InteractiveFly: GeneBrief

Sterile20-like kinase: Biological Overview | References

Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. A previous study reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. This study also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, a genome-wide RNAi screen was conducted, looking for new regulators of moesin activity. Slik was identified as a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). The phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. These results implicate the Slik-STRIPAK complex in the control of multiple morphogenetic processes (De Jamblinne, 2020).

Cell morphogenesis is an important process by which cells adapt their shapes to achieve different functions. Filaments of the actin and microtubule cytoskeletons play important roles during this process. Actin filaments apply forces to the cortex to contribute to plasma membrane remodeling, whereas microtubules are important for targeted trafficking and signaling. Proteins of the ezrin, radixin, and moesin (ERM) family link actin filaments and microtubules to the plasma membrane (Solinet, 2013). They regulate important cellular processes, such as cell division, migration, and epithelial organization. The phosphorylation of a conserved threonine residue in the C-terminus of ERMs promotes their activation. ERMs cycle between an active conformation at the plasma membrane and an inactive form in the cytosol. An intramolecular interaction between the N-terminal FERM domain and C-terminal tail (C-terminal ERM association domain [CERMAD]) inactivates ERMs. Their activation involves a conformational switch through a multistep mechanism: (1) Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), a phospholipid of the plasma membrane, recruits ERMs to the cortex and slightly opens the molecules; (2) this preopening allows access to LOK, a sterile 20 (Ste20)-like Ser/Thr protein kinase that wedges between the FERM and CERMAD domains to complete full ERM opening; and (3) the kinase phosphorylates the regulatory threonine of ERMs to stabilize their open conformation. This opening unmasks the actin-binding site in the CERMAD and the microtubule-interacting site in the FERM domain and allows ERMs to link actin and microtubule filaments at the plasma membrane to regulate cell morphogenesis (De Jamblinne, 2020).

Previous work has shown that moesin controls epithelial integrity and mitotic morphogenesis. In Drosophila wing imaginal discs, it was discovered that Slik, the Drosophila orthologue of mammalian SLK and LOK, phosphorylates moesin to control epithelial tissue integrity. Cells lacking either Slik or moesin undergo an epithelial-to-mesenchymal transition (EMT), sort basally out of the wing disc epithelium, and undergo apoptosis (De Jamblinne, 2020).

In Drosophila S2 cells in culture, it was shown that moesin controls mitotic cell shape and organization of the mitotic spindle. Mitosis entry is characterized by an approximately threefold increase in moesin phosphorylation. Slik is responsible for the basal level of moesin phosphorylation in interphase and for the specific activation of moesin in mitosis. In metaphase, phosphorylated moesin (p-moesin) spreads around the cortex and increases global cortical rigidity by coupling actin forces to the plasma membrane. This promotes rounding of cells. In addition, moesin-microtubule interactions regulate mitotic spindle organization. In anaphase, the Ser/Thr phosphatase Pp1-87B dephosphorylates moesin at the poles and favors p-moesin accumulation at the equator. This redistribution drives cell elongation and cytokinesis. After abscission, Pp1-87B dephosphorylates moesin to allow cortex relaxation. Either inhibition or overactivation of moesin has damaging effects in mitosis. Overactivation of moesin overstiffens the polar cortex in anaphase, preventing cell elongation and cytokinesis. Conversely, moesin or Slik double-stranded RNA (dsRNA) depletion renders the cortex too soft; cells do not control mitotic morphogenesis properly and present abnormal cortical blebs(De Jamblinne, 2020).

In addition to Pp1-87B and Slik, this study also found that the PtdIns-4-kinase CG10260 and the PtdIns(4)P 5-kinase Skittles regulate localized PtdIns(4,5)P2 production at the plasma membrane to control moesin localization. However, it is still unknown how the enzymes of the signaling network activating moesin are themselves regulated. To this end, an unbiased genome-wide dsRNA screen was performed in Drosophila S2 cells and p-moesin levels were measured. Striatin-interacting phosphatase and kinase complex (STRIPAK) were found to be a positive regulator of moesin phosphorylation. At the core of the STRIPAK complex, there is a protein Ser/Thr phosphatase 2A (hereafter referred to as 'PP2A_STRIPAK'). PP2A_STRIPAK is a heterotrimeric enzyme that consists of a PP2A catalytic C subunit (Mts, in Drosophila), a PP2A scaffold A subunit (PP2A-29B), and a striatin regulatory B subunit (Cka). On top of this core, striatin-interacting proteins known as STRIPs (Strip) help to scaffold Ser/Thr kinases of the Ste20-like family (De Jamblinne, 2020).

STRIPAK controls the activity of its associated kinases, either by regulating phosphorylation of regulatory residues or by recruiting them to specific sites in the cell. Thereby, STRIPAK affects cell proliferation and survival, cytoskeletal regulation, and vesicle trafficking. This study shows that Slik is a new STRIPAK-associated kinase. PP2A_STRIPAK was found to promote Slik association with the cortex by regulating its phosphorylation status. Finally, PP2A_STRIPAK was shown to control Slik and moesin functions to regulate mitotic morphogenesis and epithelial integrity. These results place STRIPAK as a critical upstream regulator of cell morphogenesis through its effects on Slik and, ultimately, moesin (De Jamblinne, 2020).

This study discovered that dSTRIPAK dephosphorylates Slik to favor its association with the cortex. This regulation of Slik cortical association occurs both in interphase and in metaphase. Importantly, loss of Slik cortical association correlates with a decreased activation of moesin, the substrate of Slik throughout the cell cycle. Finally, dSTRIPAK was shown to control moesin-related biological functions such as mitotic morphogenesis and epithelial integrity during development (De Jamblinne, 2020).

dSTRIPAK is a multimolecular complex that functionally bridges PP2A phosphatase activity with kinases of the Ste20-like family (Kuck, 2019). PP2A_STRIPAK was shown to inhibit the catalytic activity of its associated kinases. For instance, PP2A_STRIPAK dephosphorylates key regulatory residues of the activation loop of Hippo and Mst3/4. This study found a new functional interaction between PP2A_STRIPAK and an Ste20-like kinase. PP2A_STRIPAK regulates Slik phosphorylation to control its association with the cortex and thereby regulates its activity toward moesin. PP2A_STRIPAK could indirectly regulate an upstream regulator of Slik or directly dephosphorylate this Ste20-like kinase. The latter hypothesis is favored because this study found that Slik associates with Cka and Strip, two major components of PP2A_STRIPAK (De Jamblinne, 2020).

It is unlikely that PP2A_STRIPAK dephosphorylates the regulatory residues of the activation loop of Slik, because no change of its kinase activity was detected upon depletion of key components of PP2A_STRIPAK. Focus was then placed on 21 other potential phosphosites, and PP2A_STRIPAK was found to favor association of Slik with the cortex by regulating some of these phosphosites. Replacing these potentially phosphorylated residues by nonphosphorylatable alanine either within the NCD or within the CTD of Slik is sufficient to promote the cortical enrichment of these two phosphodeficient mutants and to prevent their regulation by PP2A_STRIPAK. Importantly, both these mutants rescue or partially rescue the decrease in moesin phosphorylation observed after PP2A_STRIPAK depletion. Although these experiments demonstrate that global phosphorylation governs the association of Slik with the cortex, the sites regulated by PP2A_STRIPAK remain to be precisely identified (De Jamblinne, 2020).

It was previously found that the CTD of Slik is necessary and sufficient for its association with the cell cortex (Panneton, 2015). Interestingly, this domain is polybasic (with a theoretical isoelectric point of ~9.6 compared with Slik ~6.1). As previously demonstrated for other signaling proteins, these positive charges could promote the association of Slik with the negatively charged inner leaflet of the plasma membrane. It is proposed that the global negative charge brought by phosphorylation promotes the dissociation of Slik with the cell cortex by electrostatic repulsion with the negatively charged plasma membrane. In this model, a cycle of phosphorylation-dephosphorylation of Slik serves as an electrostatic switch that controls Slik cortical association. It has previously been reported that Slik can be phosphorylated by other not yet identified kinases (Panneton, 2015). Consistent with this, it was found that OA treatment promotes phosphorylation of a Slik kinase dead mutant even in the absence of endogenous Slik. Therefore the hypothesis is favored that one or more other kinases regulate the association of Slik with the cortex. Current efforts aim to identify which kinase(s) could promote the dissociation of Slik from the plasma membrane (De Jamblinne, 2020).

PP2A phosphatase activity plays important roles during mitosis. Independently of STRIPAK, PP2A regulates mitotic exit by dephosphorylating Cdk1 substrates when associated with the B55 regulatory subunit (Drosophila Tws). When associated with the B56 regulatory subunit (Drosophila Wdb), PP2A controls spindle organization by counteracting several kinases, such as Aurora-B or Plk1. However, only a few studies reported a role of PP2A_STRIPAK during mitosis. In mammalian cells, PP2A_STRIPAK regulates abscission by controlling Mink1 activity, and depletion of STRIP or striatin promotes cytokinesis failures. This study has discovered that PP2A_STRIPAK controls mitotic morphogenesis. Mitotic cell morphogenesis requires that moesin be activated at mitosis onset. PP2A_STRIPAK was found to control enrichment of Slik at the cortex of metaphase cells and subsequent moesin activation (De Jamblinne, 2020).

Moesin and Slik play an important role in promoting epithelial integrity in vivo, although the details of their regulation in this context are also poorly understood. In mitotic epithelial cells, p-moesin accumulates at the basolateral membrane and is needed for planar spindle orientation. Moesin-depleted cells frequently undergo mitoses perpendicular to the plane of the epithelium, with the daughter cells undergoing EMT and delaminating and ultimately being eliminated by apoptosis. This appears to be a major cause of the loss of epithelial tissue integrity in moesin mutants. Several observations implicate dSTRIPAK in promoting moesin activation and epithelial tissue integrity. dSTRIPAK-depleted cells undergo EMT, delaminate, and are eliminated by apoptosis, particularly those located in the wing pouch. In dSTRIPAK-depleted cells in the wing hinge region that maintained their epithelial phenotype, reduced levels were observed of p-moesin, a readout for Slik activity. This was not just correlative; the ability of overexpressed Slik to phosphorylate moesin depended on dSTRIPAK. Finally, dSTRIPAK-depleted cells showed a redistribution of Slik protein, with a reduction of protein levels particularly prominent in the basal half of the cells. Together, these results support the finding that dSTRIPAK is a general regulator of Slik localization to promote the morphogenetic functions of moesin. It is noted that the phenotypic consequences of interfering with dSTRIPAK were more severe than those observed upon depletion of Slik, with strong dSTRIPAK depletion causing early lethality. This suggests that dSTRIPAK regulates other targets, consistent with its known role in downregulating the growth-inhibitory function of Hippo. Hippo signaling is particularly important for growth of the wing blade, which may explain why depletion of dSTRIPAK components had a stronger effect in the wing pouch than in hinge regions (De Jamblinne, 2020).

In conclusion, this study identified that PP2A_STRIPAK regulates the localization of Slik by phosphorylation to control moesin activation. This work places STRIPAK as a critical upstream regulator of moesin function for mitotic morphogenesis and epithelial integrity (De Jamblinne, 2020).

Slik phosphorylation of talin T152 is crucial for proper talin recruitment and maintenance of muscle attachment in Drosophila

Talin is the major scaffold protein linking integrin receptors with the actin cytoskeleton. In Drosophila, extended talin generates a stable link between the sarcomeric cytoskeleton and the tendon matrix at muscle attachment sites. This study identified phosphorylation sites on Drosophila talin by mass spectrometry. Talin is phosphorylated in late embryogenesis when muscles differentiate, especially on T152 in the exposed loop of the F1 domain of the talin head. Localization of talin-T150/T152A is reduced at muscle attachment sites and can only partially rescue muscle attachment compared to wild type talin. Slik was identified as the kinase phosphorylating talin at T152. Slik localizes to muscle attachment sites, and the absence of Slik reduces the localization of talin at muscle attachment sites causing phenotypes similar to talin-T150/T152A. Thus, these results demonstrate that talin phosphorylation by Slik plays an important role in fine-tuning talin recruitment to integrin adhesion sites and maintaining muscle attachment (Katzemich, 2019).

Phosphorylation of the Talin FERM domain by Slik as being important for Talin function in muscles. This pathway is likely conserved in vertebrates, because SLK and talin colocalize at focal adhesions and a conditional Slk knockout in skeletal muscles results in progressive myopathy (Pryce, 2017). In platelets, the high stoichiometry phosphorylation sites of Talin are T144 and T150. The effect of their phosphorylation is somewhat inconclusive, but it appears that T144/T150A mutations in tissue culture reduce cell adhesion and increase focal adhesion turnover. A recent structural study indicates that the unstructured F1 loop does not interact with positively charged membrane phospholipids, suggesting that F1 loop phosphorylation should not disrupt membrane recruitment. This is consistent with the full rescue observed with T152E. Talin-E1777A, a mutant keeping Talin always in the extended, active conformation, fully rescues muscle detachment, and localizes more strongly to muscle attachment sites than does wild-type Talin. A multi-step mechanism of Talin activation is proposed, in which phosphorylation of threonine 150/152 is one step contributing to Talin activation and separation of the head and rod domains of Talin. Although the detailed mechanism and the sequence of events remain to be uncovered, this work identifies the first kinase involved in Talin F1 loop phosphorylation and demonstrates that this phosphorylation is crucial for the maintenance of muscle attachment (Katzemich, 2019).

Moesin is involved in polarity maintenance and cortical remodelling during asymmetric cell division

An intact actomyosin network is essential for anchoring polarity proteins to the cell cortex and maintaining cell size asymmetry during asymmetric cell division of Drosophila neuroblasts. However, the mechanisms that control changes in actomyosin dynamics during asymmetric cell division remain unclear. This study finds that the actin-binding protein, Moesin, is essential for neuroblast proliferation and mitotic progression in the developing brain. During metaphase, phosphorylated Moesin (p-Moesin) is enriched at the apical cortex and loss of Moesin leads to defects in apical polarity maintenance and cortical stability. This asymmetric distribution of p-Moesin is determined by components of the apical polarity complex and Slik kinase. During later stages of mitosis, p-Moesin localization shifts more basally, contributing to asymmetric cortical extension and myosin basal furrow positioning. These findings reveal Moesin as a novel apical polarity protein that drives cortical remodelling of dividing neuroblasts, which is essential for polarity maintenance and initial establishment of cell size asymmetry (Abeysundara, 2017).

Studies investigating ERM function have highlighted the importance of the ERM proteins in regulating the mechanical properties of the cell cortex. This study provides new insight into the role of Moesin in organizing the cortex of cells that establish intrinsic polarity and undergo asymmetric cell division (ACD) in vivo. When Moesin was knocked down in Insc-expressing cells, the larval CNS was reduced in size due to a decrease in the proportion of dividing NBs throughout larval development. Expressing Moe^dsRNA using the Insc-GAL4 driver affected overall larval development and resulted in larval lethality. However, viable progeny were obtained when Moesin levels were reduced using other NB-GAL4 drivers, asense-GAL4 and worniu-GAL4. When upstream activation sequence-GFP (UAS-GFP) was expressed using the different NB GAL4 drivers, GFP mRNA expression was ~5 fold greater using Insc-GAL4 compared with asense- or worniu-GAL4. Thus, the differences in viability are likely due to the increased strength of Insc-GAL4. Recent studies that identified the Hippo pathway as an essential regulator of NB quiescence also used Insc-GAL4 in their analyses. As it cannot be excluded that the reduced proportion of mitotic NBs may partially be due to impaired cell cycle reentry or an overall delay in larval development, the analysis focussed on the mitotic NBs that had exited quiescence. It was confirmed that defects in mitotic progression and polarity maintenance were observed at both early and late stages of the Moesin knockdown and in the late hypomorphic mutants, demonstrating a functional requirement of Moesin within the larval NBs (Abeysundara, 2017).

Proper regulation and function of the ERM proteins are required during cell division in both flies and mammals . In Drosophila S2 cells, the increased and uniform distribution of p-Moesin at the metaphase cortex enhanced cortical rigidity and cell rounding, proposed to be essential for stable spindle positioning. Drosophila Moesin was also shown to bind and stabilize microtubules at the cortex of cultured cells. Thus, an asymmetric ERM distribution during metaphase would be predicted to influence spindle position and orientation accordingly. In human colorectal Caco2 cells, polarized ezrin locally stabilized actin, providing a physical platform for astral microtubule-mediated centrosome positioning during interphase. HeLa cells cultured on L-shaped micropatterns also displayed restricted ERM activation at the cell cortex adjacent to the adhesive substrate, which was essential for LGN/NuMA polarization and guiding spindle orientation. In Drosophila wing imaginal epithelial cells, p-Moesin was enriched at the basal cortex of mitotic cells and the loss of Moesin led to defects in planar spindle orientation and recruitment of the pericentriolar material marker, Centrosomin. Thus, a role for Moesin in guiding spindle orientation and centrosome behaviour has been well documented. In Drosophila NBs, this study found that p-Moesin was apically enriched at the metaphase cortex, although the mitotic spindle has been reported to be symmetric and centrally located during metaphase. Thus, apical p-Moesin is likely not involved in generating spindle asymmetry during metaphase. The possibility of its involvement in preparing for the establishment of an asymmetric spindle during anaphase cannot be excluded. Furthermore, the loss of Moesin affected spindle orientation in only a small proportion of NBs, and the localization of the Drosophila LGN orthologue, Pins, was largely unaffected in Moesin knockdown NBs during metaphase. Thus, Moesin does not appear to play a prominent role in regulating spindle orientation in NBs. However, Moesin may affect the localization or activity of interacting partners downstream of Pins such as Mud or the heterotrimeric G protein subunit Gαi. Alternatively, the loss of both Moesin and Pins may cause more severe defects in spindle orientation and cell size asymmetry. Thus, future studies examining the loss of both Moesin and Pins may reveal a role for Moesin in maintaining centrosome positioning and spindle orientation in NBs (Abeysundara, 2017).

This study found that overall NB cell size was reduced in the Moesin knockdown. The reduced size of interphase NBs during early larval stages (48 h ALH) suggests that Moesin may be involved in NB enlargement prior to NB exit from quiescence. NB reactivation also appeared impaired in the ventral nerve cords of Moesin knockdown larvae. Previous studies have implicated Insulin/PI3K signaling in NB growth and reactivation during early larval stages. Further examination of these signaling pathways in the Moesin knockdown NBs are required to determine the mechanisms underlying its potential role in NB enlargement and reactivation. Of the NBs that had exited quiescence, a large proportion of mitotic defective NBs were observed during early and late larval stages. These NBs were not round and may reflect the importance of Moesin in cell rounding during early mitosis, as previously shown in Drosophila cell culture. Alternatively, the mitotic defective NBs may represent a population of NBs that have failed to undergo cell division. As the loss of Moesin also resulted in a reduced proportion of mitotic NBs undergoing each stage of mitosis, it is proposed that Moesin is essential for cell shape changes and mitotic progression during ACD (Abeysundara, 2017).

ERM proteins localize to the apical cortex of a wide variety of polarized cells and are essential for maintaining the apical identity and surface properties of epithelial tissues across multiple organisms. By binding directly to filamentous actin and linking membrane-associated proteins to the underlying actin cytoskeleton, the ERM proteins localize to numerous actin-rich structures. Thus, it is possible that the apical p-Moesin represents areas rich in actin filaments at the NB cortex. Although the actin cytoskeleton is important for cortical tethering of polarity complexes in NBs, filamentous actin does not display an asymmetric distribution. Thus, apical p-Moesin may correlate with enhanced cortical stability at the apical cortex necessary for polarity maintenance and integrity (Abeysundara, 2017).

Confirming a role for p-Moesin in stabilizing cortical actin, it was found that Bazooka and aPKC crescents were not observed in a proportion of Moe^dsRNA NBs undergoing prophase and actin appeared discontinuous at the cell cortex. As Bazooka and aPKC polarity is established by prophase, prior to the polar enrichment of p-Moesin, it is concluded that Moesin is involved in polarity maintenance rather than establishment. Similarly, in the Mus musculus and Caenorhabditis elegans intestinal epithelium, ERM proteins are involved in apical membrane assembly and integrity but do not appear to be required for polarity establishment. During metaphase, a proportion of Moe^dsRNA NBs lacked both Par-6 and aPKC polar crescents. However, the majority of Moe^dsRNA NBs displayed Bazooka and Pins polar crescents at the metaphase cortex. In the absence of Par-6 and aPKC, apical domains consisting of Bazooka, Inscuteable, Pins, and Discs large are still able to form. Thus, Moesin may be specifically maintaining Par-6/aPKC polarity during metaphase but have little effect on other apical polarity proteins such as Bazooka and Pins. Furthermore, the aPKC polar domain was disorganized, and cortical blebbing was observed in the Moe^G0323 mutant NBs. Thus, Moesin regulates the integrity and maintenance of the apical domain, likely through affecting cortical stability during ACD (Abeysundara, 2017).

The complex spatiotemporal regulation of Moesin activity during mitosis has been demonstrated in symmetrically dividing S2 cells and requires the coordinated activities of PP1-87B phosphatase, Slik kinase, and regulators of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) levels at the cell cortex. This study showed that Slik was uniformly distributed at the NB cell cortex. As Slik is regulated by phosphorylation, it is possible that the phosphorylated Slik is asymmetrically distributed in mitotic NBs. Furthermore, Slik was found to be essential for NB proliferation and polarity maintenance, likely through regulating Moesin phosphorylation at the NB cortex. The loss of Flapwing and PP1-87B phosphatases did not alter the apical enrichment of p-Moesin in metaphase NBs. Future studies examining other phosphatases and regulators of PI(4,5)P2 levels at the NB cortex are essential for further understanding Moesin regulation during ACD (Abeysundara, 2017).

In addition to Slik kinase, this study found that known apical polarity proteins (Cdc42, Par-6, aPKC, Lgl, and Pins) are important for the proper apical enrichment of p-Moesin during metaphase. As Moesin is also important for maintenance of the apical domain, these findings support a mutually dependent interaction among the apical polarity proteins that has been extensively reported in NBs. Components of the apical polarity complexes also mediate spindle asymmetry and asymmetric cortical extension during anaphase, leading to the generation of unequal-sized daughter cells. Similarly, this study found that Moesin was important for initial positioning of an asymmetric basal furrow during anaphase (Abeysundara, 2017).

In Drosophila NBs, a cortical polarity-induced pathway, consisting of Pins and the heterotrimeric G-proteins, is essential for apical cortical extension and formation of a Myosin-induced basal furrow, independent of the mitotic spindle. This study found that the relative fluorescent intensity (FI) of p-Moesin was reduced at the apical cortex during anaphase when compared with metaphase NBs. Furthermore, the loss of Moesin resulted in the absence of p-Myosin at the basal cortex, affecting basal furrow positioning during anaphase. In Drosophila S2 cells, reduced p-Moesin at the cell poles was shown to lead to cortical relaxation and membrane elongation. Thus, it is proposed that p-Moesin regulation at the apical cortex is important for asymmetric cortical extension and furrow positioning during early anaphase, likely along with Pins and the heterotrimeric G-proteins. However, Moesin also appeared to influence Myosin-mediated cortical contractility during metaphase as well. This study showed that with the loss of Moesin, p-Myosin and Rok-GFP displayed a nonuniform distribution at the metaphase cortex, revealing unstable actomyosin dynamics and a delay in anaphase onset. Although no observable differences were found in cortical Rho1 localization at the metaphase cortex, future studies using alternative biosensor approaches may allow for more precise visualization and analysis of Rho1 signaling. In addition, further investigation of the mechanical properties of cultured NBs will provide great insight into how Moesin function influences the mitotic cortex in the absence of physical constraint or external cues. While this work was under review, another group showed that Rok and Protein Kinase N are involved in the precise spatiotemporal regulation of Myosin flow during the establishment of physical asymmetry. Given the current findings, it will be interesting to further examine how Moesin precisely regulates Myosin dynamics, along with the other components of the polarity-induced cleavage furrow positioning pathway (Abeysundara, 2017).

Regulation of catalytic and non-catalytic functions of the Drosophila Ste20 kinase Slik by activation segment phosphorylation

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. A structure-function analysis was carried out to determine how these two distinct activities of Slik are controlled. The conserved C-terminal coiled-coil domain (CCD) 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 CCD 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 (Panneton, 2015).

Slik and the receptor tyrosine kinase Breathless mediate localized activation of Moesin in terminal tracheal cells

A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. This study shows that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. The results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway (Ukken, 2014).

The PP1 phosphatase Flapwing regulates the activity of Merlin and Moesin in Drosophila

The signalling activities of Merlin and Moesin, two closely related members of the protein 4.1 Ezrin/Radixin/Moesin family, are regulated by conformational changes. These changes are regulated in turn by phosphorylation. The same sterile 20 kinase-Slik co-regulates Merlin or Moesin activity whereby phosphorylation inactivates Merlin, but activates Moesin. Thus, the corresponding coordinate activation of Merlin and inactivation of Moesin would require coordinated phosphatase activity. Drosophila protein phosphatase type 1 β (Flapwing) fulfils this role, co-regulating dephosphorylation and altered activity of both Merlin and Moesin. Merlin or Moesin are detected in a complex with Flapwing both in-vitro and in-vivo. Directed changes in flapwing expression result in altered phosphorylation of both Merlin and Moesin. These changes in the levels of Merlin and Moesin phosphorylation following reduction of flapwing expression are associated with concomitant defects in epithelial integrity and increase in apoptosis in developing tissues such as wing imaginal discs. Functionally, the defects can be partially recapitulated by overexpression of proteins that mimic constitutively phosphorylated or unphosphorylated Merlin or Moesin. These results suggest that changes in the phosphorylation levels of Merlin and Moesin lead to changes in epithelial organization (Yang, 2012).

The results suggest that Flw would act antagonistically to the kinase Slik during the coordinate regulation of Mer, acting as a tumour suppressor protein, and Moe, required to maintain epithelial integrity. If Flw acts as a coordinate regulatory phosphatase for Mer and/or Moe, it would be expected that Flw is in a protein complex with both Mer and Moe, and this was found to be true. A reproducible increase was found in the ratio of dephosphorylated to phosphorylated Mer isoforms when flw is overexpressed, and a decrease in this ratio was found when flw expression is reduced. In addition, four distinct Mer phosphorylation isoforms were detected. Supporting these observations, the over-expression of flw increases the amount of dephosphorylated Mer signal present as compared to the wild type tissue. Flw also affects the phosphorylation of Moe. The amount of phosphorylated Moesin protein is reduced when flw is over-expressed as compared to when flw expression is reduced. Thus, Flw appears to be a phosphatase specific for both Mer and Moe (Yang, 2012).

Most importantly, using functional assays in whole animals, Flw mediated regulation of Mer and Moe has clear effects on both Mer and Moe protein localization to the plasma membrane and on epithelial organization. There is a higher intensity of staining of both Mer and phosphorylated Moe associated with the plasma membrane upon reduction of flw expression. When the levels of other typical apical domain markers as well as basolateral markers were examined by maximum intensity projection analysis, it was found that maximum projections from larval wing discs show increased brightness of p-ERM, F-actin and anti-Armadillo, within the cells in which flw expression is reduced, whereas the septate junction marker anti-Coracle staining is not changed in intensity over the whole disc. This suggests that as a result of changes in Mer and Moe phosphorylation there are changes in links to the actin cytoskeleton and adherens junctions where both Mer and Moe play roles in wild type cells. Previous studies have demonstrated that phosphorylated Mer is more tightly associated with the plasma membrane. In agreement with data from Drosophila, mammalian cells also show increased plasma membrane association of a phosphomimic form of moesin or the related protein ezrin whereas dephosphorylated ERM proteins are less associated with the plasma membrane. Following flw knockdown in selected cells in the wing epithelium, cells within the boundary between cells with reduced flw expression levels and cells with wild type flw expression levels undergo the greatest amount of change in terms of epithelial integrity. The loss of polarity leads to increased apoptosis in these cells. These effects are observed when flw expression is reduced in only a few cells such as using the ptc Gal4 driver or in the entire dorsal compartment of the wing such as using the apterous Gal4 driver. The cells along the boundary region appear to fold inwards and detach from the rest of epithelium. This is likely the direct result of the difference in adhesion between cells that have reduced flw expression and cells which express wild type levels of Flw protein. As Mer and Moe appear to be direct targets of Flw, and both Mer and Moe have roles in adhesion, the changes in the adhesion of wing epithelium upon reduction of flw are likely a result of changes in Mer and Moe phosphorylation and thus activity. The combination of excess active Moe and excess inactive Mer would affect the balance between maintenance and loss of stabilization of adherens junctions leading to the changes in adhesion and deformation of the wing epithelia that were observed. These adhesion differences could account for the formation of the large folds along the boundary of the ptc expression domain, since cells of similar adhesion are more likely to adhere to themselves (Yang, 2012).

The deformation of the wing imaginal tissue appears to be progressive, since in pre-pupal wing discs (10 h after pupariation) deep holes are observed that extend from the apical surface basally indicating that cells at the apical surface have left the epithelium and are forming balls of cells basally within the disc. In further support of the results, the loss of sds22, a PP1 regulatory subunit, in clonal analysis shows that in large clones in wing discs there is infolding of the mutant tissue with cells being extruded from the epithelium. Cells with loss of function Sds22 also exhibit Moe hyper-phosphorylation. Notably, this is reminiscent of what was observe with reduction of flw expression and overexpression of phosphomimic or nonphsophorylatable Mer or Moe (Yang, 2012).

While a likely cause of some of the changes seen in functional assays are due to changes in Mer and Moe phosphorylation as a result of changes in flw expression, the possibility remains that the level of analysis and resolution of the functional assays in both larval and pupal imaginal wing discs may be insufficient to clearly show subtle differences in the subcellular localization on the membrane of Mer, Moe and apical markers. Thus, it cannot be concluded that the defects associated with flw are due solely to defects in Mer and Moe activity (Yang, 2012).

The ability to partially recapitulate the loss of flw phenotype in ptc expressing cells by the over-expression of either a phosphomimic or nonphsophorylatable Mer or Moe also strongly suggests that this phenotype is, in part, due to the differences in the ratios of active Mer or Moe to inactive Mer or Moe which lead to the corresponding changes in apical epithelial integrity, in third instar discs. This is exemplified by the observation that often with overexpression of either the phosphomimic or nonphosphorylatable Mer or Moe, the formation of a fold is most apparent at the edge of ptc expression at the boundary where the difference in the expression of Mer or Moe within the ptc expressing cells and the neighbouring wild type cells would be greatest. In this way it is not unexpected that the overall effect on the tissue deformation and adhesion is the same with phosphomimic or nonphosphorylatable Mer or Moe, although it is possible that the underlying causes are different due to the predicted opposite activities of the transgenes (Yang, 2012).

Within or directly beside the edge of the ptc expression domain in wing imaginal discs, significantly more cells stain positively for activated Caspase 3. This suggests that cells in these affected domains are undergoing increased levels of apoptosis. These phenotypes are again reminiscent of what is observed in loss of function clones of Sds22, which exhibit an increase in the number of apoptotic cells in the wing discs (Yang, 2012).

It was also demonstrated that Flw binds to the scaffold protein Sip1. It functions with the kinase Slik to regulate Moe activity to maintain epithelial cell integrity. Therefore, the findings suggest that Mer, Moe, Flw, and Sip1 function within a protein complex. This coordinate incorporation within a regulated protein complex is necessary to coordinate cellular response to changing epithelial integrity. This might also explain why the overexpression of flw does not have a strong effect on epithelial integrity. If Mer and Moe need to be part of a complex with Flw and Sip1 in order to regulate epithelial integrity and proliferation, then expression of excess phosphatase outside the complex would have no effect on tissue morphology and growth. In contrast, loss of the phosphatase would have a direct effect since there would be reduced levels of functional protein complex (Yang, 2012).

Future studies are required to determine additional members of this regulatory complex, such as the likely candidates Sds22 and MYPT-75D. The similarity in phenotypes between Sds22 mutant cells and the results of knockdown of flw function would also support a role of Sds22 to interact with Flw in regulating Moe function (Yang, 2012).

This study has shown that the Mer and Moe proteins are direct targets of the catalytic subunit of the PP1 phosphatase Flw. This identifies another important player in the regulation of both Mer and Moe in Drosophila. This is the first identification of a phosphatase coordinately regulating both Mer and Moe activity in vivo. What remains to be determined is how Flw is targeted to regulate Mer and Moe function and what downstream pathways may be affected by these interactions (Yang, 2012).

Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. This paper identifies two novel networks that collectively control the dynamic reorganization of the mitotic cortex. Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. The Pp1-87B/Slik phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division (Roubinet, 2011).

These findings unravel how, by integrating two regulatory networks, Moe activity provides a spatiotemporal framework to control cell shape transformations during division (see Model of the spatiotemporal regulation of Moe activity throughout the successive steps of the cell cycle). The increase in cortical rigidity that drives cell shape remodeling at the interphase/mitosis transition involves a Pp1-87B/Slik molecular switch that timely regulates Moe phosphorylation (Roubinet, 2011).

PI(4,5)P₂ was further identified as a spatial cue that controls Moe distribution at the cortex. This latter aspect coordinates the spatial balance in cortical stiffness/contractility that is required for anaphase cell elongation and cytokinesis. It is proposed that the concerted action of these two regulatory networks ensures the proper series of mitotic cell shape transformations required for the fidelity of cell division (Roubinet, 2011).

A global increase in cortical actomyosin forces generate cell rounding at mitosis entry. These forces are transmitted to the plasma membrane through the activation of ERM proteins. At mitosis exit, both cortical contractions and ERM activity must be down-regulated to allow cells to go back to their interphase shape. In Drosophila cells, the Slik kinase was shown to activate Moe at mitosis entry (Carreno, 2008; Kunda, 2008). This study identifies the Pp1-87B phosphatase as essential for Moe inactivation after cytokinesis and in interphase (Roubinet, 2011).

Although Slik homogenously associates with the cell cortex in both interphase and early mitosis, Pp1-87B is cytoplasmic in interphase and relocalizes to the spindle in pro/metaphase. An attractive model would be that together with a 'constitutive' cortical association of the Slik activator in interphase and pro/metaphase, intracellular redistribution of the Pp1-87B inhibitor represents an efficient way to restrict high levels of Moe phosphorylation to mitosis entry. During anaphase, Pp1-87B concentrates near the chromosomes migrating toward the polar cortex, whereas Slik accumulates at the cleavage furrow. In this model, redistribution of both Pp1-87B and Slik after the anaphase onset contributes to enrich Moe at the equator and to decrease it at poles. Finally, relocalization of Pp1-87B in the cytoplasm after cytokinesis would contribute to relax the cortex for the next interphase by maintaining low Moe activity. A growing number of evidence supports that Pp1 phosphatases play important roles in the temporal control of cell division. Pp1-87B being required for mitotic spindle morphogenesis, this phosphatase could contribute to synchronize cell shape control operated through Moe regulation to chromosome segregation. Although additional investigations will be required to unravel how the activity and distribution of Pp1-87B and Slik are regulated, these results indicate that the Slik/Pp1-87B switch represents an important control of Moe activity during the cell cycle (Roubinet, 2011).

The results show that local levels of PI(4,5)P₂ provide an additional mechanism to regulate Moe function at the cortex of dividing cells. Several studies have established a role of PI(4,5)P₂ in the localization of ERM proteins in polarized processes of differentiated cells. This study provides evidence that during mitosis, PI(4,5)P₂-rich membrane domains act as a spatial cue that regulates both Moe distribution and activation at the cortex (Roubinet, 2011).

The distribution of PI(4,5)P₂ at the plasma membrane is tightly regulated during mitosis. As in mammalian cells, it was found that PI(4,5)P₂ is actively enriched at the equator of anaphase Drosophila S2 cells, suggesting that equatorial accumulation of PI(4,5)P₂ is a feature shared by most animal cells. Although a previous study did not detect PI(4,5)P₂ enrichment at the cleavage furrow of Drosophila spermatocytes, whether this is caused by an intrinsic difference between mitosis and meiosis or by experimental limitations in vivo remains to be established. However, how this dynamic localization is regulated remained unknown. This study shows that the equatorial enrichment of PI(4,5)P₂ relies, at least in part, on the enzymatic activity of Skittles and Pten. During cytokinesis, the equatorial accumulation of PI(4,5)P₂ plays a role in cleavage furrow formation and ingression, through controlling the activity and/or recruitment of several components of the contractile ring. PI(4,5)P₂ hydrolysis is also necessary for maintaining cleavage furrow stability and efficient cytokinesis. The current findings extend the functional repertoire of PI(4,5)P₂ during mitosis to the control of local properties of the mitotic cortex, which are required for polar relaxation and cell elongation. Through functional screenings, novel regulators of cell division were identified among the entire set of enzymes implicated in phosphoinositide biosynthesis. Two main pathways regulate PI(4,5)P₂ levels in mitotic cells, and their alterations provoke similar cortical disorganization. The first pathway involves the Pten tumor suppressor, a PI(3,4,5)P3 3-phosphatase. Pten was shown to accumulate at the septum of dividing yeast cells, as well as at the cleavage furrow in Dictyostelium discoideum. The results of living Drosophila cells show a progressive delocalization of Pten from the polar cortex to the equator after anaphase onset, suggesting that Pten dynamics rely on mechanisms conserved throughout evolution. Furthermore, depletion of Pten leads to a significant enrichment of PI(3,4,5)P3 at the cortex, especially at the cleavage furrow. These results show that Pten uses PI(3,4,5)P3 to spatially control PI(4,5)P₂ levels at the mitotic cortex (Roubinet, 2011).

The second pathway relies on Skittles, a PI(4)P 5-kinase that plays a major role in regulating the levels and localization of PI(4,5)P₂ during mitosis. Skittles switches from an isotropic cortical distribution in pro/metaphase to equatorial enrichment after the anaphase onset. Depletion of Skittles results in a phenotype similar to the mitotic cortical defects observed after inducible PI(4,5)P₂ hydrolysis. It was also found that CG10260, a phosphoinositide 4-kinase, contributes to the organization of the mitotic cortex. Genetics screens have identified a role for phosphoinositide 4-kinases in the division of budding and fission yeast as well as for cytokinesis of male spermatocytes in flies. CG10260 is involved in PI(4)P synthesis, the major substrate of Skittles to produce PI(4,5)P₂. Together, these data show that Skittles acts as a key regulator of PI(4,5)P₂ levels and Moe activation at the mitotic cortex. Interestingly, Skittles is required for Moe activation in Drosophila oocytes, suggesting that this enzyme plays a broad role in the regulation of ERM proteins (Roubinet, 2011).

An important question is how Skittles and Pten are enriched at the equator in anaphase. It has been reported that activated RhoA stimulates a PI(4)P 5-kinase activity and promotes PI(4,5)P₂ synthesis in mammalian cells. During anaphase, activated RhoA localizes at the equatorial cortex, where it could recruit and/or activates Skittles to promote PI(4,5)P₂ production. This anisotropy in PI(4,5)P₂ distribution might be in turn reinforced by the localized activity of Pten, whose membrane association is itself dependent on PI(4,5)P₂. Together, the activity of Skittles and Pten could therefore provide a feed-forward regulatory loop of local PI(4,5)P₂ levels at the cortex of dividing cells (Roubinet, 2011).

The metaphase/anaphase transition is characterized by a break in cortical symmetry, with concomitant relaxation of the polar cortex and contraction of the equator. The anisotropic distribution of Moe participates in coordinating this differential in cortical tension. Overactivation of Moe impairs cell elongation and causes cytokinesis failure, suggesting that the polar cortex is too rigid for cell division. Accumulation of F-actin at the cleavage furrow can be attributed, at least in part, to a cortical flow of F-actin filaments from polar regions to the equator. Overactivation of Moe at the poles could block this actin cortical flow, through an excessive bridging of the actin cytoskeleton with the plasma membrane, leading to an abnormal stiffness of the polar cortex. Therefore, redistribution of activated Moe from the polar cortex to the equator participates in polar relaxation, anaphase cell elongation, and cytokinesis fidelity (Roubinet, 2011).

Contraction of the equatorial actomyosin ring increases the cytoplasmic pressure exerted on the plasma membrane. Relaxation of the polar cortex is thus required to dissipate this extra pressure by increasing the cellular volume, a process that was proposed to involve short-lived polar blebs. These polar blebs were recently found to play important roles during cell division. Perturbation of their dynamics triggers anaphase spindle rocking and destabilization of cleavage furrow positioning. Although recent studies have addressed how cortical blebs are regulated in interphase, understanding of the signalization that controls dynamics of cortical blebs in mitosis has poorly progressed since pioneering studies. The results show that a transient recruitment of Moe at the mitotic bleb membrane is required for efficient polar bleb retraction, as are the functions of the Moe positive regulators Slik, Skittles, and Pten. Active Moe contributes to cortical bleb organization because alteration of Moe function (or regulation) disrupts actin organization and efficient bleb retraction. This leads to disorganization of the mitotic cortex, characterized by giant blebs that continue growing in an unregulated manner. Therefore, although a global decrease in Moe activity at the polar cortex contributes to cell elongation and cytokinesis, transient and local association of Moe at the rim of polar blebs is important for their retraction. If the binding of Moe to PI(4,5)P₂ is required at both the equator and bleb membrane, the influence of the Slik kinase on Moe activation appears different between these two regions of the anaphase cortex. Although Slik depletion abolishes Moe recruitment to polar blebs, remnants of cortical Moe are still visible at the equator, likely as a result of high PI(4,5)P₂ levels at the furrow (Roubinet, 2011).

Although these mechanisms synergistically contribute to the cortical contractility at the equator, they also allow cortical relaxation at the polar cortex through control of transient anaphase blebs. It is proposed that this dual mechanism of Moe regulation is exploited by animal cells to ensure proper cell division (Roubinet, 2011).

Sip1, the Drosophila orthologue of EBP50/NHERF1, functions with the sterile 20 family kinase Slik to regulate Moesin activity

Organization of the plasma membrane in polarized epithelial cells is accomplished by the specific localization of transmembrane or membrane-associated proteins, which are often linked to cytoplasmic protein complexes, including the actin cytoskeleton. This study identified Sip1 as a Drosophila orthologue of the ezrin-radixin-moesin (ERM) binding protein 50 (EBP50; also known as the Na⁺/H⁺ exchanger regulatory factor NHERF1). In mammals, EBP50/NHERF1 is a scaffold protein required for the regulation of several transmembrane receptors and downstream signal transduction activity. In Drosophila, loss of Sip1 leads to a reduction in Slik kinase protein abundance, loss of Moesin phosphorylation and changes in epithelial structure, including mislocalization of E-cadherin and F-actin. Consistent with these findings, Moesin and Sip1 act synergistically in genetic-interaction experiments, and Sip1 protein abundance is dependent on Moesin. Co-immunoprecipitation experiments indicate that Sip1 forms a complex with both Moesin and Slik. Taken together, these data suggest that Sip1 promotes Slik-dependent phosphorylation of Moesin, and suggests a mechanism for the regulation of Moesin activity within the cell to maintain epithelial integrity (Hughes, 2010).

Previous studies of EBP50/NHERF1 suggested a key role for this protein in regulating transmembrane protein localization and retention at the plasma membrane. In this study, advantage was taken of the genetic tools available in Drosophila to identify and characterize some of the in vivo functions of the EBP50/NHERF1 orthologue Sip1. Searches of the Drosophila genome suggest that Sip1 is the single fly orthologue of EBP50/NHERF1, whereas in mammals there are two paralogues, EBP50/NHERF1 and NHERF2. Thus studies in Drosophila should have fewer potential genetic redundancy problems than those in mammalian systems (Hughes, 2010).

Previous studies of EBP50/NHERF1 suggested a key role for this protein in regulating transmembrane protein localization and retention at the plasma membrane. This study has taken advantage of the genetic tools available in Drosophila to identify and characterize some of the in vivo functions of the EBP50/NHERF1 orthologue Sip1. Searches of the Drosophila genome suggest that Sip1 is the single fly orthologue of EBP50/NHERF1, whereas in mammals there are two paralogues, EBP50/NHERF1 and NHERF2. Thus studies in Drosophila should have fewer potential genetic redundancy problems than those in mammalian systems (Hughes, 2010).

Collectively, the data suggest a novel activity for Sip1, namely that it facilitates Slik-dependent phosphorylation and activation of Moesin. Both genetic and biochemical evidence support this conclusion. Clonal analysis indicates that Sip1 is required for normal subcellular localization and abundance of Slik and normal phosphorylation of Moesin without affecting overall Moesin abundance. In addition, Sip1 appears to be upregulated in slik mutant cells, suggesting that Slik and Sip1 have an interdependent relationship. Co-immunoprecipitation experiments in pupae and in cultured S2 cells indicated that Sip1 forms complexes with both Slik and Moesin, although at the moment it is unclear whether all three proteins are present in a single complex. Taken together, these results suggest that Sip1 functions to bring Slik and Moesin in close proximity, possibly by acting as a scaffold that links these proteins together and leading to phosphorylation of Moesin. Although yeast two-hybrid data indicate that Moesin and Sip1 interact directly, this study has not distinguished between the possibilities of direct versus indirect interactions with Slik. It is interesting to note that EBP50/NHERF1 has been shown to form a complex with Ste20-related kinase SLK, the mammalian orthologue of Slik, via the adapter protein PDZK1 (Hughes, 2010).

One potential problem with this model is that previous work has suggested that EBP50/NHERF1 is unable to bind the folded, inactive form of ezrin, raising the question of how Sip1 can facilitate activation of Moesin if it cannot bind the inactive, folded form. A possible answer to this question comes from recent observations that ERMs are regulated by several factors that might operate in a step-wise fashion. Specifically, previous studies have suggested either a two-step mechanism involving binding of PIP2 to the FERM domain, followed by phosphorylation of the conserved C-terminal Thr, or a 'rheostat'-like mechanism that allows for several intermediate states of unfolding and activation. Proposed components of the rheostat mechanism include binding of the FERM domain to PIP2 and phosphorylation, not only of the C-terminal Thr, but of other residues as well. Thus, it is proposed that interaction of a Sip1-Slik complex with Moesin probably requires previous partial unfolding of Moesin through one or more of these mechanisms (Hughes, 2010).

Although a role for EBP50/NHERF1 in promoting ERM phosphorylation has not been proposed previously, several aspects of the current findings and conclusions are consistent with previous studies in mammalian systems. Sip1 binds Moesin in both two-hybrid and co-immunoprecipitation analyses, as has been observed for EBP50/NHERF1 in mammalian cells. Consistent with this result, loss of Moesin and/or ezrin results in mislocalization of Sip1/EBP50 in both the fly and in the mouse. In Ebp50 mutant mice, there is a decrease in phosphorylated ERM proteins in the kidney and intestinal epithelia. EBP50 is suggested to be necessary to stabilize active phosphorylated ERM proteins at the apical membrane, but this observation would also be consistent with the model whereby EBP50/Sip1 promotes ERM phosphorylation. Assuming that this is true, what remains unclear is the identity of the responsible kinase in mammalian cells. Although Rho kinase has been proposed to phosphorylate this residue, subsequent studies have raised questions about this proposal, and no alternative kinase has been identified. The overall conservation of ERMs and Sip1/EBP50/NHERF1 between flies and humans strongly suggests that there is an as yet unidentified sterile 20 kinase family member involved in ERM phosphorylation in mammalian cells (Hughes, 2010).

The requirement of Sip1 for promotion of Moesin activity is also supported by the observation that reduction of the Rho1 gene dosage partially suppresses the lethality of the Sip1 mutant animal. Previously, it has been shown that Moesin regulates epithelial integrity via downregulation of Rho1 signaling. Similarly, Slik hypomorphic defects are rescued by a reduced Rho1 gene dosage, suggesting that Slik regulates Rho1 signaling via phosphorylation and activation of Moesin. Taken together, these results suggest that Sip1, Slik and Moesin function synergistically to negatively regulate Rho1 pathway activity, and emphasize the importance of the functional interaction between Sip1 and Moesin for proper epithelial morphogenesis (Hughes, 2010).

Support for this model comes from the genetic interaction that were observed between Sip1 and Moe. Co-expression of an activated Moe transgene strongly enhances the effects of Sip1 expression, whereas expression of the inactive, non-phosphorylatable MoeT559A allele suppresses Sip1 hypermorphic phenotypes. These results are consistent with Sip1 and Moe acting synergistically. Furthermore, the observation that the non-phosphorylatable T559A allele suppresses Sip1 expression phenotypes could suggest that co-expression of this form inactivates Sip1, possibly through direct interaction, although it was not possible to demonstrate increased complex formation in co-immunoprecipitation experiments. In addition, the strong synergistic genetic interactions between Sip1 and the phosphomimetic form of MoeT559D suggests that Sip1 either interacts specifically with phosphorylated Moesin, or promotes Moesin phosphorylation (Hughes, 2010).

It is interesting to note that either reduced or increased levels of Sip1 expression have dramatic effects on epithelial integrity. Loss of function Sip1 clones in follicle cell epithelia result in reorganization of the actin cytoskeleton and loss of E-cadherin localization, strongly suggesting that adherens junction integrity is affected. In addition, ectopic expression of Sip1 in the adult wing leads to the formation of vesicles, which is indicative of defects in cell adhesion. In mammalian cells it has also been shown that reduced ezrin levels result in the loss of cell adhesion complexes. This suggests that the production of Sip1 protein must be tightly regulated within epithelial cells to regulate epithelial integrity, probably through regulation of the interaction between Slik and Moesin (Hughes, 2010).

Strong effects of loss of Sip1 function were consistently observed within the follicle cell epithelia, but not in the columnar epithelial cells of third imaginal wing discs, indicating a differential cell requirement for Sip1 activity. This might be linked to changes in cell morphology, because follicle epithelial cells are initially cuboidal, but undergo dramatic changes in cell shape beginning at stage 9. At this point, the follicle cells located over the growing oocyte migrate posteriorly and become columnar, whereas the follicle cells over the nurse cells become squamous. The majority of the defects that were observed in the follicle cells, whether located over the oocyte or nurse cells, occur at or following, stage 9. These changes in cell type would require alteration in both cell adhesion and the organization of the actin cytoskeleton, which contribute to the force required for the migration. Thus, these cells might be particularly susceptible to perturbations that affect regulation of cytoskeletal dynamics or linkage between the plasma membrane and the cytoskeleton. Another, not mutually exclusive, factor is that follicle cells are no longer mitotically active at the stages observed, whereas the wing imaginal discs undergo mitosis throughout their development. Perhaps there are independent mechanisms for regulating the interactions with Slik and Moesin in actively dividing cells. A third possibility is that an unknown protein acts redundantly to Sip1 to regulate the activity of Slik and Moesin in the third instar imaginal disc tissue (Hughes, 2010).

A crucial question that remains to be answered regarding Sip1, Moesin and Slik is how their activities are co-regulated in developing epithelia. Previous work clearly suggests that regulation of Slik kinase activity is important in developing epithelial cells. The results presented in this study indicate that the effect of Slik on Moesin activity, and therefore on epithelial integrity, could be regulated by Sip1. Furthermore, because previous studies have shown that Slik also regulates the tumour suppressor Merlin, these results raise the possibility that Sip1 could control proliferation in epithelial tissues. Since it is now possible to study the EBP50/NHERF family in Drosophila, it should be possible to identify other gene pathways that are involved in the activity and localization of Sip1 (Hughes, 2010).

Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells

Cell division requires cell shape changes involving the localized reorganization of cortical actin, which must be tightly linked with chromosome segregation operated by the mitotic spindle. How this multistep process is coordinated remains poorly understood. This study shows that the actin/membrane linker moesin, the single ERM (ezrin, radixin, and moesin) protein in Drosophila melanogaster, is required to maintain cortical stability during mitosis. Mitosis onset is characterized by a burst of moesin activation mediated by a Slik kinase-dependent phosphorylation. Activated moesin homogenously localizes at the cortex, a specialized layer of cytoplasmic proteins on the inner face of the cell membrane, in prometaphase and is progressively restricted at the equator in later stages. Lack of moesin or inhibition of its activation destabilized the cortex throughout mitosis, resulting in severe cortical deformations and abnormal distribution of actomyosin regulators. Inhibiting moesin activation also impaired microtubule organization and precluded stable positioning of the mitotic spindle. It is proposed that the spatiotemporal control of moesin activation at the mitotic cortex provides localized cues to coordinate cortical contractility and microtubule interactions during cell division (Carreno, 2008).

Phosphorylation and activity of the tumor suppressor Merlin and the ERM protein Moesin are coordinately regulated by the Slik kinase

Merlin and Moesin are closely related members of the 4.1 Ezrin/Radixin/Moesin domain superfamily implicated in regulating proliferation and epithelial integrity, respectively. The activity of both proteins is regulated by head to tail folding that is controlled, in part, by phosphorylation. Few upstream regulators of these phosphorylation events are known. This study demonstrates that in Drosophila melanogaster, Slik, a Ste20 kinase, controls subcellular localization and phosphorylation of Merlin, resulting in the coordinate but opposite regulation of Merlin and Moesin. These results suggest the existence of a novel mechanism for coordinate regulation of cell proliferation and epithelial integrity in developing tissues (Hughes, 2006).

A previous study has shown that the Slik kinase positively regulates Moesin activity via phosphorylation near the C terminus, thereby inhibiting activation of the Rho small GTPase and promoting epithelial integrity (Hipfner, 2004). Overexpression of Slik in imaginal tissues results in the hyperphosphorylation of Merlin, suggesting that in addition to Moesin, Slik regulates the phosphorylation state of Merlin. Interestingly, in mammalian cells, Merlin phosphorylation is affected by PAK, which, like Slik, is a member of the Ste20 family of kinases. Current models of Merlin function predict that hyperphosphorylated Merlin is inactive, which is consistent with the observation that slik functions antagonistically to Merlin in genetic interaction tests. In accord with this notion, slik was originally identified in a misexpression screen by its ability to cause overproliferation when expressed ectopically in imaginal epithelia (Hipfner, 2003). Collectively, the data presented in this study leads to a prediction that activity of the Slik kinase coordinately regulates both epithelial morphology and, at the same time, cell proliferation. This is the first demonstration of a single mechanism with the potential to regulate both processes simultaneously in developing tissues (Hughes, 2006).

It is speculated that the observed coordinate regulation of Merlin and Moesin may be important in the developing imaginal discs during larval and pupal development. In larval stages, most imaginal epithelia proliferate rapidly and at the same time maintain a highly structured epithelial monolayer. At this stage, Slik activity could allow high rates of proliferation and simultaneously promote epithelial integrity that is necessary to prevent the unregulated growth or invasive cell behavior. At the end of larval life and at the onset of metamorphosis, the cell cycle slows dramatically, and, at the same time, the imaginal discs radically change shape during a morphogenetic process termed eversion. Previous studies have shown that these shape changes require rearrangements of local contacts between cells, suggesting that epithelial integrity must be modulated. It is predicted that at this stage, Slik function may be decreased to coordinate these changes in the imaginal epithelium. Further studies to examine Slik expression and the regulation of its function will be of interest in this regard (Hughes, 2006).

This study also provides the first genetic evidence that Moesin and Merlin functionally interact through competition for Slik kinase activity, although previous studies have shown physical interactions between these proteins. It is interesting to note that in mammalian Schwann RT4 cell lines, expressing constitutively phosphorylated Merlin not only impairs the ability of Merlin to suppress proliferation and motility but also induces a novel ERM-like phenotype. This phenotype was attributed to the conversion of Merlin to an ERM-like molecule. However, if Merlin and Moesin are also coordinately regulated in mammalian cells, an alternative possibility is that overexpression of a phosphomimetic Merlin could affect the phosphorylation state of endogenous ERM proteins, thereby increasing their level of activity (Hughes, 2006).

This study found that the loss of slik function results in a dramatic shift in Merlin localization from the apical plasma membrane to punctate cytoplasmic structures. It was previously shown that Merlin traffics from the plasma membrane with endocytic vesicles in cultured cells, raising the possibility that in the absence of Slik, activated Merlin is more stably associated with endocytic compartments than in normal cells. If this is so, inactive Merlin may reside at the plasma membrane and, in response to activation, traffics internally, presumably in association with transmembrane proteins. If this model is correct, it suggests that Merlin may function in tumor suppression by facilitating removal from the plasma membrane of receptors that promote cell proliferation. This model fits well with the recent observation that several receptors, including Notch and the EGF receptor, accumulate to abnormal levels on the surface of cells that are mutant for Merlin and the functionally redundant related tumor suppressor expanded (Hughes, 2006).

Several important questions remain regarding the regulation of Moesin and Merlin are described in this study. It remains unclear whether Slik itself can directly phosphorylate either protein or whether there are one or more kinases operating downstream of Slik. Additionally, the dual functions described in this study may provide novel insights into the role of the mammalian orthologues of Slik, such as PAK, in the malignant transformation of epithelial cells. Equally important will be to elucidate how Slik activity is itself controlled. Given its ability to simultaneously regulate epithelial integrity and proliferation in developing epithelial tissues, Slik may function as a central integrator of the multitude of signals that converge to regulate growth and morphology during development (Hughes, 2006)

Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth

The Drosophila Sterile-20 kinase Slik (FlyBase name: Polo kinase kinase 1) promotes tissue growth during development by stimulating cell proliferation and by preventing apoptosis. Proliferation within an epithelial sheet requires dynamic control of cellular architecture. Epithelial integrity fails in slik mutant imaginal discs. Cells leave the epithelium and undergo apoptosis. The abnormal behavior of slik mutant cells is due to failure to phosphorylate and activate Moesin, which leads to excess Rho1 activity. This is distinct from Slik's effects on cell proliferation; these effects are mediated by Raf. Thus Slik acts via distinct pathways to coordinate cell proliferation with epithelial cell behavior during tissue growth (Hipfner, 2004).

The wing imaginal disc is an epithelial sac composed of two distinct but continuous epithelial layers enclosing a central lumen. The portion of the disc that will form the wing is a pseudostratified columnar epithelium. In optical cross-section, the dense packing of the cells is visible, with nuclei appearing stacked in layers. Overlying the columnar epithelium is the squamous peripodial epithelium. The apical surface of both epithelial layers is oriented toward the lumen of the disc, as seen by the concentration of F-actin near the adherens junctions. In wing discs lacking Slik activity, the columnar epithelium is abnormally thin. Many cells lost their capacity to remain integrated in the epithelium and are extruded basally to form a disorganized mass. Many of these cells undergo apoptosis as evidenced by pyknotic nuclei and by TUNEL labeling, though clusters of cells survived. A similar but milder defect was seen in discs with reduced Slik activity. Many apoptotic cells with activated Caspase are seen in optical sections below the epithelial layer. Some of the extruded slik mutant cells are alive and appeared mesenchymal, having lost their polarized epithelial character. These cells produce F-actin-rich filopodia and appear to acquire motile behavior. Thus slik activity helps cells to maintain epithelial integrity (Hipfner, 2004).

The observation that many live slik mutant cells are extruded from the epithelium suggests that loss of epithelial integrity is not a consequence of apoptosis. This was confirmed by producing clones of slik mutant cells that expressed the baculovirus caspase inhibitor p35. p35-expressing slik mutant cells also lose epithelial integrity and are extruded from the epithelium, but remain alive. This suggests that apoptosis is a consequence of the loss of epithelial organization, perhaps due to loss of survival signaling by Raf (Hipfner 2004; Hipfner, 2004).

To investigate whether slik is essential for epithelial polarity per se, the subcellular localization of junctional complex proteins was compared in normal cells and clones of slik¹ null mutant cells. In wild-type cells, Slik protein is concentrated apically and colocalizes with cortical actin, in addition to a diffuse cytoplasmic staining. Slik is concentrated apical to septate junctions (marked by Discs large) and adherens junctions (marked by E-Cadherin and anti-phosphotyrosine) and to the apical-most marginal zone complexes containing the PDZ-domain protein Patj, indicating that Slik is at or near the apical membrane rather than in the junctions. Junctional complexes appear normal in slik mutant cells that remain in the disc epithelium, but are lacking in mutant cells that have left the epithelium. Thus, although it is not essential for apical-basal polarity, slik contributes to maintaining epithelial organization. Cells lacking slik often lose epithelial polarity and leave the epithelium. Many of these cells undergo apoptosis (Hipfner, 2004).

slik¹ mutant clones generated in the eye disc early in development and provided with a growth advantage can grow to large sizes. As in the wing disc, many cells are extruded from the eye disc and undergo apoptosis, whereas others remain integrated in the epithelium and show normal expression of the neuronal marker ELAV in photoreceptors. Adult eyes with large slik¹ mutant clones show moderate external roughness. Although many properly organized ommatidial clusters in these eyes are found, mutant photoreceptors show defects during subsequent differentiation. Each photoreceptor normally projects a stack of actin-based microvilli, or rhabdomere, from the apical membrane domain that provides the increased membrane surface for harvesting light. Rhabdomeres form during pupal development (PD). By 70% PD, the eight photoreceptors that comprise a single facet in the wild-type eye have begun to form rhabdomeres consisting of short bundles of microvilli. The cells are connected to each other by adherens junctions, which separate the apical from the basolateral membrane domains. At 95% PD the microvilli have extended to form regular bundles. The arrangement of photoreceptors is largely normal in complete ommatidia containing slik mutant clones, but many slik mutant photoreceptors had patches of apical membrane devoid of microvilli. Where present, the microvilli in slik mutant photoreceptors do not form organized stacks. Mutant photoreceptors are present in eyes from 18-day-old adult flies, indicating that their survival as postmitotic cells is not impaired. These defects resemble those described recently (Karagiosis, 2004) in photoreceptors lacking Moesin (Hipfner, 2004).

The similarity in slik and moesin mutant phenotypes prompted a comparison of the defects in imaginal discs in more detail. Loss of Moesin causes defects in epithelial integrity due to Rho1-induced changes in the actin cytoskeleton (Speck, 2003). Interestingly, cells extruded from moesin mutant discs also undergo massive apoptosis. The organizational changes in the actin cytoskeleton are strikingly similar in the two genotypes. These defects in moesin mutant discs can be rescued by reduction of rho1 gene dosage (Speck, 2003). Similarly, the defects in epithelial integrity and cell survival in hypomorphic slik mutants are strongly suppressed when rho1 gene dosage is reduced, suggesting that some defects in slik mutants are due to excessive Rho1 activity and that Slik acts in the same pathway as Moesin (Hipfner, 2004).

Moesin is activated by phosphorylation on a conserved Threonine residue in the C-terminal domain. Thr 556-phosphorylated Moesin (P-Moesin) colocalizes with apical Slik protein in wild-type cells, in addition to a diffuse distribution of both proteins. P-Moesin levels are considerably reduced in slik¹ clones, compared to adjacent Slik-expressing cells in the wing disc. Immunoblot analysis showed that Thr 556-phosphorylation is reduced in slik mutant wing discs; the level of total Moesin is unchanged. Likewise, Slik RNAi in S2 cells results in a dose-dependent decrease in endogenous Slik protein and in Moesin phosphorylation. Expression of the kinase domain of Slik is sufficient to induce PMoesin in the wing disc, whereas a catalytically inactive version of this domain has no effect. Activated Moesin has been shown to stabilize F-actin (Speck, 2003), and increased F-actin staining is observed in Slik kinase domain-expressing cells. These data indicate that Slik activity is required for Moesin Thr 556-phosphorylation in vivo. However, it was not possible to direct phosphorylation of Moesin by Slik in vitro, suggesting that Slik may act indirectly to promote Moesin phosphorylation in vivo (Hipfner, 2004).

Speck (2003) showed that activated Moesin limits Rho activity in vivo. Experiments in cultured mammalian cells have suggested that Rho might also act upstream of Moesin to promote its phosphorylation, forming a feedback loop. To assess the involvement of Rho in Moesin phosphorylation, the effects of removing Slik, Rho1, and the Rho effector kinase Rok from S2 cells were compared by RNAi. Depletion of endogenous Slik strongly reduces Moesin phosphorylation. Most cells depleted of Slik show low levels of Moesin phosphorylation. Expression of the Slik kinase domain in the Slik-depleted cells restores Moesin phosphorylation to an extent that is consistent with the transfection efficiency in the experiment. Depletion of Rho1 causes a modest reduction in Moesin phosphorylation, and expression of the Slik kinase domain increases the level of Moesin phosphorylation in Rho1-depleted cells. Many cells depleted of Rho1 show levels of Moesin phosphorylation similar to those of control S2 cells, and cells expressing the Slik kinase domain are always among those with the higher level of Moesin phosphorylation. This indicates that Rho1 is not required for Slik-induced phosphorylation of Moesin in S2 cells. The mammalian kinase Rock has been suggested to serve as an effector of Rho in mediating ERM protein phosphorylation, though this is controversial. In this study, depletion of the Drosophila ortholog, Rok, from S2 cells had little or no effect on the level of Moesin phosphorylation, and did not prevent Slik kinase domain-induced Moesin phosphorylation. These observations suggest that Rho1 is not required for Moesin phosphorylation in S2 cells. Although the level of Rho activity is elevated in slik mutants, the level of Moesin phosphorylation is very low, suggesting that Rho activity cannot compensate for the lack of Slik activity (Hipfner, 2004).

Next it was asked whether the epithelial defects in slik mutants could be rescued by expression of a constitutively active, phosphomimetic form of Moesin (Moesin^TD; Speck, 2003). moe^GO323 mutants served as a control. Moesin protein was nearly undetectable in moe^GO323 discs by immunoblot. In moe^GO323 discs, arm^GAL4 drove ubiquitous Moesin^TD expression to a level lower than endogenous Moesin. This level of activated Moesin had no effect on the development of wild-type animals. moe^GO323 mutants grew more slowly than wild-type larvae, reached pupal stages later, and died as pupae. More than half of the mutant larvae were rescued to pharate adult (25%) or adult stages (28%) by expression of Moesin^TD (vs. 0% and 1% for moe^GO323). Moesin^TD expression also partially rescued slik¹ mutants. Nearly half of the slik¹ animals survived to pupation, but all died shortly thereafter. In contrast, 9% of slik¹ mutants expressing Moesin^TD reached the pharate adult stage. One small rescued slik¹ mutant fly survived to adulthood. Discs from rescued slik¹ mutants showed some improvement in overall structure and actin organization. Basal extrusion of cells was reduced and in some cases nearly eliminated (Hipfner, 2004).

Moesin^TD expression did not rescue slik¹ animals to the same extent as moe^GO323 mutants. Despite improvements in epithelial integrity and the stage of lethality, Moesin^TD had little or no effect on the slow rate of development and growth of slik¹ mutants. One explanation for this may be that Moesin acts downstream of Slik to regulate epithelial integrity but not to promote growth. Thus the requirement for Moesin in Slik-driven tissue growth was examined. The effect of Slik on cell proliferation is easily observed in the peripodial cells overlying the columnar wing disc epithelium. Expression of Slik in the columnar epithelium induces nonautonomous proliferation of peripodial cells, detectable as an increase in nuclear density and BrdU incorporation. The same effect was observed when Slik was expressed in moe^GO323 mutants. Activated Moesin^TD does not affect cell proliferation. Thus Moesin is not necessary for Slik-induced cell proliferation, nor is Moesin activity sufficient to stimulate cell proliferation (Hipfner, 2004).

It is concluded that Slik regulates epithelial integrity via Moesin activation, independent of its effects on tissue growth. Slik-induced tissue growth is Raf-dependent. Expression of a kinase inactive form of Slik is capable of promoting Raf-dependent overproliferation, suggesting that the growth effect may be mediated by protein-protein interactions rather than by Slik kinase activity (Hipfner, 2003). Consistent with this notion, expression of the kinase domain of Slik alone does not induce cell proliferation or tissue overgrowth. In contrast, Moesin phosphorylation is dependent on Slik kinase activity. Taken together, these observations suggest two distinct effector pathways for Slik -- a pathway controlling growth, involving Raf, and a separate pathway controlling epithelial integrity involving Moesin phosphorylation (Hipfner, 2004).

What might be the purpose of the dual activities of Slik? Slik is not essential for cells to grow and divide, but it does control the rate of cell proliferation. Slik activity must be maintained within a defined range; too much or too little activity results in apoptosis (Hipfner, 2003). Mitogenic signaling through Slik can promote tissue growth and at the same time reinforce cellular architecture by maintaining Moesin in an active state. Proliferation-induced apoptosis prevents excessive Slik signaling from deregulating proliferation, as has been suggested for certain oncogenes. Under conditions of reduced mitogenic signaling, decreased Slik activity would result in a lower rate of cell proliferation and, by reducing Moesin activity, make cells more likely to be extruded from the disc and thus to undergo apoptosis. A more direct function of ERM proteins in regulating apoptosis may also be involved (Gautreau, 1999; Parlato, 2000). Thus, Slik activity may serve as a switch between pro-proliferative and pro-apoptotic states. Consistent with this idea, loss of ERM protein phosphorylation and activity has been shown to be an early event in apoptosis (Kondo, 1997). Interestingly, it is known that slowly dividing cells in Drosophila imaginal discs undergo apoptosis as a result of reduced ability to compete for survival factors. The dual activity of Slik may help to ensure elimination of less fit cells (Hipfner, 2004).

The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development

Cell proliferation and programmed cell death are closely controlled during animal development. Proliferative stimuli generally also induce apoptosis, and anti-apoptotic factors are required to allow net cell proliferation. Genetic studies in Drosophila have led to identification of a number of genes that control both processes, providing new insights into the mechanisms that coordinate cell growth, proliferation, and death during development and that fail to do so in diseases of cell proliferation. This study presents evidence that the Drosophila Sterile-20 kinase Slik promotes cell proliferation and controls cell survival. At normal levels, Slik provides survival cues that prevent apoptosis. Cells deprived of Slik activity can grow, divide, and differentiate, but have an intrinsic survival defect and undergo apoptosis even under conditions in which they are not competing with normal cells for survival cues. Like some oncogenes, excess Slik activity stimulates cell proliferation, but this is compensated for by increased cell death. Tumor-like tissue overgrowth results when apoptosis is prevented. Evidence is presented that Slik acts via Raf, but not via the canonical ERK pathway. Activation of Raf can compensate for the lack of Slik and support cell survival, but activation of ERK cannot. It is suggested that Slik mediates growth and survival cues to promote cell proliferation and control cell survival during Drosophila development (Hipfner, 2003).

Search PubMed for articles about Drosophila Slik

Abeysundara, N., Simmonds, A. J. and Hughes, S. C. (2017). Moesin is involved in polarity maintenance and cortical remodelling during asymmetric cell division. Mol Biol Cell 29(4):419-434. PubMed ID: 29282284

Carreno, S., Kouranti, I., Glusman, E. S., Fuller, M. T., Echard, A. and Payre, F. (2008). Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells. J. Cell Biol. 180: 739-746. PubMed Citation: 18283112

De Jamblinne, C. V., Decelle, B., Dehghani, M., Joseph, M., Sriskandarajah, N., Leguay, K., Rambaud, B., Lemieux, S., Roux, P. P., Hipfner, D. R. and Carreno, S. (2020). STRIPAK regulates Slik localization to control mitotic morphogenesis and epithelial integrity. J Cell Biol 219(11). PubMed ID: 32960945

Hipfner, D. R. and Cohen, S. M. (2003). The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development. PLoS Biol 1(2): E35. PubMed ID: 14624240

Hipfner, D. R., Keller, N. and Cohen, S. M. (2004). Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth. Genes Dev 18: 2243-2248. PubMed ID: 15371338

Hughes, S. C. and Fehon, R. G. (2006). Phosphorylation and activity of the tumor suppressor Merlin and the ERM protein Moesin are coordinately regulated by the Slik kinase. J Cell Biol 175(2): 305-313. PubMed ID: 17060498 .

Hughes, S. C., Formstecher, E. and Fehon, R. G. (2010). Sip1, the Drosophila orthologue of EBP50/NHERF1, functions with the sterile 20 family kinase Slik to regulate Moesin activity. J Cell Sci 123(Pt 7): 1099-1107. PubMed ID: 20215404

Karagiosis, S. A. and Ready, D. F. (2004). Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis. Development 131(4): 725-732. PubMed ID: 14724125

Katzemich, A., Long, J. Y., Panneton, V., Fisher, L., Hipfner, D. and Schock, F. (2019). Slik phosphorylation of talin T152 is crucial for proper talin recruitment and maintenance of muscle attachment in Drosophila. Development. PubMed ID: 31511253

Kschonsak, Y. T. and Hoffmann, I. (2018). Activated ezrin controls MISP levels to ensure correct NuMA polarization and spindle orientation. J Cell Sci 131(10). PubMed ID: 29669740

Kuck, U., Radchenko, D. and Teichert, I. (2019). STRIPAK, a highly conserved signaling complex, controls multiple eukaryotic cellular and developmental processes and is linked with human diseases. Biol Chem. PubMed ID: 31042639

Panneton, V., Nath, A., Sader, F., Delaunay, N., Pelletier, A., Maier, D., Oh, K. and Hipfner, D. R. (2015). Regulation of catalytic and non-catalytic functions of the Drosophila Ste20 kinase Slik by activation segment phosphorylation. J Biol Chem 290(34):20960-20971. PubMed ID: 26170449

Pelaseyed, T., Viswanatha, R., Sauvanet, C., Filter, J. J., Goldberg, M. L. and Bretscher, A. (2017). Ezrin activation by LOK phosphorylation involves a PIP2-dependent wedge mechanism. Elife 6. PubMed ID: 28430576

Pelaseyed, T. and Bretscher, A. (2018). Regulation of actin-based apical structures on epithelial cells. J Cell Sci 131(20). PubMed ID: 30333133

Pryce, B. R., Al-Zahrani, K. N., Dufresne, S., Belkina, N., Labreche, C., Patino-Lopez, G., Frenette, J., Shaw, S. and Sabourin, L. A. (2017). Deletion of the Ste20-like kinase SLK in skeletal muscle results in a progressive myopathy and muscle weakness. Skelet Muscle 7(1): 3. PubMed ID: 28153048

Quizi, J. L., Baron, K., Al-Zahrani, K. N., O'Reilly, P., Sriram, R. K., Conway, J., Laurin, A. A. and Sabourin, L. A. (2013). SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration. Oncogene 32(39): 4656-4663. PubMed ID: 23128389

Roubinet, C., et al. (2011). Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis. J. Cell Biol. 195(1): 99-112. PubMed Citation: 21969469

Solinet, S., Mahmud, K., Stewman, S. F., Ben El Kadhi, K., Decelle, B., Talje, L., Ma, A., Kwok, B. H. and Carreno, S. (2013). The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex. J Cell Biol 202(2): 251-260. PubMed ID: 23857773

Speck, O., Hughes, S. C., Noren, N. K., Kulikauskas, R. M. and Fehon, R. G. (2003). Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature 421(6918): 83-87. PubMed ID: 12511959

Ukken, F. P., Aprill, I., JayaNandanan, N. and Leptin, M. (2014). Slik and the receptor tyrosine kinase Breathless mediate localized activation of Moesin in terminal tracheal cells. PLoS One 9(7): e103323. PubMed ID: 25061859

Yang, Y., et al. (2012). The PP1 phosphatase flapwing regulates the activity of Merlin and Moesin in Drosophila. Dev. Biol. 361(2): 412-26. PubMed ID: 22133918

date revised: 7 March 2021

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