not have microvilli or contain Ezrin, they do appear to use a related pathway to control aspects of cell polarization. The
homolog of Mst4 in budding yeast is Kic1, which binds the Mo25 homolog Hym1, and loss of this pathway compromises polarized cell growth (Nelson et al., 2003). In fission yeast, the respective homologs of Mst4 and Mo25 are Nak1 and Pmo25, and they too are implicated in the polar accumulation of actin at growth zones (Leonhard and Nurse, 2005). In both nematodes and yeast, it will be fascinating to find the substrates of the Mst4 homologs to see how this polarity pathway has been utilized through evolution.
REFERENCES Alessi, D.R., Sakamoto, K., and Bayascas, J.R. (2006). Annu. Rev. Biochem. 75, 137–163. Baas, A.F., Boudeau, J., Sapkota, G.P., Smit, L., Medema, R., Morrice, N.A., Alessi, D.R., and Clevers, H.C. (2003). EMBO J. 22, 3062–3072. Baas, A.F., Kuipers, J., van der Wel, N.N., Batlle, E., Koerten, H.K., Peters, P.J., and Clevers, H.C. (2004). Cell 116, 457–466.
492 Developmental Cell 16, April 21, 2009 ª2009 Elsevier Inc.
Boudeau, J., Baas, A.F., Deak, M., Morrice, N.A., Kieloch, A., Schutkowski, M., Prescott, A.R., Clevers, H.C., and Alessi, D.R. (2003). EMBO J. 22, 5102–5114. Bretscher, A., Edwards, K., and Fehon, R.G. (2002). Nat. Rev. Mol. Cell Biol. 3, 586–599. Leonhard, K., and Nurse, P. (2005). J. Cell Sci. 118, 1033–1044. Nelson, B., Kurischko, C., Horecka, J., Mody, M., Nair, P., Pratt, L., Zougman, A., McBroom, L.D., Hughes, T.R., Boone, C., et al. (2003). Mol. Biol. Cell 14, 3782–3803. Saotome, I., Curto, M., and McClatchey, A.I. (2004). Dev. Cell 6, 855–864. Sonnichsen, B., Koski, L.B., Walsh, A., Marschall, P., Neumann, B., Brehm, M., Alleaume, A.M., Artelt, J., Bettencourt, P., Cassin, E., et al. (2005). Nature 434, 462–469. 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., and Clevers, H. (2009). Dev. Cell 16, this issue, 551–562.
Previews Epithelial Polarity: Dual Lkb1 Pathways Regulate Apical Microvilli Anthony Bretscher1,* 1Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2009.03.012
The Lkb1/Strad/Mo25 complex can polarize single epithelial cells, leading to the formation of a brush border containing microvilli on the apical surface. In this issue of Developmental Cell, ten Klooster et al. show that the Lkb1/Strad/Mo25 complex mediates two separate pathways, and that both are required for assembly of apical microvilli. Cells have regulatory mechanisms for placing the appropriate proteins and lipids in specific domains at the cell surface to achieve a state known as cell polarity. A classical example is the polarized epithelial cell, with its microvilli-studded apical membrane—comprising the brush border—segregated by tight junctions from the distinct basolateral domain. The signaling pathways that polarize cells have been studied in a diversity of systems, including yeasts, nematodes, flies, and vertebrate systems, and many of these pathways have been conserved through evolution. For example, a search for factors important for the first asymmetric division in the nematode embryo led to the discovery of the PAR proteins, many of which are now known to be critical factors in the establishment of polarity in multiple systems. In epithelial cells, cues from adjacent cells and the extracellular matrix were generally believed to provide the polarity input. Thus the report that single epithelial cells could be induced to polarize spontaneously upon induction of the Lkb1 kinase, the human homolog of nematode PAR-4, was a fascinating finding (Baas et al., 2004). The authors showed that upon activation of Lkb1, single epithelial cells become polarized with a morphologically distinct apical brush border containing microvilli, and with apical proteins segregated from the basolateral membrane by rudimentary complexes containing tight junction markers. At the time, Lkb1 kinase was known to regulate cell proliferation and to be mutated in Peutz-Jeghers cancer syndrome, and to also have many other roles, including responding to low levels of energy metabolism by phosphorylating AMPK (Alessi et al., 2006), but its role in
cell polarity was novel. A paper in this issue of Developmental Cell (ten Klooster et al., 2009) begins to dissect the pathways from Lkb1 that set up this polarity. Active Lkb1 exists in a complex with the pseudokinase Strad and an adaptor protein, Mo25 (Baas et al., 2003; Boudeau et al., 2003). To test the role of Mo25, the authors knocked down expression of Mo25 in cells expressing Lkb1 and Strad. The cells were unable to polarize: no actin-rich brush borders were formed and apical and basolateral proteins were not segregated. A key feature of microvilli on polarized epithelial cells is the presence of the membrane-microfilament linking protein Ezrin (Bretscher et al., 2002). Ezrin is required for normal microvilli on these cells (Saotome et al., 2004), and its activity is regulated by phosphorylation of threonine 567. In single epithelial cells polarized by the expression of Lkb1/Strad/Mo25, phosphorylated Ezrin is enriched in apical microvilli, whereas in Mo25-depleted cells, Ezrin phosphorylation drops dramatically. Moreover, overexpression of a nonphosphorylatable mutant, Ezrin T567A, inhibits Lkb1/Strad/Mo25-induced actin polarization. This suggests that Ezrin phosphorylation is a critical aspect of the polarization process, but how is it linked to Lkb1 activation? Two strategies to identify the relevant kinase converged on the Ste20-related Mst4. First, the authors undertook a twohybrid screen with Mo25 as bait that yielded Strad, as expected, and Mst4. Second, they assayed a panel of 80 kinases to ask which can phosphorylate the appropriate Ezrin peptide. Along with kinases already known to phosphorylate Ezrin on threonine 567, they identified members of the Mst family of kinases. In
cells expressing Lkb1/Strad/Mo25, knockdown of Mst4 eliminated Ezrin phosphorylation and the polarized distribution of actin. These findings implicated Mst4 downstream of Lkb1/Strad/Mo25 in the polarization pathway. Mst4 is activated by autophosphorylation, but the level of this phosphorylation is independent of the presence of active Lkb1, so how does Lkb1/Strad/Mo25 regulate Mst4’s function? Mst4 is normally localized to the Golgi in fibroblasts through binding of GM130; however, the authors show that upon Lkb1/Strad/ Mo25 activation in epithelial cells, Mst4 becomes partially translocated to the apical domain, where it presumably phosphorylates Ezrin. What aspects of epithelial cell polarization does the Mst4 kinase control? As originally shown by Baas et al. (2004), active Lkb1 polarizes both the actin cytoskeleton to form microvilli containing the actin cross-linking protein villin, and the asymmetric distribution of membrane markers to define the apical and basolateral membranes. When Mst4 is depleted in the presence of active Lkb1/Strad/Mo25, membrane proteins are still polarized, but Ezrin is not phosphorylated and no villincontaining apical microvilli are assembled. In these Mst4-depleted cells, apical microvilli can be restored by expression of the phosphomimetic Ezrin T567D. Since expression of Ezrin T567D without active Lkb1/Strad/Mo25 cannot polarize the cells, full polarization depends on both Ezrin phosphorylation by Mst4 and on other aspects of the Lkb1/Strad/Mo25dependent pathway to polarize apical and basolateral membrane proteins. This pathway has been elucidated in cultured epithelial cells, but does it relate
Developmental Cell 16, April 21, 2009 ª2009 Elsevier Inc. 491
Developmental Cell
Previews to epithelial cell polarization in tissues? As ten Klooster et al. show, Mst4 is enriched in the apical aspect of epithelial cells that line the small intestine. However, while intestinal epithelial cells in mice lacking Ezrin have less ordered microvilli, microvilli are present and they still contain villin (Saotome et al., 2004). Thus, it seems likely that in the context of tissues, additional signaling pathways independent of Ezrin contribute to drive apical microvilli assembly or maintenance. The Lkb1/Strad/Mo25/Mst4 pathway seems to be widely conserved in regulating cell polarity. Nematodes contain homologs of these proteins (PAR-4/STRD-1/ MOP-25/GCK-1) and RNAi knockdown of gck-1 reduces cortical ruffling in the onecell embryo (Sonnichsen et al., 2005), a process that in vertebrates involves Ezrin-related proteins. Although yeasts do not have microvilli or contain Ezrin, they do appear to use a related pathway to control aspects of cell polarization. The
homolog of Mst4 in budding yeast is Kic1, which binds the Mo25 homolog Hym1, and loss of this pathway compromises polarized cell growth (Nelson et al., 2003). In fission yeast, the respective homologs of Mst4 and Mo25 are Nak1 and Pmo25, and they too are implicated in the polar accumulation of actin at growth zones (Leonhard and Nurse, 2005). In both nematodes and yeast, it will be fascinating to find the substrates of the Mst4 homologs to see how this polarity pathway has been utilized through evolution.
REFERENCES Alessi, D.R., Sakamoto, K., and Bayascas, J.R. (2006). Annu. Rev. Biochem. 75, 137–163. Baas, A.F., Boudeau, J., Sapkota, G.P., Smit, L., Medema, R., Morrice, N.A., Alessi, D.R., and Clevers, H.C. (2003). EMBO J. 22, 3062–3072. Baas, A.F., Kuipers, J., van der Wel, N.N., Batlle, E., Koerten, H.K., Peters, P.J., and Clevers, H.C. (2004). Cell 116, 457–466.
492 Developmental Cell 16, April 21, 2009 ª2009 Elsevier Inc.
Boudeau, J., Baas, A.F., Deak, M., Morrice, N.A., Kieloch, A., Schutkowski, M., Prescott, A.R., Clevers, H.C., and Alessi, D.R. (2003). EMBO J. 22, 5102–5114. Bretscher, A., Edwards, K., and Fehon, R.G. (2002). Nat. Rev. Mol. Cell Biol. 3, 586–599. Leonhard, K., and Nurse, P. (2005). J. Cell Sci. 118, 1033–1044. Nelson, B., Kurischko, C., Horecka, J., Mody, M., Nair, P., Pratt, L., Zougman, A., McBroom, L.D., Hughes, T.R., Boone, C., et al. (2003). Mol. Biol. Cell 14, 3782–3803. Saotome, I., Curto, M., and McClatchey, A.I. (2004). Dev. Cell 6, 855–864. Sonnichsen, B., Koski, L.B., Walsh, A., Marschall, P., Neumann, B., Brehm, M., Alleaume, A.M., Artelt, J., Bettencourt, P., Cassin, E., et al. (2005). Nature 434, 462–469. 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., and Clevers, H. (2009). Dev. Cell 16, this issue, 551–562.