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: Precomputed BLAST | Entrez Gene | UniGene |
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.


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).


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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