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Address correspondence to Sonsoles Campuzano, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas and Univerisidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Tel.: 34-91-497-5072. Fax: 34-91-497-4799. email: scampuzano{at}cbm.uam.es
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Abstract |
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Key Words: DaPKC; Crumbs; Drosophila; epithelial cell polarity; signal transduction
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Introduction |
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The mechanisms that underlie the establishment and maintenance of epithelial polarity have been extensively studied in Drosophila. Thus, three membrane-associated multiprotein complexes, the Bazooka (Baz), Crumbs, and Discs large (Dlg) complexes, are required to organize apicobasal polarity in the epithelial cells of the Drosophila embryo (for reviews see Müller, 2000; Ohno, 2001; Tepass et al., 2001; Knust and Bossinger, 2002; Henrique and Schweisguth, 2003; Roh and Margolis, 2003). The Baz complex is formed by the PSD-95/Dlg/ZO-1 domain containing proteins Baz and DPar-6, and the Drosophila atypical PKC (DaPKC), a Ser/Thr kinase (Kuchinke et al., 1998; Wodarz et al., 2000; Petronczki and Knoblich, 2001). This complex, which is localized at the marginal zone of the apico-lateral cell membrane, above the ZA, is formed stepwise during early embryogenesis. Thus, at blastoderm stage, Baz and DaPKC are found at the apical membrane domain (Wodarz et al., 2000; Bilder et al., 2003). Slightly later, at the onset of gastrulation, DPar-6 is incorporated to the BazDaPKC complex, where it binds directly to both Baz and DaPKC (Petronczki and Knoblich, 2001). Embryos deficient in Baz or DPar-6 cannot properly assemble the ZA and lack several apical markers of the cell membrane (Müller and Wieschaus, 1996; Petronczki and Knoblich, 2001). Similarly, epithelial cells of DaPKC mutant imaginal discs display disrupted apicobasal polarity (Rolls et al., 2003). Thus, each of the components of the Baz complex is essential to establish epithelial polarity.
The Crumbs complex, which is assembled at gastrulation, is also localized at the marginal zone. It consists of the transmembrane (TM) protein Crumbs (Crb) and the cytoplasmic PDZ-containing proteins Stardust (Sdt) and Pals1-associated TJ protein (Patj; formerly known as Discs lost; Tepass et al., 1990; Bhat et al., 1999; Bachmann et al., 2001; Hong et al., 2001; Pielage et al., 2003). Crb contains 30 epidermal growth factorlike and four laminin A G-domainlike repeats in its extracellular region and a short intracellular domain (Tepass et al., 1990). Expression of this intracellular domain partially normalizes mutant crb embryos (Wodarz et al., 1995; Klebes and Knust, 2000). This suggests that Crb exerts its function, at least in part, by proteinprotein interactions mediated by this domain. The intracellular domain has been subdivided into proximal juxtamembrane and COOH-terminal subdomains. The latter contains the binding site for Sdt (Bachmann et al., 2001; Hong et al., 2001; Roh et al., 2002), which on its turn binds to Patj (Roh et al., 2002). The juxtamembrane domain is required for Crb to form a complex with DMoesin and ß-heavy spectrin and thus mediates the interaction of the Crb complex with the apical spectrin cytoskeleton (Medina et al., 2002). Genetic studies have shown that the Crb complex is, similarly to the Baz complex, indispensable for the establishment of epithelial apicobasal polarity and stabilization of the ZA (Knust et al., 1993; Grawe et al., 1996; Müller and Wieschaus, 1996; Tepass, 1996; Bachmann et al., 2001; Hong et al., 2001).
The third complex, formed by the PDZ proteins Dlg (Woods and Bryant, 1991) and Scribble (Scrib; Bilder and Perrimon, 2000), and the Myosin type II binding protein Lethal giant larvae (Lgl; Mechler et al., 1985), is found at the basolateral domain of the cell membrane, basal to the ZA. These proteins are required to restrict the Baz and Crb complexes to the apical cell membrane and for correct positioning of the ZA (Bilder et al., 2000, 2003; Bilder and Perrimon, 2000; Tanentzapf and Tepass, 2003).
The three polarity complexes are evolutionarily conserved (for reviews see Ohno, 2001; Roh and Margolis, 2003; Macara, 2004). Thus, in mammalian epithelial cells, the homologues of DaPKC, Baz, and DPar-6, namely aPKC, Par-3/atypical PKC isotype-specific interacting protein (ASIP), and Par-6, and the corresponding homologues of Crb, Sdt, and Patj, that is, Crb3, Pals1 (protein associated with Lin seven 1), and Patj, respectively, form two complexes that are localized at the TJ and regulate the assembly of these junctions. The basolateral proteins Scrib, mDlg, and mLgl also play important roles in epithelial cell polarity (Izumi et al., 1998; Gao et al., 2002; Hirose et al., 2002; Lemmers et al., 2002; Roh et al., 2002, 2003; Suzuki et al., 2001, 2002; Hurd et al., 2003; Straight et al., 2004).
In Drosophila, the Baz and Crb complexes not only colocalize in the apicolateral region of the cytocortex (Hong et al., 2001; Bilder et al., 2003), but they interact physically by means of DPar-6 and Patj (Nam and Choi, 2003). Furthermore, absence of either complex similarly disrupts epithelial polarity (for review see Tepass et al., 2001), which suggests a functional relationship between them. Here, we report a novel physical connection between the Baz and Crb complexes mediated through direct binding of DaPKC to both Crb and Patj. We find that the kinase activity of DaPKC is necessary to maintain the apical localization of the components of the Crb complex and to prevent the apical localization of basolateral markers. Furthermore, Crb is a phosphorylation target of DaPKC. These results provide a molecular mechanism for the functional interaction between the Crb and Baz complexes based on DaPKC-dependent phosphorylation of Crb.
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Results |
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Crb is a phosphorylation target of DaPKC
Because the kinase activity of DaPKC was required for the apical localization of the Crb complex and DaPKC directly binds Crb and Patj, we analyzed if these proteins were phosphorylation substrates of DaPKC. We found that in an in vitro assay recombinant aPKC, the human homologue of DaPKC, phosphorylated a GST-Crbi fusion protein, either with or without the ERLI domain (Fig. 3 A, arrow). Interestingly, Patj, which was not phosphorylated by aPKC
, strongly decreased Crb phosphorylation when present in the reaction mixture (Fig. 3 A). Note however that Patj was not a general inhibitor of aPKC
, as it did not block aPKC
autophosphorylation (Fig. 3 A, asterisk) or phosphorylation of another substrate, the myelin basic protein (not depicted).
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In vivo activity of Crb is modulated by its state of phosphorylation
To examine the functional relevance of Crb phosphorylation, we compared the in vivo activity of wild-type and nonphosphorylatable Crb proteins. Wild-type and mutated Crb intracellular domains, either CrbiT6AT9AS11AS13A or CrbiT6AT9A, were fused to Crb signal peptide (SP) and TM domains (see Materials and methods). These proteins were overexpressed in embryos under the control of the Ubx-Gal4 driver line. Use of this driver enables direct comparison, in a single embryo, of the wild-type nonexpressing and the Crb-overexpressing domains (Fig. 4 A). As described previously (Wodarz et al., 1995; Klebes and Knust, 2000), overexpression of membrane-tethered Crbi conferred partial apical character to the whole cell membrane, as shown by the distribution of DaPKC and Patj around the whole cell contour, coincident with the ectopic distribution of Crb (Fig. 4 B and not depicted). However, localization of the basolateral markers Nrt and Scrib was not affected (Fig. 4 C and not depicted). In contrast, in embryos overexpressing nonphosphorylatable Crbi constructs (either UAS-CrbiT6AT9AS11AS13A or UAS-CrbiT6AT9A), both the endogenous and overexpressed Crb as well as Patj and DaPKC failed to associate with the membrane and were found diffusely in the cytoplasm (Fig. 4, D and E; not depicted). In addition, the basolateral marker Scrib appeared mislocalized in the apical membrane (Fig. 4 E). Thus, overexpression of a nonphosphorylatable Crbi had the opposite effect than overexpression of wild-type Crbi and resembles the polarity phenotype of strong crb mutants, where Patj and DaPKC are lost from the plasma membrane (Bhat et al., 1999; Klebes and Knust, 2000; Bilder et al., 2003). This suggested that the nonphosphorylatable Crbi acted as a DN. Indeed, nonphosphorylatable Crbi was still able to pull down DaPKC, Patj, and DMoesin from embryonic extracts (Fig. 2 C and not depicted) and thus it may compete with the endogenous Crb for its interacting partners.
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As a further functional test, we analyzed the consequences of overexpressing the different forms of Crb in the imaginal disc epithelia using the en-Gal4 line. This line drives expression of UAS transgenes in the posterior compartment of the wing disc, which gives rise to the posterior wing (Fig. 4 H). Overexpression of Crbiwt caused severe tissue disorganization in the wing, an effect that has been attributed to apicalization of the wing disc cells (Fig. 4 J; Bilder et al., 2003; Tanentzapf and Tepass, 2003). Although the overexpression of nonphosphorylatable CrbiT6AT9AS11AS13A generally lead to embryonic lethality, in a few cases, surviving flies were obtained and displayed only mild defects in the posterior crossvein (not depicted). In contrast, overexpression of CrbiT6DT9D in the imaginal discs disrupted the wing epithelium in a way similar to that caused by Crbiwt (Fig. 4 K). This supports the results obtained in the embryo.
The above experiments indicate that the phosphorylation of Crb by DaPKC is important for its function. Hence, reducing the DaPKC activity should decrease the effect of the overexpression of Crbiwt. This was the case. Expression of UAS-DaPKCCAAXDN largely normalized the effect caused by overexpression of wild-type Crbi (Fig. 4, L and M). As expected, the effect of overexpressing CrbiT6DT9D was less sensitive to the decrease of DaPKC activity. Thus, whereas 100% of flies overexpressing simultaneously UAS-Crbiwt and UAS-DaPKCCAAXDN showed essentially normal wings (Fig. 4 M), 40% of the flies with concomitant expression of UAS-CrbiT6DT9D and UAS-DaPKCCAAXDN still displayed strong cuticular defects (Fig. 4 N).
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Discussion |
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Requirement of aPKC kinase activity in epithelial polarity
We have addressed the question of whether the kinase activity of DaPKC is required for epithelial polarity and have found that this is indeed the case. Thus, diminishing the activity of DaPKC by overexpressing a DN, kinase-dead DaPKCCAAXDN, causes the loss of membrane associated Crb and Patj concomitant with an apical expansion of basolateral markers. Conversely, membrane tethered wild-type DaPKC, located all around the cell cortex, targets apical proteins (Crb, Patj, and Baz) to the basolateral domain. These polarity defects are not due to nonspecific effects of DaPKCCAAXDN because its overexpression strongly normalized the cuticular malformations associated with the overexpression of UAS-DaPKCCAAXWT (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200311031/DC1).
In mammalian epithelial cells in culture, overexpression of kinase-inactive aPKC disrupts the establishment of epithelial polarity (Suzuki et al., 2001). Our results fully agree with these data. We have also found that overexpression in Drosophila embryos of Xenopus kinase-dead aPKC causes the same effects than overexpression of kinase-dead DaPKC (unpublished data). aPKC kinase activity is also needed to establish epithelial polarity in zebra fish. Thus, the zebra fish mutant Heart and soul encodes an aPKC
mutated in a domain critical for the activation of its kinase function and this results in a defective ZA (Horne-Badovinac et al., 2001). All these results show that the kinase activity of aPKC is necessary to establish epithelial polarity in different animal phila and poses the question as to the identification of the phosphorylation targets of DaPKC in epithelial cells.
Crb is a phosphorylation target of DaPKC in epithelial cells
The identified targets of aPKC belong to two groups: basolateral proteins, negatively regulated by aPKC (Lgl, both in vertebrate and invertebrate cells [Betschinger et al., 2003; Plant et al., 2003; Yamanaka et al., 2003] and mammalian Par-1 [Hurov et al., 2004]), and apical proteins, namely Par-3 (Hirose et al., 2002) and Crb (this work), which require such phosphorylation for their proper activity.
Our results strongly suggest that interaction of DaPKC with Crb and the subsequent phosphorylation of the latter are most relevant for epithelial polarity. This conclusion is based in several experimental observations. First, aPKC phosphorylates Crb in vitro at its intracellular domain. Second, this phosphorylation appears to be essential in vivo because mutation of the putatively phosphorylatable Thr/Ser of the Crb intracellular domain into Ala inactivates Crb. Nonphosphorylatable Crbi still binds DaPKC, Patj, and DMoesin. Thus, it should act as a DN form competing with wild-type Crb and we find that this is indeed the case. Third, mutation of the putatively phosphorylatable residues of Crbi into negatively charged Asp maintains its biological function. This suggests that the DN character of the CrbiT6AT9A is not due to a mere change of T6 and T9 to Ala but to the conversion of Crbi into a nonphosphorylatable protein. Fourth, the phenotypic effects of overexpressing Crbiwt were suppressed by a reduction of DaPKC kinase activity, supporting that DaPKC is required for Crb activity. CrbiT6DT9D was less sensitive to a reduction in DaPKC activity than CrbiWT. However, there was still a partial recovery of the cuticular malformations caused by overexpression of CrbiT6DT9D when DaPKC activity was compromised. This suggested that conversion of both Thr into Asp did not render Crb into a completely constitutively active protein. Note also that CrbiT6DT9D still retains Ser11 and Ser13. Although our in vitro experiments do not point at these residues as DaPKC-dependent phosphorylation sites, we cannot totally exclude their functionality in vivo, which could be modified by reduced DaPKC activity. Other alternative, but not mutually excluding explanations, may be that the partial rescue of CrbiT6DT9D by DAPKCCAAXDN results from the interference of DAPKCCAAXDN with the endogenous Crb and/or may be caused by the functional antagonism between Crb and the basolateral proteins that colocalize on the whole cytocortex in these experimental conditions.
Together, these results indicate that DaPKC-dependent phosphorylation of Crb is essential for its wild-type activity. Because the putatively aPKC-dependent phosphorylatable residues of Crb are conserved in mammalian homologues, one of the main functions of aPKC in epithelial cells may be to maintain apicobasal polarity through posttranslational modification of Crb. Note, however, that the genetic control of epithelial polarity may be different in C. elegans, because the putatively phosphorylatable residues are absent from C. elegans Crb-1 and inactivation of crb-1 by dsRNAi did not disturb formation of the C. elegans apical junction (Bossinger et al., 2001).
Additionally, and although it has not been demonstrated, it is conceivable that DaPKC-dependent phosphorylation of Baz may be required for apicobasal polarity in Drosophila, as it is the case in cultured mammalian epithelial cells where phosphorylation of the mammalian homologue of Baz, ASIP/Par-3, by PKC-3 is indispensable for the formation of the TJ (Hirose et al., 2002).
aPKC plays a dual role in the control of epithelial polarity. In addition to its requirement for the proper activity of the apical proteins, aPKC negatively regulates the activity of the basolateral proteins. Thus, in mammalian epithelial cells aPKC-dependent phosphorylation of Lgl and Par-1b prevents the association of these proteins with the plasma membrane (Yamanaka et al., 2003; Hurov et al., 2004). In the apical side of the Drosophila neuroblasts, DaPKC counteracts the activity of Lgl releasing it from its association with the cell membrane (Betschinger et al., 2003). A similar antagonistic effect between DaPKC and Lgl and Dlg appears to take place in the imaginal discs (Rolls et al., 2003). In agreement with these observations, we have found that the kinase activity of DaPKC is required to exclude Scrib from the apical region of the embryonic epithelial cells. This function of DaPKC would be independent of its role in Crb activation because ectopic basolateral localization of Crb on its own did not affect the localization of basolateral markers (Fig. 4 C). Although ectopic accumulation of Crb is associated with relocalization of endogenous DaPKC to the whole cell contour, conceivably that amount of DaPKC is insufficient to affect the localization of basolateral markers.
A molecular mechanism for the functional interaction between the apical complexes
It has been proposed that the Baz, Crb, and Dlg complexes act sequentially to direct the maturation of epithelial cell polarity, the Baz complex being the first to be assembled and the most critical apical regulator (Bilder et al., 2003). Our findings provide a mechanistic framework for the regulation of the Crb complex by the Baz complex. The functional interaction between the Drosophila apical complexes relies in physical contact between some of their components. Thus, DPar-6 and Patj directly interact in vitro (Nam and Choi, 2003). Now we find that DaPKC directly binds to Crb and Patj. Although in vitro DaPKC interacts with and is able to phosphorylate a Crbi protein devoid of the COOH-terminal ERLI domain (Fig. 3 A), pull-down experiments of embryonic extracts show that this domain is necessary for the interaction between the Baz and Crb complexes. This suggests that in vivo the interaction may be reinforced by proteins that, directly or indirectly, are bound to the ERLI domain. Sdt is a likely candidate because it binds to this domain of Crb (Bachmann et al., 2001; Hong et al., 2001; Roh et al., 2002) and its mammalian homologue Pals-1 binds Par-6 (Hurd et al., 2003). Additionally, human Crb-3 and Par-6 interact (Lemmers et al., 2004), but it is not yet known whether this also occurs in Drosophila.
The interaction between the apical complexes results in DaPKC-dependent phosphorylation and activation of Crb. Although our results show that the phosphorylation state of Crb does not affect its interaction with several known binding partners, we speculate that such phosphorylation may be necessary for Crb to adopt a correct conformation and/or to interact with proteins required for Crb stabilization at the apical domain. Moreover, the finding that Patj inhibits the DaPKC-dependent phosphorylation of Crb, suggests the existence of alternative and excluding complexes. During early embryonic stages, Patj and DaPKC may directly interact, although such interaction would not affect the kinase activity of DaPKC (in fact, DaPKC kinase activity is required for the apical localization of Patj at blastoderm stage; unpublished data). At gastrulation, when the Crb complex accumulates at the plasma membrane, we hypothesize that a greater affinity of Patj for Sdt (bound to Crb) than for DaPKC would release Patj from its interaction with DaPKC and allow the phosphorylation of Crb. Note that the Baz complex may perform additional roles in relation to the Crb complex. Thus, previous data suggested the existence of Crb-dependent and -independent mechanisms for the localization of Patj and Sdt (Tanentzapf et al., 2000; Nam and Choi, 2003). The interaction between components of the apical complexes, as stated by Nam and Choi (2003), would account for these results.
In summary, the present results stress the intricacy of the relationships between the evolutionarily conserved proteins that establish and maintain epithelial polarity. There is a feedback mechanism between the Baz and Crb complexes. Thus, phosphorylation of Crb by DaPKC is a requisite for the ulterior Crb complex-dependent maintenance of the apical localization of the Baz complex. The wealth of data demonstrating the physical and functional interactions between both complexes allows them to be considered as part of a unique apical determinant complex, whose components are added stepwise. Additionally, there is a functional antagonism among the apical and the basolateral domain determining proteins (Bilder et al., 2003, Tanentzapf and Tepass, 2003; this work). Clearly, identification of additional targets of DaPKC and comprehensive molecular and genetic analyses of the functional interactions among the different complexes will allow further insight into the establishment and maintenance of epithelial polarity.
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Materials and methods |
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Mis-expression studies
Mis-expression experiments were performed using the Gal4/UAS system (Brand and Perrimon, 1993) and en-Gal4, Ubx-Gal4 (Calleja et al., 1996; a gift from M. Calleja, CBMSO, Madrid, Spain) and the maternal Gal4 line MatVP16V67-Gal4 (generated by D. St. Johnston, University of Cambridge, Cambridge, UK). Embryos expressing UAS-DaPKC, UAS-DaPKCCAAXWT, UAS-DaPKCCAAXDN, or UAS-XaPKCCAAXDN driven by the MatVP16V67-Gal4 line were collected after 616 h at 18 or 25°C, fixed, and stained. Embryos expressing UAS-Crbi, UAS-CrbiT6DT9D, UAS-CrbiT6AT9A, and UAS-CrbiT6AT9AS11AS13A driven by Ubx-Gal4 were collected for 16 h at 18 or 25°C. To assay the effect of a reduction of DaPKC signaling on the activity of Crbi, either wild-type or mutated, en-Gal4 females were mated with UAS-DaPKCCAAXDN/If; UAS-Crbi/MKRS males. This cross scheme allows unambiguous identification of en-Gal4/UAS-DaPKCCAAXDN; en-Gal4; UAS-Crbi and en-Gal4/UAS-DaPKCCAAXDN; UAS-Crbi flies in the progeny. The progeny of the above mentioned cross was maintained at 18°C during embryonic development and then transferred to 25°C for subsequent development in order to minimize deleterious effects associated to the expression of Crb constructs at the embryonic stages. The effect of concomitant expression of UAS-DaPKCCAAXWT and UAS-DaPKCCAAXDN was examined in the progeny of the cross of en-Gal4 females with UAS-DaPKCCAAXDN/If; UAS-DaPKCCAAXWT/MKRS males.
Immunohistochemistry
Embryos were fixed as in Tepass et al. (1990) and stained with rabbit anti-Patj and rat antiCrb-intra (Bhat et al., 1999; a gift from H. Bellen, Baylor College of Medicine, Houston, TX), mouse Mab cq4 anti-Crb (DSHB), rabbit anti-Baz (a gift from A. Wodarz, Heinrich-Heine-Universität Düsseldorf, Dusseldorf, Germany), rabbit anti-aPKC C20 (Santa Cruz Biotechnology, Inc.), mouse Mab BP106 anti-Nrt (Hortsch et al., 1990), rabbit anti-Scrib (a gift from C. Doe, University of Oregon, Eugene, OR), rabbit antiß-galactosidase (Cappel) and mouse antiß-galactosidase (Promega). FITC and rhodamine red-X secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. Embryos were mounted in Vectashield (Vector Laboratories). Staining of en-Gal4/UAS-lacZ wings with X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) was performed as described in Calleja et al. (1996).
Confocal images were acquired in a MicroRadiance 2000 microscope (Bio-Rad Laboratories). Images of Fig. 1 were collected with a 63x/1.4 oil Plan-Apochromat objective and magnified 2x. Images of Fig. 4 were collected with 40x/1.3 oil Plan-Neofluar objective and magnified 2.4x. The acquisition software was Lasersharp 2000 BioRad 5.0. Wing images shown in Fig. 4 and Fig. S1 were obtained in an Axiophot microscope (Carl Zeiss MicroImaging, Inc.) with 2.5x/0.075 Plan-Neofluar objective, optovar 2x, and acquired with a camera (model DFC300; Leica) using Fire Cam software (Leica). Images were imported into Adobe Photoshop 7.0 for cropping and contrast adjustment before being assembled into figures using the same software.
Protein binding and in vitro phosphorylation assays
GST-fusion proteins were obtained from subclones in pGEX-3X (Amersham Biosciences) of full-length Patj (Bhat et al., 1999), a gift from H. Bellen, and Crb wild-type or mutated intracellular domain (see Site directed mutagenesis) and CrbiERLI (Hong et al., 2001), a gift from Y.N Jan (University of San Francisco, San Francisco, CA). To perform pull-down experiments, protein extracts were prepared from 0-16h Drosophila embryos by homogenizing them in lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EGTA; 2 mM EDTA, 1% Triton X-100 containing protease inhibitors). Debris were removed by centrifugation at 12,000 g for 15 min at 4°C. 300 µg of Drosophila protein extract were incubated for 16 h at 4°C with 30 µg of GST or of the different GST-fusion proteins bound to glutathione beads as described in Sanz et al. (1999). Bound proteins were separated by SDS-PAGE and immunoblotted with anti-aPKC
C20, anti-Baz (a gift from A. Wodarz), anti-DMoesin (from D. Kiehart, Duke University, Durham, NC) or anti-Patj antibodies. For in vitro binding assays, His-tagged DaPKC was obtained from a subclone in a pRSET (Invitrogen) that also tagged the expressed protein with the Xpress epitope. 100 µg of the different purified GST-fusion proteins were mixed with 40 µl of His-tagged DaPKC immobilized in Ni-NTA-agarose and incubated over night in lysis buffer at 4°C. Bound proteins were separated by SDS-PAGE and immunoblotted with anti-GST (Santa Cruz Biotechnology, Inc.). Control of His-tagged DaPKC loading was performed by immunoblotting with anti-Xpress epitope antibody (Invitrogen). In vitro kinase assay were performed as in (Leitges et al., 2001) using 30 ng of baculovirus-expressed recombinant human aPKC
(Calbiochem) and 3 µg of the indicated GST-fusion proteins or of myelin basic protein. This phosphorylation depended on aPKC activity because it was inhibited by previous denaturation of the kinase. Note that although a human aPKC was used, the high similarity between human aPKC and DaPKC (79% of identity and 90% of similarity in the kinase domain) makes it unlikely the possibility of obtaining different results with DaPKC instead of human aPKC.
Site directed mutagenesis
Mutagenesis were performed by the site directed Quick-Change system (Stratagene). Full length DaPKC cDNA, isolated from a cDNA Drosophila ZAP library (a gift from P. Hurban, Paradigm Genetics, Research Triangle Park, NC), was used as a template to incorporate to DaPKC the CAAX motif with the following primers: CAAX-forward: 5'-ATATGGTGCACGGATCGTTT-3' and CAAX-reverse: 5'-GCGAATTCTCAGGACAGCACACATTTCGAGCTCATGCAGCCAGGACCACTCTCATCGGGTGGGTTCA-GTTTGACGCAATCCTCCAGAGACATCAGCAAGG-3'.
The DaPKCCAAX clone was used to generate a DaPKCCAAX kinase inactive (K293W) with the following primers: DN forward: 5'-CGCATCTACGCCATGTGGGTGATCAAGAAGG-3' and DN reverse: 5'-CCTTCTTGATCACCCACATGGCGTAGATGCG-3'.
Generation of the UAS-Crb i construct was essentially as described previously (Wodarz et al., 1995). crb cDNA (a gift from A. Wodarz) was used as a template to amplify the SP and TM domains using the following pairs of primers: CRB SP fwd: 5'-GCGAATTCTAGCCAAAAGCAACCC-3' and CRB SP rev: 5'-GCAGATCTGGAGCCATTAAAGTACG-3'; and CRB TM fwd: 5'-GCAGATCTTCGACCACAGACATTG-3' and CRB TM rev: 5'-CGTCTAGAAGGTGTATATGGGCGG-3'.
PCR products were subcloned in the pGEMT-easy (Promega). pBluescript or pGEX-3X subclones containing the wild-type Crbi sequence (Wodarz et al., 1995), were used as templates to obtain CrbiT6A with the following primers: 5'-CAGGAACAAGCGAGCAGCGAGGGGCACC-3' and 5'-GTGCCCCTCGCTGCTCGCTTGTTCCTGG-3'. Crbi T6A was used as a template to generate CrbiT6A S13A with the following primers: 5'-CCTATAGCCCGGCCGCGCAAGAGTACTGC-3' and 5'-GCAGTACTCTTGCGCGGCCGGGCTATAGG-3'. Crbi T6A was used as a template to generate CrbiT6A T9A with the following primers: 5'-CAGCAGGGGCGCGTATAGCCCGAGC-3' and 5'-GCTCGGGCTATACGCGCCCCTCGCTG-3'. Crbi T6A S13A was used as a template to obtain CrbiT6A T9A S11A S13A with the following primers: 5'-GCGAGGGGCGCGTATGCCCCGGCCGCG-3' and 5'-CGCGGCCGGGGCATACGCGCCCCTCGC-3'. T6, T9, S11, and S13 were added back to CrbiT6A T9A S11A S13A using the following primers, respectively: ABT6: 5'-CAGGAACAAGCGAGCAACCAGGGGCGCG-3' and 5'-GCGCCCCTGGTTGCTCGCTTGTTCCTG-3'; ABT9: 5'-GCAGCGAGGGGCACCTATGCCCCGG-3' and 5'-CCGGGGCATAGGTGCCCCTCGCTGC-3'; ABS11: 5'-GGGGCGCGTATAGCCCGGCCGCGC-3' and 5'-GCGCGGcCGGGCTATACGCGCCCC-3'; and ABS13: 5'-CCTATGCCCCGAGCGCGCAAGAGTACTGC-3' and 5'-GCAGTACTCTTGCGCGctCGGGgcATAGG-3'.
pBluescript or pGEX-3X subclones containing the wild-type Crbi sequence (Wodarz et al., 1995), were used as templates to obtain CrbiT6D with the following primers: 5'-CCAGGAACAAGCGAGCAGACAGGGGCACC-3' and 5'-GGTGCCCCTGTCTGCTCGCTTGTTCCTGG-3'. Crbi T6D was used as a template to obtain CrbiT6DT9D with the following primers: 5'-GCAGACAGGGGCGACTATAGCCCGAGCG-3' and 5'-CGCTCGGGCTATAGTCGCCCCTGTCTGC-3'. All mutations were confirmed by sequencing. Wild-type and mutated Crbi domains were ligated with the SP and TM domains and subcloned in pUAST.
Online supplemental material
An overexpression assay was used to exclude the possibility of nonspecific effects of DN-DaPKC expression. Fig. S1 shows that the polarity defects, found in wings and associated with the overexpression of DaPKCCAAXWT are normalized by overexpression of UAS-DaPKCCAAXDN. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200311031/DC1.
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Acknowledgments |
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This work was supported by grants from CAM (08.5/0030/2000) to S. Campuzano; Dirección General de Investigación Científica y Técnica (PB98-0682) and MCYT (BMC2002-00411) to J. Modolell; SAF1999-0053, 2FD97-1429, and SAF2000-0175 from MCYT, 08.1/0060/2000 from CAM, and "II Ayuda de la Fundación Juan March a la Investigación básica" to J. Moscat. An institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa is acknowledged.
Submitted: 6 November 2003
Accepted: 6 July 2004
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References |
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Ashburner, M. 1989. Drosophila. Transformation. A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 10171063.
Bachmann, A., M. Schneider, B. Theilenberg, F. Grawe, and E. Knust. 2001. Drosophila stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature. 414:638643.[CrossRef][Medline]
Benton, R., and D.S. Johnston. 2003. Drosophila PAR-1 and 14-3-3 inhibit Bazooka/PAR-3 to establish complementary cortical domains in polarized cells. Cell. 115:691704.[CrossRef][Medline]
Berra, E., M.T. Diaz-Meco, I. Dominguez, M.M. Municio, L. Sanz, J. Lozano, R.S. Chapkin, and J. Moscat. 1993. Protein kinase C zeta isoform is critical for mitogenic signal transduction. Cell. 74:555563.[Medline]
Betschinger, J., K. Mechtler, and J.A. Knoblich. 2003. The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl. Nature. 422:326330.[CrossRef][Medline]
Bhat, M.A., S. Izaddoost, Y. Lu, K.O. Cho, K.W. Choi, and H.J. Bellen. 1999. Discs Lost, a novel multi-PDZ domain protein, establishes and maintains epithelial polarity. Cell. 96:833845.[Medline]
Bilder, D., and N. Perrimon. 2000. Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature. 403:676680.[CrossRef][Medline]
Bilder, D., M. Li, and N. Perrimon. 2000. Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science. 289:113116.
Bilder, D., M. Schober, and N. Perrimon. 2003. Integrated activity of PDZ protein complexes regulates epithelial polarity. Nat. Cell Biol. 5:5358.[CrossRef][Medline]
Bossinger, O., A. Klebes, C. Segbert, C. Theres, and E. Knust. 2001. Zonula adherens formation in Caenorhabditis elegans requires dlg-1, the homologue of the Drosophila gene discs large. Dev. Biol. 230:2942.[CrossRef][Medline]
Brand, A.H., and N. Perrimon. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118:401415.
Calleja, M., E. Moreno, S. Pelaz, and G. Morata. 1996. Visualization of gene expression in living adult Drosophila. Science. 274:252255.
Campos-Ortega, J.A., and V. Hartenstein. 1985. The embryonic development of Drosophila melanogaster. Springer-Verlag, Berlin Heidelberg, Germany. 227 pp.
Drubin, D.G., and W.J. Nelson. 1996. Origins of cell polarity. Cell. 84:335344.[Medline]
Gao, L., G. Joberty, and I.G. Macara. 2002. Assembly of epithelial tight junctions is negatively regulated by Par6. Curr. Biol. 12:221225.[CrossRef][Medline]
Grawe, F., A. Wodarz, B. Lee, E. Knust, and H. Skaer. 1996. The Drosophila genes crumbs and stardust are involved in biogenesis of adherens junctions. Development. 122:951959.
Henrique, D., and F. Schweisguth. 2003. Cell polarity: the ups and downs of the Par6/aPKC complex. Curr. Opin. Genet. Dev. 13:341350.[CrossRef][Medline]
Hirose, T., Y. Izumi, Y. Nagashima, Y. Tamai-Nagai, H. Kurihara, T. Sakai, Y. Suzuki, T. Yamanaka, A. Suzuki, K. Mizuno, and S. Ohno. 2002. Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation. J. Cell Sci. 115:24852495.
Hong, Y., B. Stronach, N. Perrimon, L.Y. Jan, and N.J. Jan. 2001. Drosophila stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature. 414:634638.[CrossRef][Medline]
Horne-Badovinac, S., D. Lin, S. Waldron, M. Schwarz, G. Mbamalu, T. Pawson, Y. Jan, D.Y. Stainier, and S. Abdelilah-Seyfried. 2001. Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis. Curr. Biol. 11:14921502.[CrossRef][Medline]
Hortsch, M., N.H. Patel, A.J. Bieber, Z.R. Traquina, and C.S. Goodman. 1990. Drosophila neurotactin, a surface glycoprotein with homology to serine esterases, is dynamically expressed during embryogenesis. Development. 110:13271340.[Abstract]
Hurd, T.W., L. Gao, M.H. Roh, I.G. Macara, and B. Margolis. 2003. Direct interaction of two polarity complexes implicated in epithelial tight junction assembly. Nat. Cell Biol. 5:137142.[CrossRef][Medline]
Hurov, J.B., J.L. Watkins, and H. Piwinica-Worms. 2004. atypical PKC phosphorylates Par-1 kinases to regulate localization and activity. Curr. Biol. 14:736741.[CrossRef][Medline]
Izumi, Y., T. Hirose, Y. Tamai, S. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J. Cell Biol. 143:95106.
Klebes, A., and E. Knust. 2000. A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila. Curr. Biol. 10:7685.[CrossRef][Medline]
Knust, E., and O. Bossinger. 2002. Composition and formation of intercellular junctions in epithelial cells. Science. 298:19551959.
Knust, E., U. Tepass, and A. Wodarz. 1993. crumbs and stardust, two genes of Drosophila required for the development of epithelial polarity. Dev Suppl. 261268.
Kuchinke, U., F. Grawe, and E. Knust. 1998. Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr. Biol. 8:13571365.[Medline]
Leitges, M., L. Sanz, P. Martin, A. Duran, U. Braun, J.F. Garcia, F. Camacho, M.T. Diaz-Meco, P.D. Rennert, and J. Moscat. 2001. Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaß pathway. Mol. Cell. 8:771780.[Medline]
Lemmers, C., E. Medina, M.H. Delgrossi, D. Michel, J.P. Arsanto, and A. Le Bivic. 2002. hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells. J. Biol. Chem. 277:2540825415.
Lemmers, C., D. Michel, L. Lane-Guermonprez, M.H. Delgrossi, E. Medina, J.P. Arsanto, and A. Le Bivic. 2004. CRB3 binds directly to Par6 and regulates the morphogenesis of the tight junctions in mammalian epithelial cells. Mol. Biol. Cell. 15:13241333.
Macara, I.G. 2004. Parsing the polarity code. Nat. Rev. Mol. Cell Biol. 5:220231.[CrossRef][Medline]
Mechler, B.M., W. McGinnis, and W.J. Gehring. 1985. Molecular cloning of lethal(2)giant larvae, a recessive oncogene of Drosophila melanogaster. EMBO J. 4:15511557.[Abstract]
Medina, E., J. Williams, E. Klipfell, D. Zarnescu, G. Thomas, and A. Le Bivic. 2002. Crumbs interacts with moesin and beta(Heavy)-spectrin in the apical membrane skeleton of Drosophila. J. Cell Biol. 158:941951.
Müller, H.A., and E. Wieschaus. 1996. armadillo, bazooka and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134:149163.[Abstract]
Müller, H.J. 2000. Genetic control of epithelial cell polarity: Lessons from Drosophila. Dev. Dyn. 218:5267.[CrossRef][Medline]
Nam, S.C., and K.W. Choi. 2003. Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila. Development. 130:43634372.
Ohno, S. 2001. Intercellular junctions and cellular polarity: the PARaPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr. Opin. Cell Biol. 13:641648.[CrossRef][Medline]
Petronczki, M., and J.A. Knoblich. 2001. DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3:4349.[CrossRef][Medline]
Pielage, J., T. Stork, I. Bunse, and C. Klambt. 2003. The Drosophila cell survival gene discs lost encodes a cytoplasmic Codanin-1-like protein, not a homolog of tight junction PDZ protein Patj. Dev. Cell. 5:841851.[CrossRef][Medline]
Plant, P.J., J.P. Fawcett, D.C. Lin, A.D. Holdorf, K. Binns, S. Kulkarni, and T. Pawson. 2003. A polarity complex of mPar-6 and atypical PKC binds, phosphorylates and regulates mammalian Lgl. Nat. Cell Biol. 5:301308.[CrossRef][Medline]
Roh, M.H., and B. Margolis. 2003. Composition and function of PDZ protein complexes during cell polarization. Am. J. Physiol. Renal Physiol. 285:F377F387.
Roh, M.H., O. Makarova, C.-J. Liu, K. Shin, S. Lee, S. Laurinec, M. Goyal, R. Wiggins, and B. Margolis. 2002. The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J. Cell Biol. 157:161172.
Roh, M.H., S. Fan, C.J. Liu, and B. Margolis. 2003. The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J. Cell Sci. 116:28952906.
Rolls, M.M., R. Albertson, H.P. Shih, C.Y. Lee, and C.Q. Doe. 2003. Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia. J. Cell Biol. 163:10891098.
Sanz, L., P. Sanchez, M.J. Lallena, M.T. Diaz-Meco, and J. Moscat. 1999. The interaction of p62 with RIP links the atypical PKCs to NF-kappaß activation. EMBO J. 18:30443053.
Straight, S.W., K. Shin, V.C. Fogg, S. Fan, C.J. Liu, M. Roh, and B. Margolis. 2004. Loss of PALS1 expression leads to Tight Junction and polarity defects. Mol. Biol. Cell. 15:19811990.
Suzuki, A., T. Yamanaka, T. Hirose, N. Manabe, K. Mizuno, M. Shimizu, K. Akimoto, Y. Izumi, T. Ohnishi, and S. Ohno. 2001. Atypical protein kinase C is involved in the evolutionarily conserved PAR protein complex and plays a critical role in establishing epithelia-specific junctional structures. J. Cell Biol. 152:11831196.
Suzuki, A., C. Ishiyama, K. Hashiba, M. Shimizu, K. Ebnet, and S. Ohno. 2002. aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization. J. Cell Sci. 115:35653573.
Tanentzapf, G., and U. Tepass. 2003. Interactions between the crumbs, lethal giant larvae and bazooka pathways in epithelial polarization. Nat. Cell Biol. 5:4652.[CrossRef][Medline]
Tanentzapf, G., C. Smith, J. McGlade, and U. Tepass. 2000. Apical, lateral, and basal polarization cues contribute to the development of the follicular epithelium during Drosophila oogenesis. J. Cell Biol. 151:891904.
Tepass, U. 1996. Crumbs, a component of the apical membrane, is required for zonula adherens formation in primary epithelia of Drosophila. Dev. Biol. 177:217225.[CrossRef][Medline]
Tepass, U., C. Theres, and E. Knust. 1990. crumbs encodes an EFG-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell. 61:787799.[Medline]
Tepass, U., G. Tanentzapf, R. Ward, and R. Fehon. 2001. Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35:747784.[CrossRef][Medline]
Wodarz, A., U. Hinz, M. Engelbert, and E. Knust. 1995. Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia in Drosophila. Cell. 82:6776.[Medline]
Wodarz, A., A. Ramrath, A. Grimm, and E. Knust. 2000. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelial neuroblasts. J. Cell Biol. 150:13611374.
Woods, D.F., and P.J. Bryant. 1991. The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell. 66:451464.[Medline]
Yamanaka, T., Y. Horikoshi, Y. Sugiyama, C. Ishiyama, A. Suzuki, T. Hirose, A. Iwamatsu, A. Shinohara, and S. Ohno. 2003. Mammalian Lgl forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity. Curr. Biol. 13:734743.[Medline]