Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3-Par-6-aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs-Stardust (Sdt)-Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. By overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones is demonstrated. A novel physical interaction occurs between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and Crumbs is identified as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3-Par-6-aPKC and Crumbs-Sdt-Patj complexes based in the posttranslational modification of Crb by DaPKC (Sotillos, 2004; full text of article ).
The C tail of Fz receptors regulates their localization and signaling activity. A short Fz Cterm governs apical localization, which is critical for effective Fz-PCP signaling. In contrast, a long Cterm (like that of Fz2) governs baso-lateral localization, promoting β-catenin signaling and preventing PCP activity. Thus, a striking feature of all core PCP proteins, including Fz1, is their apical localization within imaginal disc cells. Fz1 colocalizes partially with several components that regulate A/B polarity such as the Crumbs/Sdt/Patj and Baz/aPKC/Par-6 complexes within the marginal domain, even though it is also present more basally relative to these complexes (Djiane, 2005).
Detailed sequence analysis of the Fz1 Cterm has revealed the presence of two clustered conserved PKC phosphorylation sites (Ser554 and Ser560 in Fz1). Given that aPKC expression in the apical domain overlaps with Fz1, a test was performed to see if aPKC can phosphorylate the Fz1 Cterm on the two conserved PKC sites in an in vitro kinase assay. Purified human aPKC protein phosphorylates in vitro a GST::Fz1 Cterm fusion protein. Furthermore, mutations of the two PKC consensus sites (Ser to Ala) prevent aPKC-mediated Fz1 phosphorylation, confirming that these sites are targets of aPKC (Djiane, 2005).
To investigate the importance of these phosphorylation sites in vivo, flies were generated carrying UAS-inducible transgenes of Fz1 mutant derivatives with either both serines mutated to alanine (Fz1-AA), inactivating the two prospective PKC sites, or both Serines mutated to Glutamic acid (Fz1-EE), mimicking phosphorylation. These transgenes were analyzed under sevenless (sev)-Gal4 control, which is expressed specifically in R3/R4 precursor cells just posterior to the MF during PCP establishment. Overexpression of wild-type Fz1 provides too much activity and interferes with the balance of Fz1 regulation within the R3/R4 pair, resulting in ommatidia with random R3/R4 cell fate decision and chirality, as well as symmetrical R3/R3 type ommatidia. Similarly, overexpression of Fz1-AA (with both aPKC sites inactivated; sev>Fz1-AA) induces ommatidia with random chirality and symmetrical clusters. In contrast, the phosphomimetic Fz1-EE (sev>Fz1-EE) shows hardly any effect. These data suggest that aPKC-mediated Fz1 phosphorylation inhibits Fz-PCP signaling activity (Djiane, 2005).
Since apical Fz1 localization is critical for its proper PCP signaling activity (Wu, 2004), it was hypothesized that the Fz1-EE mutation could affect the localization of the receptor. To investigate this possibility, the expression of the different myc tagged Fz1 transgenes was examined in imaginal discs (under en-Gal4 or dpp-Gal4 control). No difference between the expression of either Fz1-AA or Fz1-EE with that of wild-type Fz1 was found. These mutant Fz1 isoforms were expressed at similar levels and colocalized apically with aPKC, indicating that phosphorylation of Fz1 by aPKC does not affect Fz1's localization (Djiane, 2005).
A second feature critical to Fz1 signaling activity is its ability to recruit Dsh to the membrane. Interestingly, the aPKC sites partly overlap with the region of the Fz Cterm known to bind Dsh, raising the possibility that phosphorylation by aPKC could interfere with Dsh recruitment. Thus tests were performed to see whether Dsh recruitment is affected by phosphorylation of the Fz1 aPKC sites. S2 cells, which have no endogenous Fz, were transfected with Dsh-GFP and the different Fz1 mutants. In this assay, wild-type and both mutant forms of Fz1 recruit Dsh-GFP efficiently to the membrane. Then, whether overexpression of Fz1-AA and Fz1-EE can recruit Dsh-GFP to apical membranes like wild-type Fz1 in vivo (expressed with en-Gal4 in the posterior compartment of wing discs, where there is a sharp boundary between expressing and nonexpressing cells) was examined. Both Fz1-AA and Fz1-EE behave like wild-type Fz1, sequestering Dsh to the apical cell membrane in imaginal disc cells (Djiane, 2005).
In summary, it was shown that Fz1 can be phosphorylated in vitro by aPKC. Together with the in vitro results, the in vivo analysis of a phosphomimetic Fz1 mutant suggests that aPKC phosphorylation regulates Fz1 activity negatively and that this effect is not mediated by affecting Fz1 localization or Fz1-mediated Dsh membrane recruitment (Djiane, 2005).
In light of the importance of the aPKC sites in Fz1, it was of interest to determine how aPKC is recruited to the receptor. This could be mediated by direct binding or through a bridging factor, the most likely candidates being the A/B determinants that bind aPKC. Thus a two-hybrid interaction screen was conducted using the Fz1 Cterm as bait and components of different A/B protein complexes as prey. The closely related Fz2 Cterm was included as well as the Stbm Cterm as control baits. All components of the aPKC/Par-6/Bazooka apical complex were tested. For Baz, three different fragments were used: an N-terminal fragment involved in Baz dimerization (BazA), a central fragment with three PDZ domains involved in Par-6 binding (BazB), and a C-terminal fragment that binds aPKC (BazC). Similarly, all components of the Crb/Sdt/Patj apical complex were tested except Crb (since Crb is a transmembrane protein) and the components of the more baso-lateral Scrib/Dlg/Lgl complex were analyzed. No direct interaction was detected between the Fz1 Cterm and aPKC, but, interestingly, Patj was found to be a specific binding partner of the Fz1 Cterm. No other protein was found to interact with the Fz1 Cterm, and in turn Patj did not interact with the Fz2 or Stbm Cterms. The interaction between the Stbm Cterm and Dlg was confirmed as was the interaction of aPKC with Par-6 and BazC (Djiane, 2005)
To confirm the Patj-Fz1 interaction in vivo, coimmunoprecipitation (CoIP) experiments were performed from Drosophila S2 cell extracts transfected with GFP fusion proteins with the Fz1 or Fz2 Cterms (GFP::Fz1 and GFP::Fz2, respectively). Patj could be co-immunoprecipitated from cells transfected with GFP::Fz1 but importantly not with GFP::Fz2 or GFP alone, demonstrating that Fz1 and Patj interact in Drosophila cells. A weak interaction was found between Fz1 and endogenous Baz and aPKC, suggesting the existence of one or several multiprotein complexes among Fz1, Patj, Baz, and aPKC. In contrast, other components of A/B protein complexes, such as Par-6 or Dlg, did not CoIP with either GFP::Fz1 or GFP::Fz2 (Djiane, 2005).
To map the Patj interaction domain with the Fz1 Cterm, GST pull-down experiments were performed. Patj is a modular protein containing a N-terminal L27 domain (previously referred to as MRE), mediating its interaction with Sdt, and four PDZ domains. Consistent with the yeast two-hybrid and CoIP results, in vitro translated full-length Patj bind the GST-Fz1 Cterm protein. The fourth PDZ domain of Patj is sufficient for direct binding to the Fz1 Cterm (Djiane, 2005).
Using the CoIP approach, the residues in the Fz1 Cterm required for Patj interaction were also mapped. Whereas the full-length Fz1 Cterm interacts with Patj, removing the last three residues (Fz1ΔBS) abolishes this interaction. The removal of an internal Cterm motif, encompassing the tryptophan critical for Dsh binding, retains Fz1 ability to bind Patj albeit to a lesser extent (Djiane, 2005).
These results support a direct interaction between the apical determinant Patj and the Fz1 Cterm and suggest that Patj could provide a link between Fz1 and aPKC, since Patj was shown in vitro to bind to aPKC either directly or indirectly through Par-6. The Fz1/Patj interaction is mediated by the fourth PDZ domain of Patj, requires the last three residues of Fz1, and is largely independent of the Fz motif that mediates Dsh binding (Djiane, 2005).
A prediction from these results that Patj negatively regulates Fz1 activity by recruiting aPKC to Fz1 is that Patj and aPKC should be either downregulated or absent in cells where Fz-PCP signaling is active. To investigate this, immunostainings were performed with Patj- and aPKC-specific antibodies in third instar larval eye discs during PCP establishment. Patj is expressed at the most apical lateral membrane of third instar eye imaginal disc cells anterior to the MF. Posterior to the furrow, as photoreceptor preclusters emerge and begin to differentiate, Patj is still detected apically in all intercluster cells but shows a complex pattern within the preclusters. In developing preclusters, Patj is enriched in R2/R5 precursors and dramatically downregulated in R3/R4 precursors between rows 1 and 7. This reduction in expression is complementary to an increase in apical Fz1 localization (monitored by Fz-GFP staining), which shows the typical double horseshoe pattern specific for PCP factors in early R3/R4 pairs. Posterior to row 7, Patj is found in R3/R4 as well but remains enriched in R2 and R5. Similarly, although not as dramatic as for Patj, aPKC expression is weaker apically in R3/R4 cells as compared to the neighboring R2 and R5 (Djiane, 2005).
The downregulation of aPKC and Patj from the R3/R4 cell border during PCP establishment raised the possibility that the PCP determinants could control expression or localization of aPKC/Patj. Clonal analyses of PCP genes revealed, however, that the Patj and aPKC characteristic expression patterns in the preclusters are unaffected in fmi, dgo, pk, stbm, and fz mutant clones. The aPKC and Patj expression patterns are therefore independent of PCP signaling, consistent with an upstream early role of these A/B polarity determinants (Djiane, 2005).
In the CoIP experiments in S2 cells, Baz was recovered together with Fz1, Patj, and aPKC (although at lower levels than Patj), raising the possibility that Baz might be in a complex with aPKC and Patj to regulate Fz1 signaling. In order to investigate this, endogenous Baz expression was examined in third instar eye discs. Although expressed apically in all cells anterior and posterior to the MF, Baz becomes enriched in R3/R4 cells during PCP establishment, showing a complementary pattern to Patj and aPKC. Subsequently, Baz is enriched in the polar R4 cell, similar to core PCP determinants such as Fz1, Dsh, and Fmi. Next, Baz expression and localization was examined in PCP mutants to determine whether this pattern is under the control of PCP signaling. Interestingly, the only significant difference was found in fz mutant clones, where the Baz pattern was less resolved. In particular, the late accumulation in only one of the two cells of the R3/R4 pair, reflecting the R4 cell fate, was missing. Thus, Baz seems to have two expression phases in the PCP context; an initial accumulation in both R3 and R4 that is likely independent of PCP establishment, and a later enrichment in R4 which is downstream of Fz1 signaling (Djiane, 2005).
The reciprocal expression pattern of aPKC/Patj and Baz and the evidence that Par-3, the vertebrate Baz homolog, can act as an inhibitor of aPKC kinase activity suggested that Baz could play an antagonizing role to the negative regulation of Fz1 by aPKC in cells where Fz1 should be active. Therefore a baz requirement in eye PCP establishment was tested by performing loss-of-function (LOF) and GOF experiments. Although baz mutant clones are very disorganized in adult eye sections, probably due to a general Baz requirement for photoreceptor differentiation, they can be analyzed for PCP defects in eye imaginal discs where the integrity of cells is maintained and large clones could be obtained. Interestingly, baz mutant clones have ommatidial clusters with inverted chirality and some that show symmetrical features, as shown with the R4 specific mδ-lacZ marker that highlights ommatidial chirality in the eye disc. Furthermore, it was found that removal of one copy of baz suppressed the PCP GOF phenotype of sev-Fz1, but not sev-Dsh or sev-Fmi, suggesting that Baz acts specifically on Fz1 as a positive regulator. Finally, sev-Gal4-driven overexpression of Baz (sev>Baz) induces eye PCP defects, with misrotated and symmetric clusters where the R3 and R4 fates have not been resolved. This effect of Baz is unlikely to be a direct effect on Patj localization, since Baz overexpression (sev>Baz or GMR>Baz; GMR-Gal4 drives expression in all cells of the eye disc posterior to MF) does not change the Patj expression pattern (Djiane, 2005).
Taken together, these results suggest that Baz-aPKC-Patj and Fz1 form a complex before the MF, but during PCP establishment, Baz localizes in a complementary pattern to Patj/aPKC and antagonizes the negative regulation of Fz1 by aPKC (Djiane, 2005).
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