atypical protein kinase C: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - atypical protein kinase C

Synonyms - CG10261

Cytological map position - 51D6--8

Function - signaling

Keywords - asymmetric cell division

Symbol - aPKC

FlyBase ID: FBgn0261854

Genetic map position -

Classification - atypical protein kinase C

Cellular location - cytoplasmic

NCBI links: | Entrez Gene
Recent literature
Hosono, C., Matsuda, R., Adryan, B. and Samakovlis, C. (2015). Transient junction anisotropies orient annular cell polarization in the Drosophila airway tubes. Nat Cell Biol 17: 1569-1576. PubMed ID: 26551273
Tubular organs exhibit a striking orientation of landmarks according to the physical anisotropy of the 3D shape, in addition to planar cell polarization. However, the influence of 3D tissue topography on the constituting cells remains underexplored. This study identified a regulatory network polarizing cellular biochemistry according to the physical anisotropy of the 3D tube geometry (tube cell polarization) by a genome-wide, tissue-specific RNAi screen. During Drosophila airway remodelling, each apical cellular junction is equipotent to establish perpendicular actomyosin cables, irrespective of the longitudinal or transverse tube axis. A dynamic transverse enrichment of atypical protein kinase C (aPKC) shifts the balance and transiently targets activated small GTPase RhoA, myosin phosphorylation and Rab11 vesicle trafficking to longitudinal junctions. It is proposed that the PAR complex translates tube physical anisotropy into longitudinal junctional anisotropy, where cell-cell communication aligns the contractile cytoskeleton of neighbouring cells.

Bailey, M. J. and Prehoda, K. E. (2015). Establishment of par-polarized cortical domains via phosphoregulated membrane motifs. Dev Cell 35: 199-210. PubMed ID: 26481050
The Par polarity complex creates mutually exclusive cortical domains in diverse animal cells. Activity of the atypical protein kinase C (aPKC) is a key output of the Par complex as phosphorylation removes substrates from the Par domain. This study investigate how diverse, apparently unrelated Par substrates couple phosphorylation to cortical displacement. Each protein contains a basic and hydrophobic (BH) motif that interacts directly with phospholipids and also overlaps with aPKC phosphorylation sites. Phosphorylation alters the electrostatic character of the sequence, inhibiting interaction with phospholipids and the cell cortex. Overlapping BH and aPKC phosphorylation site motifs (i.e., putative phosphoregulated BH motifs) were sought in several animal proteomes. Candidate proteins with strong PRBH signals associated with the cell cortex but were displaced into the cytoplasm by aPKC. These findings demonstrate a potentially general mechanism for exclusion of proteins from the Par cortical domain in polarized cells.
Soriano, E. V., Ivanova, M. E., Fletcher, G., Riou, P., Knowles, P. P., Barnouin, K., Purkiss, A., Kostelecky, B., Saiu, P., Linch, M., Elbediwy, A., Kjaer, S., O'Reilly, N., Snijders, A. P., Parker, P. J., Thompson, B. J. and McDonald, N. Q. (2016). aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization. Dev Cell 38: 384-398. PubMed ID: 27554858
Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). This study shows that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/alphaB/alphaC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. These results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.
Hannaford, M. R., Ramat, A., Loyer, N. and Januschke, J. (2018). aPKC-mediated displacement and actomyosin-mediated retention polarize Miranda in Drosophila neuroblasts. Elife 7. PubMed ID: 29364113
Cell fate assignment in the nervous system of vertebrates and invertebrates often hinges on the unequal distribution of molecules during progenitor cell division. This study addresses asymmetric fate determinant localization in the developing Drosophila nervous system, specifically the control of the polarized distribution of the cell fate adapter protein Miranda. A step-wise polarization of Miranda occurs in larval neuroblasts, and it was found that Miranda's dynamics and cortical association are differently regulated between interphase and mitosis. In interphase, Miranda binds to the plasma membrane. Then, before nuclear envelope breakdown, Miranda is phosphorylated by aPKC and displaced into the cytoplasm. This clearance is necessary for the subsequent establishment of asymmetric Miranda localization. After nuclear envelope breakdown, actomyosin activity is required to maintain Miranda asymmetry. Therefore, phosphorylation by aPKC and differential binding to the actomyosin network are required at distinct phases of the cell cycle to polarize fate determinant localization in neuroblasts.
Hannaford, M., Loyer, N., Tonelli, F., Zoltner, M. and Januschke, J. (2019). A chemical-genetics approach to study the role of atypical protein kinase C in Drosophila. Development. PubMed ID: 30635282
Studying the function of proteins using genetics in cycling cells is complicated by the fact that there is often a delay between gene inactivation and the timepoint of phenotypic analysis. This is particularly true when studying kinases, that have pleiotropic functions and multiple substrates. Drosophila neuroblasts are rapidly dividing stem cells and an important model system to study cell polarity. Mutations in multiple kinases cause neuroblast polarity defects, but their precise functions at particular time points in the cell cycle are unknown. This study used chemical genetics and reports the generation of an analogue-sensitive (as) allele of Drosophila atypical protein kinase C (aPKC). The resulting mutant aPKC kinase can be specifically inhibited in vitro and in vivo. Acute inhibition of aPKC during neuroblast polarity establishment abolishes asymmetric localization of Miranda while its inhibition during NB polarity maintenance does not in the time frame of normal mitosis. However, aPKC contributes to sharpen the pattern of Miranda, by keeping it off the apical and lateral cortex after nuclear envelope breakdown.
Durney, C. H., Harris, T. J. C. and Feng, J. J. (2018). Dynamics of PAR proteins explain the oscillation and ratcheting mechanisms in dorsal closure. Biophys J 115(11): 2230-2241. PubMed ID: 30446158
This study presents a vertex-based model for Drosophila dorsal closure that predicts the mechanics of cell oscillation and contraction from the dynamics of the PAR proteins. Based on experimental observations of how aPKC, Par-6, and Bazooka translocate from the circumference of the apical surface to the medial domain, and how they interact with each other and ultimately regulate the apicomedial actomyosin, a system of differential equations was formulated that captures the key features of dorsal closure, including distinctive behaviors in its early, slow, and fast phases. The oscillation in cell area in the early phase of dorsal closure results from an intracellular negative feedback loop that involves myosin, an actomyosin regulator, aPKC, and Bazooka. In the slow phase, gradual sequestration of apicomedial aPKC by Bazooka clusters causes incomplete disassembly of the actomyosin network over each cycle of oscillation, thus producing a so-called ratchet. The fast phase of rapid cell and tissue contraction arises when medial myosin, no longer antagonized by aPKC, builds up in time and produces sustained contraction. Thus, a minimal set of rules governing the dynamics of the PAR proteins, extracted from experimental observations, can account for all major mechanical outcomes of dorsal closure, including the transitions between its three distinct phases.
Xu, C., Tang, H. W., Hung, R. J., Hu, Y., Ni, X., Housden, B. E. and Perrimon, N. (2019). The septate junction protein Tsp2A restricts intestinal stem cell activity via endocytic regulation of aPKC and Hippo signaling. Cell Rep 26(3): 670-688.e676. PubMed ID: 30650359
Hippo signaling and the activity of its transcriptional coactivator, Yorkie (Yki), are conserved and crucial regulators of tissue homeostasis. In the Drosophila midgut, after tissue damage, Yki activity increases to stimulate stem cell proliferation, but how Yki activity is turned off once the tissue is repaired is unknown. From an RNAi screen, the septate junction (SJ) protein tetraspanin 2A (Tsp2A) was identified as a tumor suppressor. Tsp2A undergoes internalization to facilitate the endocytic degradation of atypical protein kinase C (aPKC), a negative regulator of Hippo signaling. In the Drosophila midgut epithelium, adherens junctions (AJs) and SJs are prominent in intestinal stem cells or enteroblasts (ISCs or EBs) and enterocytes (ECs), respectively. When ISCs differentiate toward ECs, Tsp2A is produced, participates in SJ assembly, and turns off aPKC and Yki-JAK-Stat activity. Altogether, this study uncovers a mechanism allowing the midgut to restore Hippo signaling and restrict proliferation once tissue repair is accomplished.
Dong, W., Lu, J., Zhang, X., Wu, Y., Lettieri, K., Hammond, G. R. and Hong, Y. (2020). A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity. J Cell Biol 219(7). PubMed ID: 32580209
Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. This study shows that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. The data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.
Zelhof, A. C., Mahato, S., Liang, X., Rylee, J., Bergh, E., Feder, L. E., Larsen, M. E., Britt, S. G. and Friedrich, M. (2020). The brachyceran de novo gene PIP82, a phosphorylation target of aPKC, is essential for proper formation and maintenance of the rhabdomeric photoreceptor apical domain in Drosophila. PLoS Genet 16(6): e1008890. PubMed ID: 32579558
The Drosophila apical photoreceptor membrane is defined by the presence of two distinct morphological regions, the microvilli-based rhabdomere and the stalk membrane. The subdivision of the apical membrane contributes to the geometrical positioning and the stereotypical morphology of the rhabdomeres in compound eyes with open rhabdoms and neural superposition. This study describes the characterization of the photoreceptor specific protein PIP82. PIP82's subcellular localization demarcates the rhabdomeric portion of the apical membrane. It was further demonstrated that PIP82 is a phosphorylation target of aPKC. PIP82 localization is modulated by phosphorylation, and in vivo, the loss of the aPKC/Crumbs complex results in an expansion of the PIP82 localization domain. The absence of PIP82 in photoreceptors leads to misshapped rhabdomeres as a result of misdirected cellular trafficking of rhabdomere proteins. Comparative analyses reveal that PIP82 originated de novo in the lineage leading to brachyceran Diptera, which is also characterized by the transition from fused to open rhabdoms. Taken together, these findings define a novel factor that delineates and maintains a specific apical membrane domain, and offers new insights into the functional organization and evolutionary history of the Drosophila retina.

In Drosophila, the multi-PDZ domain protein Bazooka (Baz) is required for establishment of apico-basal polarity in epithelia and in neuroblasts, the stem cells of the central nervous system. In neuroblasts, Baz anchors Inscuteable in the apical cytocortex, which is essential for asymmetric localization of cell fate determinants and for proper orientation of the mitotic spindle. Baz directly binds to the Drosophila Atypical protein kinase C (aPKC) and both proteins are mutually dependent on each other for correct apical localization. Loss-of-function mutants of the Drosophila aPKC show loss of apico-basal polarity, multilayering of epithelia, mislocalization of Inscuteable and abnormal spindle orientation in neuroblasts. Together, these data provide strong evidence for the existence of an evolutionary conserved mechanism that controls apico-basal polarity in epithelia and neuronal stem cells. This study is the first functional analysis of an atypical protein kinase C isoform using a loss-of-function allele in a genetically tractable organism (Wodarz, 2000).

Double mutants lacking zygotic expression of the genes stardust (sdt) and bazooka (baz) fail to establish plasma membrane polarity after cellularization of the Drosophila embryo. This phenotype is characterized by expression of the basolateral marker Neurotactin (Nrt) on the whole cell surface and mislocalization of the zonula adherens (ZA) component Armadillo (Arm). Moreover, in sdt;baz double mutants, the monolayered organization of the blastoderm epithelium is lost and cells acquire irregular shapes. These morphological changes are reminiscent of those seen during epithelial-mesenchymal transitions. Essentially, the same phenotype as in sdt;baz double mutants is observed in baz mutants lacking maternal and zygotic Bazooka (Baz), whereas zygotic sdt and baz single mutants show a weaker phenotype later in development. These data suggest that baz is absolutely required for establishment of plasma membrane polarity and epithelial morphology, whereas the early function of sdt may be partially redundant with that of baz (Wodarz, 2000 and references therein).

baz is also required for establishment of apico-basal polarity and asymmetric division of neuroblasts in the developing central nervous system (CNS). Neuroblasts delaminate from the neuroectodermal epithelium and undergo several rounds of asymmetric cell division, generating a ganglion mother cell and another neuroblast in each division. Before division, the mitotic spindle rotates by 90° and localization of the cell fate determinants Prospero and Numb becomes restricted to the basal cortex of the neuroblast. These events are prerequisites for proper segregation of Prospero and Numb into the ganglion mother cell. From delamination to early anaphase, Baz is localized in the apical cortex of neuroblasts, where it forms a complex with Inscuteable, a protein required for rotation of the mitotic spindle and correct localization of Prospero and Numb. In the absence of Baz, asymmetric cortical localization of Insc is abolished, leading to randomized spindle orientation and mislocalization of cell fate determinants. These data have led to the conclusion that apico-basal polarity in neuroblasts depends on maintenance of apical Baz expression and is thus inherited from the neuroectodermal epithelium (Wodarz, 2000 and references therein).

baz encodes a protein with three PDZ domains that shows significant sequence similarity along its entire length to Par-3 (Caenorhabditis elegans) and ASIP (rat). In the early C. elegans embryo, Par-3 is asymmetrically localized in the anterior cortex of the zygote and the cortex of blastomeres that undergo asymmetric cell divisions. In these cells, Par-3 controls spindle orientation and asymmetric localization of cell fate determinants. Later on, Par-3 is also expressed in the apical cortex of the embryonic gut epithelium. Par-3 binds to PKC-3, an atypical protein kinase C (aPKC) isoform (Tabuse, 1998 and Wu, 1998). Both proteins are mutually dependent on each other for correct cortical localization. Moreover, embryos depleted of PKC-3 by RNA interference show a very similar phenotype to par-3 mutant embryos (Tabuse, 1998). ASIP was isolated as a binding partner of the mammalian aPKC isoforms, PKClambda and PKCzeta (Izumi, 1998). Intriguingly, ASIP and PKClambda colocalize at the tight junction (TJ) in vertebrate epithelial cells (Izumi, 1998). The TJ is considered to be the boundary between apical and basolateral plasma membrane domains, and TJs create a paracellular seal that prevents the free diffusion of macromolecules in the extracellular space between cells. These observations suggest that the association of ASIP/Par-3 with aPKCs and their roles in cell polarity are functionally important and evolutionarily conserved (Wodarz, 2000 and references therein).

aPKC from Drosophila shows very high sequence similarity to PKClambda and PKCzeta from vertebrates and PKC-3 from C. elegans. Drosophila aPKC and Baz coimmunoprecipitate and directly bind to each other in a yeast two-hybrid assay. In embryos, both proteins colocalize in the apical cortex of almost all epithelial tissues and in neuroblasts. Apical localization of DaPKC in epithelia and neuroblasts is abolished in baz mutants, and vice versa: Baz is delocalized in DaPKC mutants. The phenotype of aPKC loss-of-function mutants resembles that of baz mutants, consistent with a close functional interdependence of both proteins. Together, these data provide in vivo evidence for an essential role of an atypical protein kinase C isoform in establishment and maintenance of epithelial and neuronal polarity (Wodarz, 2000).

To test whether aPKC and Baz colocalize, double-label immunofluorescence stainings of embryos was performed. aPKC and Baz are clearly colocalized in the epidermis and in neuroblasts. To determine the precise subcellular localization of aPKC and Baz with respect to the ZA, double-label immunofluorescence stainings were performed with antibodies against Arm, a component of the ZA and Baz. The merged image shows that Baz is localized apically to Arm. The same is true for aPKC. At the resolution of the confocal microscope, the possibility that the localization of Baz and DaPKC partially overlaps with Arm in the ZA cannot be ruled out (Wodarz, 2000).

Binding studies showing a physical association of aPKC and Baz, and colocalization of these two proteins suggests that they may functionally interact with each other. In stainings of baz mutant embryos derived from germ line clones (baz null embryos) with anti-aPKC antibody, apical localization of aPKC could not be detected in epithelia and neither could apical localization be detected in neuroblasts. Instead, aPKC was distributed in a diffuse fashion in the cytoplasm. baz null embryos also show a loss of membrane polarity that is evident by mislocalization of the basolateral transmembrane protein Nrt. In contrast to wild type, Nrt is not excluded from the apical plasma membrane. Moreover, the monolayered structure of the epidermis is lost and cells pile up on top of each other, as has been described before for sdt;baz double mutants (Wodarz, 2000).

To test whether mislocalization of Baz is sufficient to induce mislocalization of aPKC, Baz was overexpressed by means of the GAL4 system. Under these conditions, Baz is not confined to the apical cytocortex anymore and is found in more lateral and basal positions in epithelia and neuroblasts. Concomitantly, aPKC is also mislocalized and colocalized in ectopic positions with ectopic Baz, confirming that ectopic Baz can recruit aPKC to ectopic sites in the cytocortex (Wodarz, 2000).

It has been shown before that Baz is required for apical localization of Insc in neuroblasts and that Insc is required for stabilization of Baz in neuroblasts after delamination. A test was performed to see whether Baz and Insc are also required for localization of aPKC in neuroblasts. aPKC localization is indistinguishable from wild type in neuroblasts of inscP49/CyO heterozygous embryos, but is neither cortical nor apical in neuroblasts of inscP49 homozygous mutant embryos. In embryos lacking maternal Baz but carrying a paternal wild-type allele of baz (partial paternal rescue), asymmetric cortical localization of aPKC is detected in most neuroblasts at metaphase. However, aPKC crescents and metaphase plates are often misoriented with respect to the surface of the embryo, a phenotype that has also been observed at low penetrance in embryos lacking only zygotic expression of Baz. In embryos lacking both maternal and zygotic expression of Baz (baz null), aPKC is completely delocalized in neuroblasts and epithelial tissues. These results indicate that Baz is absolutely required for apical localization of aPKC in neuroblasts and epithelial tissues, while Insc is required for localization of aPKC only in neuroblasts. Baz levels are strongly reduced in neuroblasts of insc mutant embryos, most likely because Insc is required for stabilization of Baz. Thus, the effect of Insc on DaPKC localization is probably indirect and can be explained by the loss of Baz in insc mutant neuroblasts (Wodarz, 2000).

To investigate the role of aPKC in the control of epithelial organization and polarity, DaPKCk06403 mutant embryos were stained with antibodies against Baz, Nrt, and Arm, the Drosophila ß-catenin homolog. Most homozygous DaPKCk06403 embryos from heterozygous mothers arrest very early in development and die before or during cellularization. Those that develop further show dramatic defects in epithelial organization and polarity. The blastoderm epithelium of these embryos is multilayered; cell shapes are extremely irregular and apico-basal polarity of the epithelium is lost. Instead of being localized to the apical cortex, Baz is found in randomly scattered aggregates. The basolateral marker Nrt is abnormally localized on the whole cell surface in most cells (Wodarz, 2000).

A significant fraction of embryos derived from DaPKCk06403/CyO heterozygous mothers that possess at least one zygotic wild-type allele of aPKC show characteristic defects in the head region. While epithelial structure and distribution of Baz and Nrt is normal in the trunk region of these embryos, the epithelium at the anterior tip of the embryos is multilayered, and shows a delocalized distribution of Baz and expression of Nrt on the whole cell surface. Thus, the defects observed in the head region of these embryos are very similar to the defects observed in the whole blastoderm epithelium of homozygous DaPKCk06403 embryos from heterozygous mothers. Most likely, these defects reflect an early requirement for aPKC before the onset of zygotic transcription and are caused by insufficient maternal supply of aPKC. Consistent with this interpretation, homozygous DaPKCk06403 embryos with the wild-type maternal contribution of DaPKC develop further than homozygous mutant embryos derived from heterozygous mothers and do not show obvious defects before germ band extension. At this stage, patches devoid of apical Baz and Arm staining appear, especially in the ventral neuroectoderm and in the head. Optical cross sections of these regions reveal defects in epithelial organization and polarity (Wodarz, 2000).

To study the effect of aPKC loss-of-function on asymmetric division of neuroblasts in the embryonic CNS, DaPKCk06403 mutant embryos that received the full maternal dosage of DaPKC were stained with antibodies against Baz and Insc. In most metaphase neuroblasts of these embryos, Baz is not detectable and Insc staining is diffuse, instead of forming a tight apical crescent. In addition, the orientation of metaphase plates often deviates from the normal orientation parallel to the surface of the embryo, reflecting abnormal orientation of the mitotic spindle (Wodarz, 2000).

These findings are reminiscent of the situation in the early C. elegans embryo, where PKC-3, Par-3 and another PDZ domain protein, Par-6 (see Drosophila par-6), are mutually dependent on each other for correct localization in the anterior cytocortex (Watts, 1996; Tabuse, 1998; Hung, 1999). Consistent with these results, the phenotype of embryos depleted of PKC-3 by RNA interference is very similar to the phenotype of par-3 and par-6 mutants (Etemad-Moghadam, 1995; Watts, 1996; Tabuse, 1998; Hung, 1999. Interestingly, a Drosophila homologue of par-6 does exist (Tabuse, 1998), raising the possibility that the interaction of Par-3/Baz, PKC-3/DaPKC and Par-6 has been evolutionarily conserved (Wodarz, 2000).

Another example for a close functional interaction between a PDZ domain protein and protein kinase C has recently been uncovered in Drosophila. The multi-PDZ domain protein InaD binds to the eye-specific, conventional isoform of PKC and is required for its proper localization in photoreceptors. InaD contains five PDZ domains and distinct binding partners have been identified for each of them. Intriguingly, all of the proteins that bind to InaD are part of the phototransduction cascade in the Drosophila eye. Thus, it has been proposed that InaD provides a scaffold for the assembly of a signaling complex, a so called 'transducisome' (Wodarz, 2000 and references therein).

In the case of aPKC and Baz, the situation is more complicated. Consistent with a function as a scaffold, Baz is required for localization of the signaling protein aPKC. However, Baz itself is not properly localized in the absence of aPKC. It is easy to imagine how a structural multi-PDZ domain protein like InaD or Baz can localize a protein kinase, but how can aPKC be responsible for localization and stabilization of Baz? Baz possesses a PKC consensus phosphorylation site that is conserved between Baz, Par-3, and ASIP. Phosphorylation of this site by aPKC could be important to regulate binding of Baz to other proteins or to protect Baz from proteolytic degradation. It is also possible that aPKC binds simultaneously to Baz and another protein that may be required for localization of Baz. A detailed structure-function analysis of both Baz and aPKC will be necessary to clarify this issue (Wodarz, 2000).

Analysis of the aPKC loss-of-function phenotype reveals that aPKC is already required very early during embryogenesis, before the onset of zygotic transcription. Most homozygous DaPKCk06403 embryos with a reduced maternal dosage of DaPKC die before cellularization is completed. What could be the reason for this early death? aPKCs have been implicated in the control of apoptotic cell death in vertebrate tissue culture cells. Inhibition of aPKCs induces apoptosis. Treatment of cells with UV irradiation also triggers apoptosis and rapidly inhibits aPKC kinase activity, suggesting that inhibition of aPKCs is an early event in the apoptotic signaling cascade. In accordance with these data, aPKCs have been implicated in the transduction of survival signals downstream of growth factor receptors. In contrast to conventional and novel PKC isoforms, aPKCs can be activated by phosphatidylinositol(3,4,5)trisphosphate and ceramide, two second messengers that are generated in response to inflammatory cytokines and growth factors. The observation that aPKC mutant embryos show premature cell death and strongly increased TUNEL labeling, which is a hallmark of apoptosis, is consistent with a function of aPKC in the transmission of survival signals (Wodarz, 2000).

The loss-of-function phenotype of aPKC mutants in epithelia is very similar to the phenotypes described for baz null mutants and zygotic sdt, baz double mutants. The most striking abnormalities in these mutants are loss of the monolayered epithelial organization, irregular cell shapes, and loss of plasma membrane polarity. Multilayering of epithelia and abnormal cell shapes are most likely caused by defects in cell adhesion. Indeed, formation of the zona adherens (ZA), a region of intense, cadherin-mediated cell contact, is defective in aPKC, baz, and sdt mutants. Another gene, crumbs, is also required for correct positioning and maintenance of the ZA. aPKC, Baz, and Crb are all localized apically to the ZA, so how can they control formation of the ZA? This complex could be involved in the formation of a protein scaffold in the apical cytocortex that prevents ZA components from moving further apically. A similar function can be envisioned for Baz, since it is also a multi-PDZ domain protein with the capacity to interact with several partners at the same time (Wodarz, 2000 and references therein).

How does aPKC fit into this model? aPKC is required for localization and stabilization of Baz, but this may not be its only function in ZA formation. Several reports show that PKCs are involved in the assembly of adherens junctions and TJs. The majority of these studies used cultured cell lines and analyzed the effects of different inhibitors and agonists of PKCs on localization and phosphorylation of junctional proteins, cell adhesion, and cell morphology. Although these studies provided compelling evidence for an involvement of PKCs in junction formation, in most cases neither the specific PKC isoforms responsible for the observed phenotypes nor the targets of these PKCs have been unambiguously identified. In one interesting study, inhibition of aPKCs induced epithelial-mesenchymal transformation in quail neural tube explants, while inhibitors of conventional or novel PKCs had little or no effect in this assay (Minichiello, 1999; Wodarz, 2000 and references therein).

In addition to their effects on epithelial organization and cell shape, mutations in aPKC, baz, sdt, and crb also affect plasma membrane polarity. Establishment and maintenance of plasma membrane polarity requires the separation of apical and basolateral membrane domains by a diffusion barrier in the plane of the membrane. In vertebrate epithelia, this diffusion barrier is created by the TJ. In arthropod epithelia, TJs have not been found by ultrastructural analysis. It is noted, however, that the vertebrate homologs of aPKC and Baz, PKClambda, PKCzeta, and ASIP, are localized at the TJ in epithelial cells (Izumi, 1998). Moreover, aPKC and Baz are localized apically to the ZA in Drosophila epithelia, which corresponds to the position of the TJ in vertebrate epithelia. Thus, based on their localization and their mutant phenotypes, it is proposed that aPKC and Baz are components of an evolutionarily conserved protein complex that may serve similar functions as the TJ in vertebrates (Wodarz, 2000).

Neuroblasts do not possess elaborate cell junctions but clearly show cortical and, at least to some extent, plasma membrane polarity. aPKC and Baz are required for anchoring Insc in the apical neuroblast cortex and it is conceivable that aPKC and Baz may also be involved in the formation of a submembraneous protein scaffold analogous to the model proposed for epithelia. Consistent with this idea is the finding that Nrt staining is reduced precisely in those regions of the neuroblast plasma membrane where aPKC and Baz are localized beneath the membrane. Thus, aPKC and Baz may be generally responsible for the separation of membrane domains by preventing diffusion of basolateral proteins into the apical domain (Wodarz, 2000).

From the available data, it is impossible to decide whether the primary function of aPKC in neuroblasts is the stabilization of Baz or whether aPKC phosphorylates additional targets involved in asymmetric division of neuroblasts. One candidate for phosphorylation by aPKC is Miranda, an adaptor protein with six consensus PKC phosphorylation sites that binds to Prospero and Insc. Miranda colocalizes with Insc only briefly in late interphase, and then moves together with Prospero to the basal cortex of the neuroblast during prophase. It is an attractive possibility that phosphorylation of Miranda by aPKC regulates binding of Miranda to Insc and its release from the apical complex later in the cell cycle (Wodarz, 2000).

In conclusion, it has been shown that aPKC is an essential binding partner of Baz in epithelia and neuroblasts. Surprisingly, Baz does not simply function as a scaffold to anchor aPKC in the apical cytocortex, but is itself dependent on aPKC for proper localization and stability. This mutual dependence is indicative of an intimate cross-talk between structural proteins like Baz and the signaling protein aPKC. The link between signal transduction components and structural components of the cytocortex may be important to allow rapid rearrangement of cellular junctions and cell shape changes such as those occurring during delamination of neuroblasts. To fully understand the role of aPKC in the generation of cellular asymmetry, it will be essential to identify the physiological activators, inhibitors, and downstream targets of this important protein kinase (Wodarz, 2000).

aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb

In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. This study identified in vivo phosphorylation sites of mammalian Numb, and showed that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions (Smith, 2007).

To establish whether aPKC-dependent phosphorylation is a conserved mechanism for regulating the cortical membrane localization of Numb, whether a myc-tagged version of Drosophila Numb forms a complex with PKCzeta in HEK293 cells was examined. Co-immunoprecipitation of Drosophila Numb with PKCzeta indicates that this interaction is conserved. The sequence of Drosophila Numb was examined. A total of five evolutionarily conserved aPKC phosphorylation sites were revealed including Ser52 and Ser304, corresponding to Ser7 and Ser295 in murine Numb (isoform p66). Whether PKC could phosphorylate Drosophila Numb was examined in an in vitro kinase assay. Both PKCα and PKCzeta, the human orthologue of Drosophila aPKC, phosphorylated Numb in an immune-complex assay. PKCzeta also phosphorylated a GST-Numb fusion protein. Mutations of all five of the conserved aPKC sites (Numb5A) reduced the in vitro phosphorylation by PKCzeta, indicating that some of these sites are the targets of PKCzeta. A form of Numb in which Ser52 is left intact, while the other four serines were mutated to alanine (Numb4A), was still efficiently phosphorylated by PKCzeta indicating that Ser52 is one of the acceptor sites in vitro. However, mutation of Ser52 into alanine did not significantly reduce the in vitro phosphorylation of GST-Numb, suggesting that PKCzeta phosphorylates additional sites. NanoLC-MS-MS analyses of the in vitro phosphorylated GST-Numb identified a total of eight aPKC sites. Confirmation of the phosphorylated Ser52 residue was obtained from the MS-MS spectrum of m/z 497.7. Five PKCzeta phosphorylation sites that do not appear conserved were identified in this analysis (Ser31, Ser35, Ser48, Ser161, and Ser297). These sites likely account for the residual phosphorylation of GST-Numb5A. Although these analyses provide direct identification of aPKC phosphorylated residues, other potential phosphorylation sites remained elusive. For example, the early eluting tryptic peptide QMS304LR was observed only in the control sample. Its absence in the aPKC-treated sample strongly suggests that Ser304 is in fact phosphorylated and could not be detected owing to nonretention of this hydrophilic peptide during reverse phase LC. It is concluded that aPKC phosphorylates Numb at several sites in both Drosophila and mouse, including at the conserved Ser7 and Ser295 sites (Ser52 and Ser304 in Drosophila Numb) (Smith, 2007).

The localization was examined of Drosophila Numb in dividing sensory organ precursor pI cells. The pI cells divide asymmetrically within the plane of the notum epithelium and along the body axis. In these cells, Numb localizes at the anterior cortex, opposite to aPKC, which relocalizes from the apical cortex to the lateral posterior cortex upon mitosis. The possible role of phosphorylation in the regulation of Drosophila Numb localization was examined by studying the distribution of Myc-tagged versions of Numb, Numb4A, NumbS52A, and Numb5A that were expressed in pI cells using the neurPGAL4 driver. Importantly, overexpression of Numb4A, NumbS52A, or Numb5A in pI cells led to cell-fate transformation in the bristle lineage indicative of gain of Numb function. This indicates that these Numb mutant proteins are functional. Similar to endogenous Numb, Myc-Numb localized at the anterior cortex, opposite to aPKC, in all cells at prometaphase and metaphase. Consistently, myc-Numb colocalized with Pins. In contrast, the crescent formed by Numb5A appeared to extend posteriorly in 91% of the dividing pI cells at prometaphase. Interestingly, a recent study has shown that a mutant Numb protein, NumbS52F, fails to localize properly in dividing pI cells. Thus, one possible interpretation of the data is that the mislocalization of Numb5A is due to the S52A mutation. Therefore the localization of NumbS52A was studied and it was found to localize asymmetrically in 84% of the pI cells at prometaphase. Thus, the S52A mutation alone cannot be responsible for the defective localization of Numb5A. Additionally, mutations of the four other serine residues in Numb4A did not significantly change the asymmetric distribution of Numb. Therefore, it is concluded that the defects in Numb5A distribution in dividing pI cells depends on the combination of at least two mutations, S52A and a mutation in one of the four conserved aPKC consensus sites, possibly Ser304. Thus, these data support the notion that aPKC-mediated phosphorylation of Drosophila Numb contributes to the asymmetric distribution of Numb in dividing pI cells (Smith, 2007).

This study provides evidence for a conserved mechanism regulating the asymmetric distribution of the cell-fate determinant Numb. Mammalian and Drosophila Numb proteins are substrates for aPKC and phosphorylation regulates Numb localization at the cortical membrane. The data also indicate that aPKC-dependent phosphorylation regulates the polarized distribution of Numb in mammalian epithelial cells and Drosophila sensory organ precursor cells (Smith, 2007).

The aPKC/Par3/Par6 complex plays a conserved role in establishing polarity in a variety of cellular contexts, including during asymmetric cell divisions in C. elegans and Drosophila, and in apical-basal polarity of epithelial tissues. In mammalian epithelial cells, aPKC is required for the establishment and maintenance of apical-basal polarity. In this context, several targets of aPKC have been identified, including the conserved proteins, Lgl and Par1, whose activities also contribute to cell polarity. In mammalian cells, Lgl plays a role in adherens junction disassembly and phosphorylation of Lgl by aPKC restricts its localization to the lateral cell membrane. Similarily, aPKC-dependent phosphorylation of Par1 restricts its localization to the basolateral membrane of polarized MDCK cells. These data indicate that Numb is also a downstream target of aPKC in polarized cells, and that phosphorylation at Ser7 and 295 mediates exclusion from the apical domain and accumulation at the lateral domain (Smith, 2007).

A role for mammalian Numb in receptor endocytosis and recycling has been established. The current findings suggest that in polarized epithelial cells the trafficking function of Numb may be restricted to the basolateral membrane by aPKC-dependent phosphorylation. Thus, Numb may serve as a link between the Par/aPKC polarity complex and the endocytic machinery, and function to regulate the trafficking of membrane proteins at the basolateral membrane. In agreement with such a model, Numb has previously been implicated in the polarized endocytosis of the neuronal cell adhesion molecule L1. Although the relevant membrane targets of Numb in epithelial cells are currently unknown, components of the Notch pathway are attractive candidates; Numb antagonizes Notch receptor signaling pathway in both Drosophila and in mammalian cells (Smith, 2007).

In Drosophila, the Par complex has been shown to direct the asymmetric localization of Numb, Pon, and Miranda via the aPKC-mediated inhibitory phosphorylation of Lgl. However, Numb asymmetric localization could still be observed in 30% of lgl mutant pI cells, suggesting that additional mechanisms may exist to regulate the asymmetric localization of Numb. Thus, it is proposed that the aPKC-dependent phosphorylation of Numb may account for the observed Lgl-independent asymmetric localization of Numb. This proposal implicitly assumes that this Lgl-independent process is aPKC-dependent. To verify this assumption, clones of apkc mutant cells were generated. Unfortunately, large apkc mutant clones could not easily be recovered in the pupal notum, preventing studying of the distribution of Numb in apkc mutant pI cells. A mutation in one of the Numb sites shown to be phosphorylated by aPKC, Ser52, has been characterized. The mutant protein, NumbS52F, fails to localize asymmetrically in pI at mitosis. The defective localization of NumbS52F contrasts with the asymmetric localization of NumbS52A. One possible interpretation is that the S52F, but not the S52A, mutation alters the conformation of Numb such that it prevents the phosphorylation of other essential aPKC sites or inhibits the actin-dependent cortical localization of Numb that is mediated by the N-terminal region of Numb (Smith, 2007).

In addition to the aPKC-dependent regulation of Numb localization, the results raise the possibility that a hierarchy of phosphorylation sites may be responsible for controlling additional aspects of Numb localization and function. In addition to serines 7 and 295, seven additional in vivo phosphorylation sites were have identified on mammalian Numb. Several of these do not conform to PKC consensus sites yet are conserved in Drosophila. Ser276 has been described as a target of CaMK, and this site was identified in mass spectral analysis. Although the functional consequences of phosphorylation at this site were not addressed, the authors demonstrate that phosphorylation confers binding to 14-3-3 proteins suggesting this site has a regulatory role. In addition, the Drosophila Numb-associated kinase (NAK), which was isolated in a yeast two-hybrid screen as a Numb interactor (Chien, 1998), is highly related to mammalian adaptin-associated kinase (AAK), raising the possibility that members of this family of protein kinases might also phosphorylate Numb in a manner that regulates its association with α-adaptin or other endocytic proteins. Further functional analysis of Numb phosphorylation site mutants and identification of upstream kinases will yield insight into the conserved signaling pathways that regulate the localization and function of Numb and also will reveal areas of divergence (Smith, 2007).

aPKC is a key polarity molecule coordinating the function of three distinct cell polarities during collective migration

Apical-basal polarity is a hallmark of epithelia and it needs to be remodeled when epithelial cells undergo morphogenetic cell movements. This study used border cells in Drosophila ovary to address how the apical-basal polarity is remodeled and turned into front-back, apical-basal and inside-outside polarities, during collective migration. Crumbs (Crb) complex is required for the generation of the three distinct but inter-connected cell polarities of border cells. Specifically, Crb complex, together with Par complex and the endocytic recycling machinery, ensures a strict distribution control of two distinct populations of aPKC at the inside apical junction and near the outside lateral membrane respectively. Interestingly, aPKC distributed near the outside lateral membrane interacts with Tiam1/Sif and promotes the Rac-induced protrusions, whereas alteration of the aPKC distribution pattern changed protrusion formation pattern, leading to disruption of all three polarities. Therefore, this study demonstrates that aPKC, spatially controlled by Crb complex, is a key polarity molecule coordinating the generation of three distinct but inter-connected cell polarities during collective migration (Wang, 2018).

This study demonstrates that the Crb complex is required for the collective migration of border cells. Loss of function of Crb, Sdt or Patj each delayed border cell migration, which was likely to be a result of the combined effect of disrupting three distinct cell polarities. Most importantly, the front-back polarity of the border cell cluster was disrupted, as demonstrated by the ectopic formation of large actin-rich protrusions in border cells located at the side and back of the cluster. Furthermore, Patj RNAi or sdt RNAi caused border cell clusters to extend major protrusions at random angles relative to the apical-basal axis, unlike the wild-type clusters that restrict protrusion formation to the lateral region, thus extending the protrusions perpendicular to their inherent apical-basal axis. Such restriction of lateral protrusion formation would ensure that protrusions are parallel to the migration direction, resulting in efficient forward movement of the entire cluster. Mutation in crb or the expression of active forms of aPKC expanded the outside membrane area, and overexpressing Crb or reducing aPKC activity suppressed the outside membrane characteristics, causing disruption in inside-outside polarity for each border cell. Interestingly, crb mutant border cells sometimes exhibited ectopic actin patches (containing large aPKC spots) between the adjacent cells, where the inside membrane is normally located. Taken together, these results raise the following question: is there a common mechanism that is affected during the disruption of all three cell polarities? In other words, are these cell polarities interconnected and coordinated by the same mechanism? (Wang, 2018).

A common feature of loss of Crb complex components is that mislocalized aPKC generates ectopic Rac-dependent protrusions in border cells at the side and back of the cluster and at the apical and inside (junctional) region of individual border cells, leading to disruption of all three cell polarities. This indicates that there is a common mechanism involving aPKC that organizes all three polarities. First, the ectopic protrusions and the loss of these three polarities as a result of loss of Patj are likely to be mediated by the ectopically localized aPKC, since reduction of aPKC was able to rescue the ectopic protrusions. Interestingly, loss of other apical polarity proteins (Crb, Sdt, Par6, Cdc42) except for aPKC and Baz also led to similar phenotypes, including disrupted aPKC localization in the apical junctions, ectopic actin patches colocalized with large aPKC spots, and increased F-actin levels and Rac activity at or near the outside membrane. By contrast, loss of aPKC resulted in few protrusions and reduced F-actin levels at the outside membrane, while overactivation of aPKC led to increased F-actin levels and Rac activity, which are mediated by the downstream Sif. These results suggest that an important role of the Crb and Par complexes is to sequester most of the aPKC in the apical junction, leaving only a moderate level near the outside membrane to promote protrusion formation. The major pool of aPKC at the apical junction (together with Crb and Par complex components) is likely to function similarly to its classical role in epithelial cells, which is to promote apical polarity and integrity of apical and subapical junctions. However, the minor aPKC pool near the outside lateral membrane might function differently in that it can activate Sif to increase Rac-mediated actin dynamics. Such a difference might arise if complexes at the apical junction restrict or inhibit the Sif-promoting activity of aPKC. Conceivably, such inhibition would not apply to aPKC near the outside lateral membrane (Wang, 2018).

A crucial function of the Crb complex and Par complex is to produce a high level of membrane-bound aPKC at the inside apical junction and a moderate level of cytoplasmic aPKC near the outside lateral membrane so that the three distinct, but related, cell polarities can be properly established. Furthermore, polarized endocytic recycling of vesicles associated with aPKC and other apical polarity molecules ensures the polarized distribution of two aPKC pools within each border cell. Finally, it is interesting to note that the front-polarized recycling and exocytosis within the wild-type cluster, as mediated by PVF-PVR guidance signaling, could cause aPKC to be much more enriched at the outside membrane of the leading edge (to promote leading protrusion) than at the outside membrane at the side and back (to promote minor side protrusions) of the border cell cluster. When cells migrate collectively under developmental, physiological and pathological contexts, the migrating sheets or clusters of cells often display part-epithelial and part-mesenchymal characteristics. It will be interesting to determine whether aPKC together with Crb and Par complexes and the endocytic recycling machinery also play conserved roles in coordinating these three cell polarities in other types of collective migration (Wang, 2018).

Mutations in ANKLE2, a ZIKA virus target, disrupt an asymmetric cell division pathway in Drosophila neuroblasts to cause microcephaly

The apical Par complex, which contains atypical protein kinase C (aPKC), Bazooka (Par-3), and Par-6, is required for establishing polarity during asymmetric division of neuroblasts in Drosophila, and its activity depends on L(2)gl. This study shows that loss of Ankle2, a protein associated with microcephaly in humans and known to interact with Zika protein NS4A, reduces brain volume in flies and impacts the function of the Par complex. Reducing Ankle2 levels disrupts endoplasmic reticulum (ER) and nuclear envelope morphology, releasing the kinase Ballchen-VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par-complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen or l(2)gl suppresses Ankle2 mutant phenotypes and restores viability and brain size. Human mutational studies implicate the above-mentioned genes in microcephaly and motor neuron disease. It is suggested that NS4A, ANKLE2, VRK1, and LLGL1 define a pathway impinging on asymmetric determinants of neural stem cell division (Link, 2019).

Proper development of the human brain requires an exquisitely coordinated series of steps and is disrupted in disorders associated with congenital microcephaly. Congenital microcephaly in humans is characterized by reduced brain size (using occipital frontal circumference [OFC] as a surrogate measure) more than two standard deviations below the mean (Z score < -2) at birth. It is associated with neurodevelopmental disorders such as developmental delay and intellectual disability and can be caused by external exposures to toxins, in utero infections, or gene mutations. Pathogenic gene variants for microcephaly have been identified through targeted genetic testing, genomic copy number studies, and exome sequencing (ES), identifying 18 primary microcephaly loci. Many syndromes significantly overlap with classic microcephaly phenotypes, and together, these disorders can be caused by defects in a wide variety of biological processes, including centriole biogenesis, DNA replication, DNA repair, cell cycle and cytokinesis, genome stability, and multiple cell signaling pathways. In flies, microcephalic phenotypes are referred to when the third instar brain lobes are reduced in size or when adult flies exhibit small heads relative to the their body size. As in humans, microcephaly in flies can be a result of mutations that affect cell division and centrosome biology as demonstrated with mutations in WDR62 and ASPM or ASP and neuroblast (NB) proliferation (Link, 2019).

A forward, mosaic screen for neurodevelopmental and neurodegenerative phenotypes associated with lethal mutations on the X chromosome in Drosophila identified 165 loci, many with corresponding human genetic disease trait phenotypes. Among them, a mutation in Ankryin repeat and LEM domain containing 2 (Ankle2) causes loss of peripheral nervous system (PNS) organs in adult mutant clones and severely reduced brain size in hemizygous third instar larvae. To identify patients with pathogenic variants in ANKLE2, the exome database of the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) was surveyed; compound heterozygous mutations were identified in ANKLE2 in two siblings. Both infants exhibited severe microcephaly, and the surviving patient displayed cognitive and neurological deficits alongside extensive intellectual and developmental disabilities. Mutations in Ankle2 led to cell loss of NBs and affected NB division in the developing third instar larval brain. Remarkably, expression of the wild-type human ANKLE2 in flies rescued the observed mutant phenotypes. This study explored the molecular pathways and proteins that are affected by Ankle2 loss (Link, 2019).

ANKLE2 belongs to a family of proteins containing LEM (LAP2, Emerin, and MAN1) domains that typically associate with the inner nuclear membrane. Conventional LEM proteins have been shown to interact with barrier to autointegration factor (BAF), which binds to both DNA and the nuclear lamina to organize nuclear and chromatin structure. However, the LEM domain in Drosophila and C. elegans Ankle2 is not obviously conserved. Studies in C. elegans indicate that a homolog of ANKLE2 regulates nuclear envelope morphology and functions in mitosis to promote reassembly of the nuclear envelope upon mitotic exit. During this process, ANKLE2 modulates the activities of Vaccina-Related Kinase 1 (VRK1) and protein phosphatase 2A (PP2A). However, all experiments in worms were performed at the embryonic two-cell stage and no other phenotypes were reported except early lethality. While mutations in ANKLE2 have been associated with severe microcephaly, human VRK1 pathogenic variant alleles can cause a neurological disease trait consisting of complex motor and sensory axonal neuropathy and microcephaly (Link, 2019).

Mutations in both Ankle2 and the fly homolog of VRK1, ballchen, cause a loss of NBs in 3rd instar larval brains in Drosophila. NBs divide asymmetrically and are often used as a model to investigate stem cell biology and asymmetric cell division. Most NBs in the larval central brain give rise to another NB and a smaller ganglion mother cell (GMC), which then divides once again to produce neurons or glia. Proper NB maintenance and regulation is essential for precise development of the adult nervous system, and misregulation of NB number or function can lead to defects in brain size (Link, 2019).

Congenital Zika virus infection in humans during pregnancy has been associated with severe microcephaly that can be as dramatic as certain genetic forms of microcephaly including phenotypes associated with biallelic mutations in MCPH16/ANKLE2. Recently, it has been showed that a Zika virus protein, NS4A, physically interacts with ANKLE2 in human cells. Expression of NS4A in larval brains causes microcephaly, induces apoptosis, and reduces proliferation. Importantly, expression of human ANKLE2 in flies that express NS4A suppresses the associated phenotypes, demonstrating that NS4A interacts with the ANKLE2 protein and inhibits its function . Interestingly, Zika virus crosses the blood brain barrier and targets radial glial cells, the neural progenitors in the vertebrate cortex (Link, 2019).

This study shows that Ankle2 is localized to the endoplasmic reticulum and nuclear envelope, similar to NS4A, and genetically interacts with ball-VRK1 to regulate brain size in flies. An allelic series at the ANKLE2 and VRK1 loci shows that perturbation of this pathway results in neurological disease including microcephaly. The data indicate that the Ankle2-Ball (VRK1) pathway is required for proper localization of asymmetric proteins and spindle alignment during NB cell division by affecting two proteins, atypical protein kinase C (aPKC) and L(2)gl, which play critical roles in the asymmetric segregation of cell fate determinants. In addition, NS4A expression in NBs mimics phenotypes seen in Ankle2 mutants, and NS4A induced microcephaly is suppressed by removing a single copy of ball or l(2)gl. Human genomics variant data and disease trait correlations extend this asymmetric cell division pathway from proteins identified in flies and reveal insights into neurological disease. In summary, NS4A hijacks the Ankle2-Ball (VRK1) pathway, which regulates progenitor stem cell asymmetric division during brain development and defines a human microcephaly pathway (Link, 2019).

This study reports six additional patients with microcephaly that carry mutations in ANKLE2 and shows that three variants identified in probands cause a loss of ANKLE2 function when tested in flies, providing compelling evidence that its loss causes reduced brain size in flies and severe microcephaly in humans. Ankle2 is a dosage-sensitive locus whose product is inhibited by the Zika virus protein NS4A. Ankle2, similar to NS4A, is localized to the ER and it targets the nuclear envelope during mitosis. Loss of Ankle2 affects the nuclear envelope and ER distribution and results in a redistribution of Ball or VRK1, a kinase that is normally localized to the nucleus except when the nuclear envelope breaks down during mitosis. Loss of Ankle2 disrupts the localization of NB apical-basal polarity determinants such as aPKC, Par-6, Baz, and Mir, and aPKC phosphorylation is reduced by Ankle2 mutations. Importantly, loss of one copy of ball or l(2)gl suppresses the reduced brain volume associated with a partial loss of Ankle2, suggesting that much of the biological function of Ankle2 is modulated by aPKC and L(2)gl. Finally, the negative influence of NS4A on the activity of ANKLE2 can also be suppressed by removal of one copy of ball or l(2)gl, suggesting the following pathway: NS4A -| ANKLE2 -| Ball-VRK1 -> L(2)gl-LLGL1 -| aPKC. This pathway, regulated by ANKLE2, plays an important role in NB stem cell divisions in flies and microcephaly and potentially other neurological disease phenotypes in humans (Link, 2019).

Interestingly, the above pathway links environmental cues with several genetic causes of sporadic and autosomal recessive microcephaly in humans; moreover, it implicates this pathway in microcephaly accompanying congenital infection. As one example of the latter, the Zika virus has been shown to cross the infant blood brain barrier and has been identified in radial glial cells as well as intermediate progenitor cells and neurons. It is proposed that NS4A affects the function of Ankle2 leading to the release of Ball-VRK1 from the nucleus. It is speculated that this in turn affects the phosphorylation of aPKC and L(2)gl directly by masking phosphorylation sites or indirectly by promoting the activity of one or more phosphatases. Loss of VRK1 has been shown to cause microcephaly and some variant alleles are also associated with pontocerebellar hypoplasia (PCH) in humans, consistent with the loss of ball in flies that causes a severe reduction in brain size. Note that ANKLE2, VRK1, LLGL1, and aPKC as well as other components of the apical complex such as PARD3 are all present in radial glial cells during cortical development. These data suggest that ANKLE2 and its partners such as LLGL1 and asymmetric determinants are important proteins during neural cell proliferation and that the proper levels and relative amounts of these proteins determine how many neurons will eventually be formed in vertebrates. These data also indicate that variant alleles at either ANKLE2 or VRK1 are responsible for some causes of embryonic lethality and severe congenital microcephaly (Link, 2019).

LLGL1 has recently been shown to play an important role in radial glia in mice during neurogenesis, and its loss in clones increases the number of divisions. In addition, aPKCζ or λ localizes at the apical membrane of proliferating neural stem cells in chicken embryos during division and has been shown to provide an instructive signal for apical assembly of adherens junctions. Mouse knockouts of aPKCλ and aPKCι are embryonic lethal; however, aPKCζ knockouts are viable, perhaps suggesting redundant functions within the aPKC family. These proteins have not been linked to microcephaly in mice, but conditional removal of an apical complex protein Pals1 in cortical progenitors resulted in complete cortex loss. Finally, Numb is asymmetrically localized by the Par complex protein in Drosophila, segregated to the daughter cell during asymmetric cell division, and essential for daughter cells to adopt distinct fates. In mice, Numb localization is also asymmetric and null mutations exhibit embryonic lethality, neural tube closure defects, and premature neuron development. These data indicate that asymmetric division may be important for vertebrate neuronal development, but microcephaly is not a phenotype that typically associates with loss of the mice homologs of asymmetric-localized determinants identified in Drosophila. However, the observations reported in this study indicate that the ANKLE2-PAR complex pathway is evolutionarily conserved from flies to humans, although the precise mechanisms remain to be determined as different cells may use this pathway in different contexts (Link, 2019).

In order to determine whether predicted deleterious biallelic variants in PAR-complex-encoding genes or their paralogs associated with a neurologic disease trait, The BHCMG database was searched for mutations associated with neurological disease. Homozygous predicted deleterious missense variants in were found PARD3B (c.1222G>A, p.G408S) in a patient that has microcephaly and compound heterozygous mutations in PARD3B (c.1654G>A, p.A552T) that are associated with other neurological defects. The human ortholog of L(2)gl, LLGL1, is deleted in Smith-Magenis syndrome (SMS), and 86%-89% of the SMS patients have brachycephaly. These observations extend the mutational load beyond ANKLE2 and VRK1 and suggest an association between congenital disease and variants within the PAR complex, potentially by a compound inheritance gene dosage model (Link, 2019).

The Aurora A (AurA) kinase has been shown to phosphorylate the Par complex as well as L(2)gl and regulates cortical polarity and spindle orientation in NBs. The aberrant localization of Ball-VRK1 in Ankle2 mutants may lead to gain-of-function phenotypes that are highly dosage sensitive, as they can be repressed by removing a single copy of Ball-VRK1 in Ankle2A. Mislocalized Ball-VRK1 may mask or interfere with the function of AurA in NB asymmetric division as they share similar kinase substrate consensus sequences. Future studies are needed to assess Ball-VRK1 redundancy or interference with AurA function (Link, 2019).

Another possible evolutionarily parallel with implications in multicellular organismal development is the genetic interaction between the C. elegans homolog of VRK1 and an ANKLE2-like protein at the two-cell stage. Whereas VRK1 in both Drosophila and humans is localized to the nucleus, except during mitosis when the nuclear envelope is broken down, the worm VRK1 protein is localized to the nuclear envelope. The worm ANKLE2-like protein, Lem-4L, also interacts with the phosphatase PP2A, and the fly PP2A regulates NB asymmetric division by interacting with aPKC and excluding it from the basal cortex. PP2A also antagonizes the phosphorylation of Baz by PAR-1 to control apical-basal polarity in dividing embryonic NBs and regulates Baz localization in other cells such as neurons. This raises the possibility that the Ankle2 pathway also acts with PP2A in NB asymmetric division (Link, 2019).

This study identified a pathway that plays a significant role in NB asymmetric division. By combining functional studies in Drosophila together with human subject data, this study has linked several microcephaly-associated genes and congenital infection to a single genetic pathway. These studies allowed the highlighting of conserved functions of the ANKLE2 pathway and provide mechanistic insight into how a Zika infection might affect asymmetric division. This ANKLE2-VRK1 gene dosage-sensitive pathway can be perturbed by genetic variants that disturb biological homeostasis resulting in neurological disease traits or by environmental insults such as Zika virus impinging on neurodevelopment. Hence, lessons learned from the study of rare diseases can provide insights into more common diseases and potential gene- environment interactions (Link, 2019).

A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity

Par-3 regulates animal cell polarity by targeting the Par complex proteins Par-6 and atypical protein kinase C (aPKC) to specific cortical sites. Although numerous physical interactions between Par-3 and the Par complex have been identified, this study discovered a novel interaction between Par-3's second PDZ domain and a highly conserved aPKC PDZ-binding motif (PBM) that is required in the context of the full-length, purified Par-6-aPKC complex. This study also found that Par-3 is phosphorylated by the full Par complex and phosphorylation induces dissociation of the Par-3 phosphorylation site from aPKC's kinase domain but does not disrupt the Par-3 PDZ2-aPKC PBM interaction. In asymmetrically dividing Drosophila neuroblasts, the aPKC PBM is required for cortical targeting, consistent with its role in mediating a persistent interaction with Par-3. These results define a physical connection that targets the Par complex to polarized sites on the cell membrane (Holly, 2020).

The catalytic activity of atypical protein kinase C (aPKC) defines mutually exclusive cortical domains in diverse animal cells. Par-6 and aPKC are recruited to specific cellular sites where aPKC phosphorylation polarizes downstream factors by displacing them from the Par cortical domain. For example, in Drosophila neural stem cells or neuroblasts, the Par complex localizes to an apical cortical domain during mitosis where it excludes neuronal differentiation factors. Apical exclusion separates these factors into a distinct cortical domain at the basal cortex, which is segregated into the basal daughter cell following cytokinesis. Par polarized factors such as Miranda and Numb contain sequences that bind the membrane but are also phosphorylation motifs for aPKC. The direct connection of aPKC's catalytic activity to the polarization of downstream factors makes the regulatory pathways that control its cortical targeting critical to animal cell polarity (Holly, 2020).

In many cellular contexts, Par-3 (Bazooka in flies) is essential for recruitment of Par-6 and aPKC to specific cortical sites. Par-3's role in regulating Par complex cortical recruitment is thought to be direct because five physical interactions have been discovered with both Par-6 and aPKC. Four of the interactions involve at least one of Par-3's three PDZ protein-interaction domains: Par-3 PDZ1 binding to the Par-6 PDZ domain, Par-3 PDZ1 and PDZ3 domain interactions with Par-6's PDZ-binding motif (PBM), and an interaction with an undefined region of aPKC that requires both Par-3 PDZ2 and PDZ3. Additionally, because Par-3 is an aPKC substrate, the aPKC kinase domain interacts with Par-3's aPKC phosphorylation motif (APM; also known as CR3). Although protein kinases are typically thought to interact transiently with their substrates, the interaction with the APM has been proposed to mediate complex assembly. Previous investigations used small fragments of the Par complex that did not contain all potential binding motifs, such that it was not possible to assess whether any of the interactions are required for binding in the context of the purified, full-length Par-6-aPKC complex. Furthermore, none of the interactions have been shown to be required for cortical targeting of aPKC in a functional context (Holly, 2020).

Par-3 interactions with the Par-6-aPKC complex were examined by reconstituting full-length Drosophila Par-6 and aPKC. Although it was possible to purify the Par-6-aPKC complex to a high degree, Par-3 is very large (157.4 kDa) and the maltose-binding protein-fused Par-3 (MBP-Par-3; total mass 199.9 kDa) significant amounts of degradation products were obtained in addition to full-length protein. Nevertheless, using this preparation, it was possible to detect an interaction with reconstituted Par complex using a qualitative affinity chromatography (i.e., 'pull-down') assay. Additionally, phosphate transfer to full-length Par-3 (and some smaller fragments with masses consistent with COOH-terminal truncations that contain the APM) were obtained using an antibody specific to the phosphorylated APM. Phosphorylation of Par-3 by aPKC has been controversial. This result contributes to the understanding of the process by demonstrating that aPKC phosphorylates Par-3 in the context of the full-length, purified Par complex in addition to the isolated catalytic domain and APM peptide (Holly, 2020).

Using the system of purified Par complex and MBP fusions of full-length Par-3 and its degradation products, attempts were made to identify Par-3 domains required for interaction with the full Par complex. The Par-6 PBM within the Par complex was tested, as it has been reported to bind both the Par-3 PDZ1 and PDZ3 domains. ATP was included in binding experiments because Par-3 is a substrate in the context of the full Par complex. Using this experimental setup, Par-3 PDZ2 was identified as a required interaction domain for binding to the full Par complex (Holly, 2020).

To determine the mechanism by which Par-3 PDZ2 mediates binding to the Par complex, attempts were made to identify the recognition site on the complex. The Drosophila aPKC COOH-terminal sequence has the characteristics of a 'class 3' PBM and is consistent with the binding specificity of the PDZ2 domain as assessed using a phage-display assay. The aPKC COOH-terminal sequence is also highly conserved among metazoan orthologs, the same evolutionary interval in which Par-3 is found. Tests were performed to see whether the aPKC COOH terminus is required for the interaction with Par-3 by purifying Par complex lacking aPKC's final six residues. The aPKC COOH terminus was shown to be required for Par-3's interaction with the Par complex (Holly, 2020).

To test whether the Par-3 PDZ2 and aPKC COOH terminus are sufficient for binding, the interaction of the isolated motifs was examined. The isolated proteins are sufficient for complex assembly. In general, PDZ-PBM interactions are strongly dependent on the identity of the terminal residue, and it was found that Par-3 PDZ2 failed to bind the aPKC COOH terminus when the final residue was mutated from valine to alanine (aPKC V606A). It is concluded that the aPKC COOH terminus is a bona fide PBM. It was also confirmed that the aPKC PBM and Par-3 PDZ2 interaction is broadly conserved across metazoans by examining orthologs from a chordate (human), a placozoan (Trichoplax), and a cnidarian (Hydra), in addition to the arthropod Drosophila. Binding was observed for each of the orthologous pairs, indicating that the interaction is conserved across diverse metazoan organisms. Together, these results indicate that the Par-3 PDZ2 and aPKC PBM are sufficient for binding and their interaction is conserved across metazoa (Holly, 2020).

To assess the role of the Par-3 PDZ2-aPKC PBM interaction quantitatively, an equilibrium supernatant-depletion assay was implemented. The affinity of the Par-3 PDZ1-APM for the Par complex was measured, as this region could be purified to a level suitable for quantitative measurements. Addition of Par-3 PDZ1-APM depleted Par-6 and aPKC from the supernatant consistent with a Kd of 0.7 μM. To determine the effect of disrupting the Par-3 PDZ2-aPKC PBM interaction on binding affinity, Par-3 PDZ1-APM binding to Par-6-aPKCΔPBM was measured. Sufficient depletion of Par-6 and aPKCΔPBM by PDZ1-APM was not observed to allow fitting to a binding isotherm, indicating the absence of the aPKC PBM substantially decreases the affinity of the Par-3 interaction with the Par complex, consistent with the results of qualitative measurements (Holly, 2020).

The results indicate that the Par-3 PDZ2 and aPKC PBM are required for Par-3's interaction with the Par complex. The requirement for these domains suggests that the Par-3 phosphorylation site (i.e., APM) does not form a persistent interaction with the Par complex. However, this conclusion appears to be in conflict with previous work showing that the Par-3 APM is sufficient for binding to the aPKC kinase domain, both with binding assays and structure determination using X-ray crystallography. Furthermore, a stable APM-kinase interaction forms the basis of a model in which the unphosphorylated Par-3 APM forms a stable, persistent interaction with the aPKC kinase domain that is not phosphorylated until an unknown activating event occurs. The finding that Par-3 is phosphorylated by the full Par complex is inconsistent with this model, but it does not fully resolve whether the Par-3 APM is sufficient for forming a stable, persistent interaction with the Par complex (Holly, 2020).

It was hypothesized that the presence of ATP could influence the binding behavior of the Par-3 APM with the Par complex. A key difference between the current experiments and previous reports is that the current experiments included ATP, whereas previous binding experiments and structural analysis lacked ATP. Without ATP, completion of the protein kinase catalytic cycle is not possible, and interactions that would otherwise form transiently could persist (Holly, 2020).

Tests were performed to see whether the Par-3 APM forms a stable, persistent interaction with the Par complex in the absence of ATP. Vinding between Par-3 and the Par complex was detected after replacing ATP with ADP in a context where the Par-3 PDZ2-aPKC PBM interaction is disrupted. This interaction requires the APM, leading to the conclusion that the Par-3 APM can form a persistent interaction with the Par complex, but only in the absence of ATP. When ATP is present the APM interacts transiently with the Par complex, because it is phosphorylated and subsequently dissociates. Under the same conditions, the Par-3 PDZ2 interaction with the Par complex is not disrupted, however. Although it is possible to form a stalled complex between the Par-3 APM and the Par complex in the absence of ATP, it is proposed that persistent binding of the APM to the kinase domain due to the lack of ATP is unlikely in vivo because ATP concentrations are high under normal cellular conditions (Holly, 2020).

Although numerous interactions have been identified between Par-3 and Par-6-aPKC, none have been demonstrated to be required for cortical targeting of the Par complex. In fact, the interactions of Par-6 with Par-3 have been shown to be dispensable for function. To determine whether the Par-3 PDZ2-aPKC PBM interaction is required for Par complex polarization, the localization of aPKC harboring the V606A PBM point mutation was investigated during neuroblast asymmetric division by expressing aPKC-V606A in larval brain neuroblasts and comparing its localization to that of wild-type aPKC. Consistent with previous observations, wild-type aPKC is polarized to a cortical crescent around the apical pole at metaphase. In contrast, aPKC-V606A remained in the cytoplasm and was not recruited to the cortex, even though the localization of Par-3 was unaffected. The aPKC-V606A protein also failed to be recruited to the apical cortex in neuroblasts lacking endogenous aPKC. It is concluded that the Par-3 PDZ2-aPKC PBM interaction is required for cortical recruitment and polarization of aPKC in neuroblasts (Holly, 2020).

The interaction of Par-3 with the full-length Par complex was investigated, and it was found that Par-3 PDZ2 and a previously unrecognized PBM at the COOH terminus of aPKC are required for complex assembly. The Par-3 phosphorylation site (APM) can also form a persistent interaction with the aPKC kinase domain, but only if phosphorylation is not allowed to occur due to the absence of ATP. Unlike the APM-kinase domain interaction, the Par-3 PDZ2 interaction with the aPKC PBM is not influenced by the presence of ATP, suggesting that additional mechanisms besides APM phosphorylation must exist to dissociate Par-3 from the Par complex, an important component of current polarity models (Holly, 2020).

The identification of the Par-3 PDZ2 domain as a key factor in recruiting the Par complex to the cortex during animal cell polarization is consistent with previous work demonstrating that whereas Par-3 PDZ1 and 3 are dispensable in C. elegans, PDZ2 is required for cortical recruitment of Par-6 and aPKC. It is also consistent with work in both C. elegans and Drosophila showing that the interaction of Par-6 with Par-3 is not required. In Drosophila, the role of PDZ2 is less clear but is known to be required for downstream effects on epithelial structure. It is suggested that the Par-3 PDZ2-aPKC PBM interaction represents an important physical connection for animal cell polarity and that the reconstitution approach used to identify this interaction will likely be useful for understanding how other regulatory molecules, such as Cdc42, control polarity (Holly, 2020).

Girdin is a component of the lateral polarity protein network restricting cell dissemination

Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. This study shows that Drosophila Girdin (girders of actin filaments) and its human ortholog GIRDIN or GIV (Galpha-interacting vesicle associated protein) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, cell dissemination was observed from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, this study discovered a core mechanism contributing to epithelial cell polarization from flies to humans. These data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination (Biehler, 2020).

The ability of epithelia to form physical barriers is provided by specialized cell-cell junctions, including the zonula adherens (ZA). The latter is a belt-like adherens junction composed primarily of the transmembrane homotypic receptor E-cadherin, which is linked indirectly to circumferential F-actin bundles through adaptor proteins such as β-catenin and α-catenin. In Drosophila embryonic epithelia, the protein Girdin stabilizes the ZA by reinforcing the association of the cadherin-catenin complex with the actin cytoskeleton (Ha, 2015). This function in cell-cell adhesion is preserved in mammals, and supports collective cell migration (Wang, 2018; Wang, 2015). Fly and human Girdin also contribute to the coordinated movement of epithelial cells through the organization of supracellular actin cables (Biehler, 2020).

In addition to creating barriers, epithelial tissues generate vectorial transport and spatially oriented secretion. The unidirectional nature of these functions requires the polarization of epithelial cells along the apical-basal axis. In Drosophila, the scaffold protein Bazooka (Baz) is crucial to the early steps of epithelial cell polarization, and for proper assembly of the ZA. Baz recruits atypical Protein Kinase C (aPKC) together with its regulator Partitioning defective protein 6 (Par-6) to the apical membrane. The small GTPase Cdc42 contributes to the activation of aPKC and p21-activated kinase (Pak1), thereby acting as a key regulator of cell polarity. Baz also contributes to apical positioning of the Crumbs (Crb) complex, which is composed mainly of Crb, Stardust (Sdt), and PALS1-associated Tight Junction protein (Patj). Once properly localized, the aPKC-Par-6 and Crb complexes promote the apical exclusion of Baz, which is then restricted to the ZA. The apical exclusion of Baz is essential to the positioning of the ZA along the apical-basal axis, and for full aPKC activation (Biehler, 2020).

The function of aPKC is evolutionarily preserved, and human atypical PKCλ (PKClambda in other mammals) and PKCζ PKCzeta)contribute to epithelial cell polarization. aPKC maintains the identity of the apical domain through phospho-dependent exclusion of lateral polarity proteins such as Yurt (Yrt) and Lethal (2) giant larvae (Lgl). In return, these proteins antagonize the Crb- and aPKC-containing apical machinery to prevent the spread of apical characteristics to the lateral domain. In combination with the function of Baz, these feedback mechanisms provide a fine-tuning of aPKC activity in addition to specifying its subcellular localization. This is crucial, as both over- and under-activation of aPKC is deleterious to epithelial polarity in fly and mammalian cells, and ectopic activation of aPKC can lead to cell transformation (Biehler, 2020).

Cell culture work has established that mammalian GIRDIN interacts physically with PAR3 -the ortholog of Baz-and PKCλ. Depletion of GIRDIN in Madin-Darby Canine Kidney (MDCK) epithelial cells delays the formation of tight junctions in Ca2+ switch experiments. GIRDIN is also an effector of AMP-activated protein kinase (AMPK) in the maintenance of tight junction integrity under energetic stress. Moreover, mammalian GIRDIN is required for the formation of epithelial cell cysts with a single lumen, supporting a role for this protein in epithelial morphogenesis as reported in flies. As cyst morphogenesis is linked to epithelial cell polarity, these studies suggest that GIRDIN is involved in establishing the apical-basal axis. However, further studies are required to clarify the role of GIRDIN in apical-basal polarity per se, as other cellular processes could explain the phenotype associated with altered GIRDIN expression. For instance, spindle orientation defects impair the formation of epithelial cysts. Of note, PAR3, aPKC, and AMPK are all required for proper spindle positioning in dividing epithelial cells. The molecular mechanisms sustaining the putative role of GIRDIN in epithelial cell polarity also need to be better deciphered. This study investigated the role of fly and human Girdin proteins in the regulation of epithelial cell polarity, and showed that these proteins are part of the lateral polarity protein network. One crucial function of Girdin proteins is to repress aPKC function. It was also discovered that loss of Girdin proteins promotes overgrowth of cell cysts, and cell dissemination from these multicellular structures. Consistent with these data, it was found that low GIRDIN expression correlates with poor overall survival in subtypes of breast and lung cancers (Biehler, 2020).

Using classical genetics in flies, this study has shown that mutation in Girdin exacerbates the polarity defects in zygotic lgl or yrt mutant embryos and concludes that Girdin is part of the lateral polarity network. It was also found that Girdin opposes the function of aPKC, which plays a crucial role in the establishment and maintenance of the apical domain by antagonizing lateral proteins such as Lgl and Yrt. Thus a model is proposed in which Girdin supports the activity of Yrt and Lgl by restricting the activity of aPKC. This work demonstrates that the role of Girdin in restricting aPKC activity is evolutionarily conserved. This function confers on human GIRDIN the ability to maintain apical-basal polarity in Caco-2 cells, and to support epithelial cyst morphogenesis. These results are in line with previous studies suggesting a role for GIRDIN in polarity and cystogenesis in MDCK and MCF10A epithelial cells. It was shown that PKCλ enhances GIRDIN expression in MDCK cells. Moreover, knockdown of aPKC or GIRDIN gives a similar phenotype characterized by defects in tight junction integrity and cyst formation. It was thus proposed that GIRDIN is an effector of PKCλ. Although cell-type-specific mechanisms may exist, the current data suggest that this hypothesis needs to be revisited in favor of a model in which the induction of GIRDIN expression by PKClambda in MDCK cells initiates a negative feedback loop instead of cooperation between these proteins. The fact that both overactivation of aPKC or inhibition of its activity is deleterious to epithelial cell polarity and cyst morphogenesis may underlie the conflicting interpretations of the data in the literature. GIRDIN is also known to modulate heterotrimeric G protein signaling-a role that seems to contribute to the formation of normal cysts by MDCK cells (Sasaki, 2015). In addition, it was demonstrated recently that GIRDIN acts as an effector of AMP-activated protein kinase (AMPK) under energetic stress to maintain tight junction function (Aznar, 2016). Of note, these two functions are not shared by fly Girdin (Ghosh, 2017; Garcia-Marcos, 2009; Ghosh, 2017), and were thus acquired by GIRDIN during evolution to fulfill specialized functions. In contrast, the discovery in this study of the Girdin-dependent inhibition of aPKC reveals a core mechanism contributing to epithelial cell polarization from flies to humans (Biehler, 2020).

GIRDIN is considered to be an interesting target in cancer due to its role in cell motility, and high levels of GIRDIN have been reported to correlate with a poor prognosis in some human cancers. Notwithstanding that GIRDIN may favor tumor cell migration, the current study indicates that inhibition of GIRDIN function in the context of cancer would be a double-edged sword for many reasons. Indeed, this study showed that knockdown of GIRDIN exacerbates the impact of aPKC overexpression, and leads to overgrowth and lumen filling of Caco-2 cell cysts. Of note, overexpression of aPKC can lead to cell transformation, and was associated with a poor outcome in several epithelial cancers. This study thus establishes that inhibiting GIRDIN in patients showing increased aPKC expression levels could worsen their prognosis. According to the data, abolishing GIRDIN function in tumor cells with decreased levels of the human Lgl protein LLGL1, as reported in many cancers, could also support the progression of the disease by altering the polarity phenotype. Cell detachment and dissemination was observed from GIRDIN knockdown cysts, thus showing that GIRDIN is required for the cohesion of multicellular epithelial structures. Of note, cells, either individually or as clusters, detaching from cysts are alive and some of them remain viable. This is analogous to what was reported in Girdin mutant Drosophila embryos in which cell cysts detach from the ectoderm and survive outside of it. Other phenotypes in Girdin mutant embryos are consistent with a role for Girdin in epithelial tissue cohesion, including rupture of the ventral midline and fragmentation of the dorsal trunk of the trachea. Mechanistically, Girdin strengthens cell-cell adhesion by promoting the association of core adherens junction components with the actin cytoskeleton. A recent study established that this molecular function is evolutionarily conserved, and that GIRDIN favors the association of β-CATENIN with F-ACTIN. Since knockdown of GIRDIN results in cell dispersion from Caco-2 cell cysts, and since weakening of E-CADHERIN-mediated cell-cell adhesion contributes to cancer cell dissemination and metastasis, it is plausible that reduced GIRDIN expression contribute to the formation of secondary tumors and cancer progression. This may explain why this study found that low mRNA expression levels of GIRDIN correlates with decreased survival in more aggressive breast cancer subtypes and lung adenocarcinoma. Future studies using xenograft in mice, and investigating the expression of GIRDIN protein in cancer patients will help validating whether GIRDIN can repress the progression of certain types of epithelial cancers (Biehler, 2020).

In conclusion, using a sophisticated experimental scheme combining in vivo approaches in D. melanogaster with 3D culture of human cells, this study defined a conserved core mechanism of epithelial cell polarity regulation. Specifically, Girdin was shown to repress the activity of aPKC to support the function of Lgl and Yrt, and ensure stability of the lateral domain. This is of broad interest in cell biology, as proper epithelial cell polarization is crucial for the morphogenesis and physiology of most organs. In addition, the maintenance of a polarized epithelial architecture is crucial to prevent various pathological conditions such as cancer progression. Importantly, this study showed that normal GIRDIN function potentially impairs the progression of epithelial cancers by preserving cell polarity whilst restricting cell growth and cell dissemination. Thus, these results place a caveat on the idea that GIRDIN could be an interesting target to limit cancer cell migration, and indicate that inhibition of GIRDIN in the context of cancer could be precarious. Potential drugs targeting GIRDIN would thus be usable only in the context of precision medicine where a careful analysis of aPKC, LLGL1, and E-CAD expression, as well as the polarity status of tumor cells would be analyzed prior to treatment. Inhibition of GIRDIN in patients carrying tumors with altered expression of these proteins would likely worsen the prognosis (Biehler, 2020).

Distinct activities of Scrib module proteins organize epithelial polarity

A polarized architecture is central to both epithelial structure and function. In many cells, polarity involves mutual antagonism between the Par complex and the Scribble (Scrib) module. While molecular mechanisms underlying Par-mediated apical determination are well-understood, how Scrib module proteins specify the basolateral domain remains unknown. This study demonstrates dependent and independent activities of Scrib, Discs-large (Dlg), and Lethal giant larvae (Lgl) using the Drosophila follicle epithelium. The data support a linear hierarchy for localization, but rule out previously proposed protein-protein interactions as essential for polarization. Cortical recruitment of Scrib does not require palmitoylation or polar phospholipid binding but instead an independent cortically stabilizing activity of Dlg. Scrib and Dlg do not directly antagonize atypical protein kinase C (aPKC), but may instead restrict aPKC localization by enabling the aPKC-inhibiting activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each required, all three together are not sufficient to antagonize the Par complex. These data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg (Khoury, 2020).

Despite being central regulators of cell polarity in numerous tissues from nematodes to mammals, the mechanisms of Scrib module activity have remained obscure. The current work highlights previously unappreciated specificity in these activities, and begins to define the molecular functions of Scrib, Dlg, and Lgl. The data focus on the Drosophila follicle epithelium, as well as in some cases Drosophila embryos, but it is important to note that tissue contexts can differ in polarity programs: For example, in the adult Drosophila midgut epithelium, where Scrib module proteins are dispensable for epithelial organization. The failure to detect phenotypic enhancement in double-mutant follicle cells, compared to single mutants, which together with the complete penetrance of single-mutant phenotypes suggest full codependence of function rather than functional overlap. Moreover, Scrib module mutants could not be bypassed in any combination by overexpression of other genes in the module, consistent with unique roles for each protein. Thus, while Scrib, Dlg, and Lgl act in a common 'basolateral polarity' pathway, they each contribute distinct functions to give rise to the basolateral domain (Khoury, 2020).

Cell polarity is particularly evident at the plasma membrane, and most polarity regulators act at the cell cortex. Therefore, a key question in the field has concerned the mechanisms that allow cortical localization of the Scrib module and Par complex proteins, which exhibit no classic membrane-association domains. A simple linear hierarchy was found for cortical localization in the follicle that places Dlg most upstream, and contrasts with that recently described in the adult midgut, where Scrib appears to be most upstream. This work highlights the requirement of Dlg for Scrib localization, and provides insight into the mechanism, in part by ruling out previous models. One model involves a direct physical interaction, mediated by the Scrib PDZ domains and Dlg GUK domain. However, in vivo analyses show that follicle cells mutant for alleles lacking either of these domains have normal polarity; these results are supported by data from imaginal discs. In contrast, this study showa that the SH3 domain is critical for Scrib cortical localization as well as polarity. The Dlg SH3 and GUK domains engage in an intramolecular 'autoinhibitory' interaction that negatively regulates binding of partners, such as Gukh and CASK. The dispensability of the GUK domain provides evidence against an essential role for this mode of regulation in epithelial polarity, and highlights the value of investigating the GUK-independent function of the Dlg SH3 (Khoury, 2020).

A second mechanism of Scrib cortical association was also excluded. Mammalian Scrib is S-palmitoylated and this modification is required for both cortical localization and function. As Drosophila Scrib was also recently shown to be palmitoylated, an appealing model would involve Dlg regulating this posttranslational modification. However, no changes to Scrib palmitoylation were detected in a dlg mutant, and chemically or genetically inhibiting Drosophila palmitoyltransferases also had no effect on Scrib localization, although the possibility that Scrib palmitoylation may be part of a multipart localization mechanism cannot be excluded. Surprisingly, palmitoylated Scrib is incapable of reaching the cortex in dlg mutants. While a constitutively myristoylated Scrib can bypass this requirement for localization, it is nevertheless insufficient to support polarity in the absence of Dlg. These results indicate that Dlg regulates additional basolateral activities beyond localizing Scrib (Khoury, 2020).

Lgl's role as an aPKC inhibitor is well-characterized, but how Scrib and Dlg influence this antagonism is not understood. This study shows that Scrib and Dlg maintain cortical Lgl by regulating its phosphorylation by aPKC, rather than by direct physical recruitment to the membrane. A contemporaneous study by Ventura (2020) supports this finding, further showing that the major factor in Lgl cortical stabilization is PIP2. The current data also suggest that the basolateral-promoting activities of Scrib and Dlg are not via direct inhibition of aPKC kinase activity or intrinsic antagonism of aPKC localization. Instead, they are consistent with models in which Scrib and Dlg regulate the three specific aPKC-targeted residues in Lgl. Previous work has demonstrated that these phosphorylated serines (656, 660, 664) are neither functionally nor kinetically equivalent, and a recent model proposes that S664 is required for basolateral polarization by mediating a phosphorylation-dependent interaction with the Dlg GUK domain. Beyond the dispensability of the GUK domain, the enhanced ability of LglAAS to inhibit aPKC and its ability to do so largely independently of Scrib and Dlg, argues against this model. Moreover, only LglAAS among the phospho-mutants can dominantly affect aPKC activity, while WT Lgl can do the same only if Scrib and Dlg are present. Together, these results suggest that S656 is the critical inhibitory residue whose phosphorylation must be limited to enable Lgl's activity (Khoury, 2020).

The mechanism by which LglS656A,LglS660A(AAS) (LglAAS) can suppress even constitutively active aPKCΔN remains unclear. aPKC substrates can act as competitive inhibitors; either an increased substrate affinity for aPKC or reduced ability to be inhibited by virtue of having fewer phosphorylation sites could make LglAAS a more effective inhibitor than WT Lgl. Supporting this idea, it was previously shown that S664, the only residue still available in LglAAS, is phosphorylated with higher kinetic preference than S656 or S660. It is also possible that some LglAAS phenotypes may be due to aPKC-independent effects resulting from reduced phosphorylation on S656 and S660. Nevertheless, a model is proposed in which Scrib and Dlg 'protect' Lgl by limiting phosphorylation of S656, thus tipping the inhibitory balance to allow Lgl to inhibit aPKC and establish the basolateral domain (Khoury, 2020).

What mechanism could underlie Scrib and Dlg protection of Lgl? One mechanism could involve generating a high phospholipid charge density at the basolateral membrane, which has been shown to desensitize Lgl to aPKC phosphorylation in vitro. However, the current data do not find evidence for regulation of phosphoinositides by Scrib and Dlg. A second possibility is that Scrib and Dlg could scaffold an additional factor, such as protein phosphatase 1, which counteracts aPKC phosphorylation of Lgl. Alternative mechanisms include those suggested by recent work on PAR-1 and PAR-2 in Caenorhabditis elegans zygotes, a circuit with several parallels to the Scrib module. In this system, PAR-2 protects PAR-1 at the cortex by shielding it from aPKC phosphorylation through physical interaction-dependent and -independent mechanisms. By analogy, binding with Scrib or Dlg could allosterically regulate Lgl to prevent phosphorylation, although this study has ruled out the Lgl-Dlg interaction documented in the literature. Scrib or Dlg might also act as a 'decoy substrate' for aPKC, as PAR-2 does in PAR-1 protection. Indeed, Scrib is phosphorylated on at least 13 residues in Drosophila embryos, although the functional relevance of this is not yet known (Khoury, 2020).

Overall, this work highlights the multifaceted nature of Scrib module function. The failure to bypass Scrib module mutants by transgenic supply of any single or double combination of other module components, including several that were constitutively membrane-tethered, suggests that every member contributes a specific activity to polarity. Nevertheless, even the simultaneous ectopic localization of all three Scrib module proteins was insufficient to disrupt the apical domain. This insufficiency in basolateral specification may reflect an inability of apical Scrib and Dlg to protect Lgl from aPKC phosphorylation, perhaps due to the distinct molecular composition of the apical and basolateral domains. This supports the idea that in addition to intrinsic activity via Lgl, the Scrib module must recruit or activate additional, as yet unidentified effectors in basolateral polarity establishment. The independent as well as cooperative activities of the Scrib module delineated in this study demonstrate previously unappreciated complexity in the determination of basolateral polarity and set the stage for future mechanistic studies of Scrib module function (Khoury, 2020).

Par complex cluster formation mediated by phase separation

The evolutionarily conserved Par3/Par6/aPKC complex regulates the polarity establishment of diverse cell types and distinct polarity-driven functions. However, how the Par complex is concentrated beneath the membrane to initiate cell polarization remains unclear. This study shows that the Par complex exhibits cell cycle-dependent condensation in Drosophila neuroblasts, driven by liquid-liquid phase separation. The open conformation of Par3 undergoes autonomous phase separation likely due to its NTD-mediated oligomerization. Par6, via C-terminal tail binding to Par3 PDZ3, can be enriched to Par3 condensates and in return dramatically promote Par3 phase separation. aPKC can also be concentrated to the Par3N/Par6 condensates as a client. Interestingly, activated aPKC can disperse the Par3/Par6 condensates via phosphorylation of Par3. Perturbations of Par3/Par6 phase separation impair the establishment of apical-basal polarity during neuroblast asymmetric divisions and lead to defective lineage development. It is proposed that phase separation may be a common mechanism for localized cortical condensation of cell polarity complexes (Li, 2020).

How the conserved Par (Par3/Par6/aPKC) complex is selectively recruited and concentrated on membranes for polarity establishment remains unclear. In this study, different from previously reported crescent localization patterning, the endogenous Par complex is revealed to exhibit cell cycle-dependent discrete puncta formation on the apical cortex in Drosophila NBs. The condensed Par puncta emerge from prophase, further condensate and enlarge as a clustered puncta structure in metaphase, then subsequently disassemble into scattered small puncta from anaphase. The cell cycle-dependent clustering of Par proteins in Drosophila NBs were also observed by two recent studies. In vitro biochemical data together with heterologous cell-based studies showed that the Par3/Par6 complex can undergo liquid-liquid phase separation (LLPS) at very low protein concentrations, and mutations of Par3 or Par6 that impair LLPS were found to alter asymmetric cell division (ACD) in Drosophila NBs. It has been recently shown that the basal condensation of Numb in dividing NBs is also regulated by LLPS of the Numb/Partner of numb complex (Shan, 2018). Thus, LLPS may be a common mechanism for the local condensation of apical and basal polarity determining protein complexes (Li, 2020).

It is important to note that the Par proteins, each at their endogenous level, can form clustered puncta via LLPS on the cortex. Though the measured endogenous Baz level in Drosophila NBs was too low to induce its LLPS in the cytoplasm, two-dimensional membrane attachment was expected to locally enrich the protein and lead to its LLPS. In return, LLPS-mediated Par complex condensates formation acts as an effective way for cells to further concentrate limited amount of Par proteins to specific cell cortices for polarity establishment. It is proposed that apical Baz/Par3 localization is a balanced result of apical anchoring and LLPS-mediated local condensation (via multivalent protein-protein interaction, self-association, protein-membrane interaction, etc.). Thus, for the knock-in mutant Baz ΔNTD, partially impaired LLPS ability due to its defective oligomerization led to its less condensed localization and significant cytoplasmic diffusion. However, the situation was different for the overexpressed Baz NTDmu (driven by UAS/GAL4) in the rescue assay, which is ectopically localized. As LLPS is very sensitive to concentrations of biological components, an overexpression of Par proteins especially Baz/Par3, the core driving factor of LLPS, may cause artificial promotion of the Par complex condensation via LLPS. Whereas the apical anchoring capacity of NBs seems to have a limitation. In UAS/GAL4-based rescue assay, the overexpressed Baz WT phase condensates may just have reached the threshold of apical anchoring capacity, whereas the LLPS deficient, overexpressed Baz NTDmu broke the balance, and led to its cortical and cytoplasmic diffusion. If the expression level goes higher, even Baz WT can not be afforded apically. Consistent with this notion, high Flag-Baz expression in a WT background (driven by insc-gal4), has a dominant-negative effect and leads to ectopic localization of endogenous Par complex throughout the cortex, and consequently disrupts localization of basal proteins. Similarly, ectopic Baz localization was observed when exogenous Baz is forcedly expressed in embryonic NBs. It was recently shown that overexpression of Par3-induced cell polarity in apolar S2 cells by forming concentrated Par-dots that further fused into amorphous Par-islands. According to a study of protein LLPS on lipid membrane bilayers, protein clusters gradually grew and fused into larger ones with irregular shapes, and finally coalesced into a mesh-like network. Thus, the amorphous structure of Par-islands in S2 cells may arise from the overexpression and overaccumulation of Par3 in the membrane region. Therefore, caution should be taken in interpreting the overexpression phenotypes of Par3 (Li, 2020).

Another key finding in this study is that aPKC can be recruited and concentrated in Par3/Par6 condensates as an inactive client. Such condensed phase droplets could be an efficient mechanism for local condensation of aPKC. Spatiotemporal activation of aPKC (e.g., by Cdc42) and consequent phosphorylation on Par3 CR3 leads to disassembly of the Par complex condensates. Another cell cycle regulator that might play a role in Par LLPS regulation is Plk1, which inhibits the oligomerization of Par3 by phosphorylating NTD in C. elegans. A critical but currently unknown point is how the autoinhibition of Par3 is relieved, as the open conformation of Par3 is critical for the Par complex condensate formation. Nonetheless, it is likely that multilayered regulatory mechanisms can act concertedly to control the spatiotemporal assembly and disassembly of the Par complex phase separation, and hence the cell polarity regulations (Li, 2020).

It is increasingly recognized that LLPS is a common strategy for cells to form membrane-less compartments by selectively recruiting and condensing proteins/RNAs/lipids. In a broader sense, the local condensation of other master polarity complexes, such as the conserved Lgl/Dlg/Scribble complex in the apical-basal polarity, and the Prickle/Vangl and Frizzled/Disheveled/Diego complexes in the planar cell polarity, may adopt a similar LLPS-driven mechanism to establish cell polarity in different tissues. Like the Par complex proteins, all these complexes share several common features: (1) these proteins contain multiple domains, which mutually interact with each other or self-associate in vitro to form complex platform, which further recruits other binding partners to assemble into higher order protein interaction network; (2) these complexes are found to form condensed patches or puncta attached to the inner surface of plasma membranes in vivo; and (3) proteins within these condensed patches or puncta are highly dynamic and rapidly exchange with corresponding proteins in the cytoplasm. The multivalent interaction-induced LLPS theory can perfectly explain above phenomena, allowing the stable existence of large concentration gradients of the proteins within the local protein condensates and those in the cytoplasm, and at the same time, keeping the proteins in the condensed phase highly dynamic. Such dynamic association may be essential for the fast assembly/disassembly of these polarity complexes in responding to extrinsic/intrinsic cues/signals to rearrange the cell polarity. It is postulated that LLPS of polarity protein complexes induced by multivalent interactions is a general mechanism for the cell polarization (Li, 2020).


Amino Acids - 606

Structural Domains

To identify an atypical protein kinase C isoform from Drosophila, the Berkeley Drosophila genome database BLASTed with sequences from mouse PKClambda and C. elegans PKC-3 (Tabuse, 1998). One EST clone (HL05754) shows significant sequence similarity to the NH2 termini of both PKClambda and PKC-3. Further sequencing of HL05754 reveals that it contains most of the coding region of aPKC, except for a few hundred basepairs that are missing at the 3' end. BLAST searches with the HL05754 cDNA fragment show that the aPKC gene is located in genomic region 51D on the right arm of chromosome 2. Based on sequence similarity to mouse and C. elegans aPKCs and on sequence analysis tools predicting exon-intron boundaries, three additional putative 3' exons were identified that are missing in HL05754. The existence of the predicted transcript was confirmed by 3' RACE analysis of embryonic mRNA. Comparison of the aPKC cDNA sequence to the genomic sequence of the aPKC locus reveals the existence of at least 10 exons. Both the first and the last exon are noncoding and the last exon contains a canonical polyadenylation signal (AATAAA). Drosophila aPKC shows the highest sequence similarity to mouse PKClambda (68% identity), rat PKCzeta (63% identity), and C. elegans PKC-3 (58% identity). In comparison, Drosophila aPKC shows significantly lower sequence similarity to two conventional PKC isoforms from Drosophila: PKC 53E (29% identity) and PKC 98F (36% identity). BLAST searches of the completed genome sequence of Drosophila reveal that aPKC is the only aPKC in Drosophila (Wodarz, 2000).

atypical protein kinase C: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 12 February 2001

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