Little is known about differential expression, functions, regulation, and targeting of 'atypical' protein kinase C (aPKC) isoenzymes in vivo. A novel cDNA has been cloned and characterized that encodes a Caenorhabditis elegans aPKC (PKC3), composed of 597 amino acids. In post-embryonic animals, a 647-base pair segment of promoter/enhancer DNA directs transcription of the 3.6-kilobase pair pkc-3 gene and coordinates accumulation of PKC3 protein in approximately 85 muscle, epithelial, and hypodermal cells. These cells are incorporated into tissues involved in feeding, digestion, excretion, and reproduction. Mammalian aPKCs promote mitogenesis and survival of cultured cells. In contrast, C. elegans PKC3 accumulates in non-dividing, terminally differentiated cells that will not undergo apoptosis. Thus, aPKCs may control cell functions that are independent of cell cycle progression and programmed cell death. PKC3 is also expressed during embryogenesis. Ablation of PKC3 function by microinjection of antisense RNA into oocytes yields disorganized, developmentally arrested embryos. Thus, PKC3 is essential for viability. PKC3 is enriched in particulate fractions of disrupted embryos and larvae. Immunofluorescence microscopy reveals that PKC3 accumulates near cortical actin cytoskeleton/plasma membrane at the apical surface of intestinal cells and in embryonic cells. A candidate anchoring/targeting protein, which binds PKC3 in vitro, has been identified (Wu, 1998).
Cell polarity is fundamental to the differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends on cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCzeta and PKClambda have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. The present study reports the identification of a protein, ASIP (atypical PKC isotype-specific interacting protein), that binds to aPKCs, and shows that ASIP colocalizes with PKClambda to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy reveals that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, these results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry (Izumi, 1998).
Asymmetric cell divisions, critically important to specify cell types in the development of multicellular organisms, require polarized distribution of cytoplasmic components and the proper alignment of the mitotic apparatus. In Caenorhabditis elegans, the maternally expressed protein, PAR-3, is localized to one pole of asymmetrically dividing blastomeres and is required for these asymmetric divisions. An atypical protein kinase C (PKC-3) is essential for proper asymmetric cell divisions and co-localizes with PAR-3. The predicted amino acid sequence of PKC-3 shows extensive similarity to atypical mammalian PKC subfamily members PKCzeta and PKClambda. The amino terminal half contains one cysteine-zinc finger motif and lacks a potential Ca 2+ binding domain conserved in the conventional PKC family members. These structural features characterize atypical PKCs, which are dependent on neither Ca 2+ nor diacylglycerol for their activation. The carboxy-terminal half of the predicted PKC-3 protein exhibits about 70% similarity to the kinase domain of atypical PKCs. A separate study (Wu, 1998) shows that purified PKC-3 protein requires phosphatidylserine, but is independent of both Ca 2+ and diacylglycerol, for its activation (Tabuse, 1998).
Several other par genes, par-2, par-5 and par-6, are required for proper localization of PAR-3. In par-6 1-cell embryos, PAR-3 distribution is cytoplasmic and uniform rather than peripheral and asymmetric. In par-2 and par-5 1-cell embryos, although PAR-3 is detected peripherally, its localization is not restricted to the anterior half and extends to the posterior part of the embryo. At the 2-cell stage, PAR-3 is distributed all around the cortex of both blastomeres of par-2 and par-5 embryos. If PKC-3 localization is dependent upon PAR-3, then PKC-3 should be mislocalized in mutant backgrounds in which PAR-3 is mislocalized. Indeed, the distribution of PKC-3 parallels the distribution of PAR-3 in these mutant backgrounds. In par-6 embryos, no peripheral staining of PKC-3 is detected although faint and uniform staining persists in the cytoplasm. In par-2 embryos, PKC-3 is present at the cortex in a gradient along the A-P axis, but its distribution extends to about 70% of the egg length during pronuclear migration and pronuclear fusion. As the cell cycle proceeds, the distribution of PKC-3 extends over 90% of the egg length by late anaphase. As a result of the uniform distribution of PKC-3 in the par-2 1-cell embryo, all blastomeres exhibit peripheral PKC-3 staining in 80% of 2-cell embryos and 92% of 4-cell embryos. The PKC-3 distribution patterns of par-5 embryos are very similar to those exhibited by par-2 embryos, though the extent of the localization defect is somewhat more severe in par-5 than par-2 background. In par-5 embryos, PKC-3 is distributed to about 80% of the egg length during pronuclear migration and fusion, and covers almost the entire cortex of the 1-cell embryo by late anaphase. All blastomeres show peripheral PKC-3 staining in 90% of 2-cell embryos and 80% of 4-cell embryos. These results are consistent with the notion that PKC-3, cooperating with PAR-3, plays a role in par gene pathway to establish early embryonic polarity (Tabuse, 1998).
The par genes are required to establish polarity in the Caenorhabditis elegans embryo. Mutations in two of these genes, par-3 and par-6, exhibit similar phenotypes. A third gene, pkc-3, gives a similar phenotype when the protein is depleted by RNA interference. PAR-3 and PKC-3 protein are colocalized to the anterior periphery of asymmetrically dividing cells of the germline lineage. The peripheral localizations of both proteins depend on the activity of par-6. The molecular cloning of par-6 is reported and the immunolocalization of PAR-6 protein. par-6 encodes a PDZ-domain-containing protein and has homologs in mammals and flies. Moreover, PAR-6 colocalizes with PAR-3 and par-3 and pkc-3 activity are required for the peripheral localization of PAR-6. The localization of both PAR-3 and PAR-6 proteins is affected identically by mutations in the par-2, par-4 (see Drosophila Lkb1) and par-5 genes. The co-dependence of PAR-3, PAR-6 and PKC-3 for peripheral localization and the overlap in their distributions leads to a proposal that they act in a protein complex (Hung, 1999).
Cellular asymmetry is critical for the development of multicellular organisms. Homologs of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C (aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. It is concluded that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signaling proteins that are implicated in mammalian cell polarity (Lin, 2000).
PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. An unexpected link between these two systems is described. A family of mammalian Par6 proteins have been identified that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42-GTP, with a human homolog of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell-cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3 (Joberty, 2000).
Atypical protein kinase C (PKC) isotype-specific interacting protein (ASIP) specifically interacts with the atypical protein kinase C isozymes PKClambda and PKCzeta. ASIP and atypical PKC, as well as their Caenorhabditis elegans counterparts (PAR-3 and PKC-3, respectively), are thought to coordinately participate in intracellular signaling that contributes to the maintenance of cellular polarity and to the formation of junctional complexes. The potential role of ASIP in other cellular functions of atypical PKC was investigated by examining the effect of overexpression of ASIP on insulin-induced glucose uptake, previously shown to be mediated through PKClambda, in 3T3-L1 adipocytes. When overexpressed in these cells, which contain PKClambda but not PKCzeta, ASIP is co-immunoprecipitated with endogenous PKClambda but not with PKCepsilon or with Akt. The subcellular localization of PKClambda is also altered in cells overexpressing ASIP. Overexpression of ASIP inhibits insulin stimulation of both glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but it does not inhibit glucose uptake induced by either growth hormone or hyperosmolarity, both of which promote glucose uptake in a PKClambda-independent manner. Moreover, glucose uptake stimulated by a constitutively active mutant of PKClambda, but not that induced by an active form of Akt, is inhibited by ASIP. Insulin-induced activation of PKClambda, but not that of phosphoinositide 3-kinase or Akt, is also inhibited by overexpression of ASIP. These data suggest that overexpression of ASIP inhibits insulin-induced glucose uptake by specifically interfering with signals transmitted through PKClambda (Kotani, 2000).
A novel effector of Rac and Cdc42, hPar-6, has been identified that is the human homolog of a cell-polarity determinant in C. elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKC, and forms a stable ternary complex with either Rac1 or Cdc42 and PKC. This association results in stimulation of PKC kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKC-dependent pathway distinct from the pathway mediated by Raf (Qui, 2000).
Many direct targets of Rac1 and Cdc42 have been identified, but none has been shown to have a direct role in cell transformation by Rac1 and Cdc42. hPar-6 is a novel effector of Rac1 and Cdc42 that promotes PKCzeta-dependent transformation by both GTPases. Although it has been suggested that PAK1 may also contribute to transformation by Rac1 in Rat1 fibroblasts, PAK1 does not enhance transformation by activated Raf or activated Rac1 in NIH-3T3 cells, and studies using effector domain mutants indicate that interaction of PAK1 with Rac1 does not correlate with cell-cycle progression or transformation. Thus, hPar-6 appears to be the first effector shown to directly mediate transformation by Rac1 and Cdc42. The identification of PKCzeta as a downstream effector of hPar-6 represents the first elucidation of a signaling pathway linking Rac1/Cdc42 to cell transformation. A model is presented depicting two separate pathways downstream of Ras that lead to cell polarity and growth control: these pathways can contribute to cell transformation. One pathway is comprised of Rac/Cdc42, hPar-6 and PKCzeta, and the other is mediated by Raf, MEK and MAP kinase (Qui, 2000).
The mechanism by which hPar-6 regulates the kinase activity of PKCzeta is currently under investigation. Subcellular targeting by interaction with specific proteins provides an attractive mechanism for PKC isozyme-specific regulation. It is possible that hPar-6 and PKCzeta are translocated by Rac1 or Cdc42 to the membrane, where PKCzeta could interact with an activator. One candidate activator is the phosphatidylinositol 3-kinase (PI3-kinase) target PDK1, since PDK1 and PKCzeta associate in vivo via their catalytic domains, and both PI3-kinase and PDK1 stimulate PKCzeta activity. Consistent with this model, it has been demonstrated that PI3-kinase can act as a link between Ras and Rac in transformation and that membrane-targeted PKCzeta is constitutively active. The observation that hPar-6 alone exhibits little, if any, transforming activity is also consistent with the membrane-targeting model. It should also be noted that although overexpression of hPar-6 alone (i.e., in the absence of Rac1[G12V]) is sufficient to activate PKCzeta kinase activity, overexpression of hPar-6 and PKCzeta only marginally promotes focus formation, suggesting that activated Rac1 is necessary to target PKCzeta to substrates involved in transformation. However, the possibility that Rac1 activates some other pathway that is also necessary for transformation cannot be ruled out. In addition to being activated by hPar-6, PKCzeta might in turn phosphorylate hPar-6. In this regard, it should be noted that there is a putative PKCzeta-phosphorylation site in mammalian Par-6 (Qui, 2000).
The mechanism underlying transformation by hPar-6 and PKCzeta is not yet clear. Stimulation of cell proliferation and inhibition of apoptosis are, however, important characteristics of cell transformation. In this regard, it has been shown that Rac1 and Cdc42 induce cyclin D1 transcription and accumulation, phosphorylation and inactivation of the tumor suppressor protein Rb, and activation of the transcription factor E2F. Inactivation of Rb may be necessary for Rac1/Cdc42 stimulation of cell proliferation, and it is possible that hPar-6 and PKCzeta have a role in this pathway. In addition, Ras, Rac1, Cdc42 and PKCzeta are all able to activate the transcription factor NF-kappaB. NF-kappaB activation is associated with mitogenesis, anti-apoptotic activity and cell transformation. Thus, the hPar-6-PKCzeta pathway might mediate NF-kappaB activation, and thereby contribute to cell transformation by Rac1 and Ras. Another possibility is that the hPar-6-PKCzeta pathway may mediate growth control by Rac1/Cdc42 by inducing downregulation of the pro-apoptotic protein Par-4 (prostate apoptosis response-4; unrelated to the C. elegans Par gene product). Par-4 interacts with PKCzeta and overexpression of PKCzeta downregulates Par-4, an event that appears important for Ras transformation and tumor progression. Thus, cyclin D1, Rb, E2F, NF-kappaB and Par-4 all warrant further investigation as possible downstream targets of the hPar-6-PKCzeta pathway (Qui, 2000).
Polarity is a fundamental feature of all eukaryotic cells. Rac, Cdc42, Par-6 and atypical PKCs appear to be conserved in diverse metazoans, including Drosophila, C. elegans, Xenopus, mouse and humans. The CRIB motif of Par-6 is also conserved, suggesting that it interacts with Rac and/or Cdc42 in these different species. In C. elegans, inhibition of Cdc42 function by RNA-mediated gene interference (RNAi) produces defects in cell polarity similar to those observed in par and pkc-3 mutants, while in mammalian cells, Par-6 is localized to tight junctions, together with atypical PKC and ASIP, the mammalian homolog of Par-3. Moreover in C. elegans, Par-6 interacts with Par-3, and in Drosophila the Par-3 homolog has an important role in the asymmetric cleavage of epithelial cells and neuroblasts. Taken together, these observations suggest that Rac or Cdc42, Par-6, atypical PKC, and perhaps Par-3, constitute a conserved pathway that regulates cell polarity. As hPar-6 and PKCzeta mediate cell transformation by Rac1 and Cdc42, there may be a link between cell-polarity signaling and growth control: aberrant cell-polarity signaling could lead to oncogenic transformation. In the light of the important roles of Rac1/Cdc42 in Ras-induced transformation, hPar-6 and PKCzeta could represent potential targets for anti-cancer therapeutics (Qui, 2000).
Generation of asymmetry in the one-cell embryo of C. elegans establishes the anterior-posterior axis (A-P), and is necessary for the proper identity of early blastomeres. Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry. The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6. The function of Cdc42 in C. elegans had not yet been investigated, however. C. elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins. Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost. CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. It is concluded that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit (Gotta, 2001).
Phenotypic and two-hybrid data suggest that CDC-42 might activate the PAR-3/PAR-6/PKC-3 complex through interaction with PAR-6. CDC-42 appears to be necessary for the activity of the complex as well as for its correct localization. It is also possible that the PAR-3/PAR-6/PKC-3 complex has a role in activating CDC-42, since its initial anterior localization seems CDC-42 independent. One way that CDC-42 and the PAR-3/PAR-6/PKC-3 complex might direct polarity is through the regulation of the actin cytoskeleton. In par-3 mutants and in cdc-42(RNAi) embryos, enrichment of actin at the anterior of early embryos is lost. Further, cosuppression of par-2 and cdc-42(RNAi) mutant phenotypes suggests that CDC-42 and PAR-2 have counterbalancing, antagonistic activities. Because PAR-2 has a RING finger, a motif that has been proposed to be involved in ubiquitin-mediated protein degradation, CDC-42 might normally activate a protein that is a target of PAR-2. Future biochemical and in vivo studies should help to reveal the nature of these interactions and identify downstream targets (Gotta, 2001 and references therein).
Epithelial tubes are a key component of organs and are generated from cells with distinct apico-basolateral polarity. A novel function during tubulogenesis is described for ZEN-4, the Caenorhabditis elegans ortholog of mitotic kinesin-like protein 1 (MKLP1; see Drosophila Pavarotti), and CYK-4, which contains a RhoGAP (GTPase-activating protein) domain. Previous studies have revealed that these proteins comprise centralspindlin (a complex that functions during mitosis to bundle microtubules), construct the spindle midzone, and complete cytokinesis. ZEN-4/MKLP1 functions postmitotically to establish the foregut epithelium. Mutants that lack ZEN-4/MKLP1 express polarity markers but fail to target these proteins appropriately to the cell cortex. Affected proteins include PAR-3/Bazooka and PKC-3/atypical protein kinase C at the apical membrane domain, and HMR-1/cadherin and AJM-1 within C. elegans apical junctions (CeAJ). Microtubules and actin are disorganized in zen-4 mutants compared to the wild-type. It is suggested that ZEN-4/MKLP1 and CYK-4/RhoGAP regulate an early step in epithelial polarization that is required to establish the apical domain and CeAJ (Portereiko, 2004).
The integrity and homeostasis of the vertebrate epidermis depend on various cellular junctions. How these junctions are assembled during development and how their number is regulated remain largely unclear. This study addressed these issues by analysing the function of Lgl2, E-cadherin and atypical Protein kinase C (aPKC) in the formation of hemidesmosomes in the developing basal epidermis of zebrafish larvae. It has been shown that a mutation in lgl2 (penner) prevents the formation of hemidesmosomes. This study shows that Lgl2 function is essential for mediating the targeting of Integrin alpha 6 (Itga6), a hemidesmosomal component, to the plasma membrane of basal epidermal cells. In addition, it was shown that whereas aPKClambda seems dispensable for the localisation of Itga6 during hemidesmosome formation, knockdown of E-cadherin function leads to an Lgl2-dependent increase in the localisation of Itga6. Thus, Lgl2 and E-cadherin act antagonistically to control the localisation of Itga6 during the formation of hemidesmosomes in the developing epidermis (Sonawane, 2009).
How do Lgl2 and E-cadherin, localised at the lateral domain, regulate the formation of hemidesmosomes formed at the basal domain in epidermal cells? It was shown at the lateral domain, Itga6 localises with Lgl2 as well as with E-cadherin. This observation indicates that after its synthesis, a fraction of Itga6 is first targeted to the lateral domain. This lateral Itga6 fraction diminishes by 5 days of development, indicating that Itga6 localisation at the lateral domain is dynamic. In early lgl2 mutant larvae (3.75 days), there is a selective loss of Itga6 localisation at the lateral membrane domain. Moreover, in lgl2 mutant larvae, Itga6 vesicles accumulate in the cytoplasm, especially near the lateral and apical domains. Thus, it is plausible that beyond 3.5 days, a fraction of the Itga6 synthesised is targeted to the lateral membrane domain first and that Lgl2 mediates this targeting. This fraction at the lateral domain then translocates to the basal domain, where it joins the existing Itga6 fraction (localised prior to 3.5 days) clustered at the intermediate filaments, and becomes assembled into functional hemidesmosomes. The translocation of the lateral Itga6 fraction to the basal domain may occur by passive diffusion or by transcytosis. In the latter case, a likely mechanism might be Rab21/Rab5-mediated endocytosis and trafficking of Itga6 from the lateral domain and Rab11-mediated delivery to the basal domain through recycling endosomes. Since, in lgl2 mutant larvae, the Itga6 fraction targeted beyond 3.5 dpf fails to reach the lateral membrane domain and thus also the basal domain, the existing levels of Itga6 at the basal domain (localised before 3.5 dpf) remain insufficient to form functional hemidesmosomes (Sonawane, 2009).
The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work little is known how exocyst function is controlled. To identify regulators for exocyst function, a targeted RNAi screen was performed in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. Six kinase and seven phosphatase genes were found that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. In line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic SNARE proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the PP2A phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular GFP signal for the same proteins. Double RNAi experiments for par-1, pkc-3 and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in post-embryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that aPKC kinase and phosphatase PP2A regulate exocyst-dependent insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process (Jiu, 2013).
During neurogenesis in Xenopus, apicobasally polarised superficial and non-polar deep cells take up different fates: deep cells become primary neurons while superficial cells stay as progenitors. It is not known whether the proteins that affect cell polarity also affect cell fate and how membrane polarity information may be transmitted to the nucleus. This study examined the role of the polarity components, apically enriched aPKC and basolateral Lgl2, in primary neurogenesis. A membrane-tethered form of aPKC (aPKC-CAAX) suppresses primary neurogenesis and promotes cell proliferation. Unexpectedly, both endogenous aPKC and aPKC-CAAX show some nuclear localisation. A constitutively active aPKC fused to a nuclear localisation signal has the same phenotypic effect as aPKC-CAAX in that it suppresses neurogenesis and enhances proliferation. Conversely, inhibiting endogenous aPKC with a dominant-negative form that is restricted to the nucleus enhances primary neurogenesis. These observations suggest that aPKC has a function in the nucleus that is important for cell fate specification during primary neurogenesis. In a complementary experiment, overexpressing basolateral Lgl2 causes depolarisation and internalisation of superficial cells, which form ectopic neurons when supplemented with a proneural factor. These findings suggest that both aPKC and Lgl2 affect cell fate, but that aPKC is a nuclear determinant itself that might shuttle from the membrane to the nucleus to control cell proliferation and fate; loss of epithelial cell polarity by Lgl2 overexpression changes the position of the cells and is permissive for a change in cell fate (Sabherwal, 2009).
The Xenopus oocyte contains components of both the planar cell polarity and apical-basal polarity pathways, but their roles are not known. This study examined the distribution, interactions and functions of the maternal planar cell polarity core protein Vangl2 and the apical-basal complex component aPKC. Vangl2 is distributed in animally enriched islands in the subcortical cytoplasm in full-grown oocytes, where it interacts with a post-Golgi v-SNARE protein, VAMP1, and acetylated microtubules. Vangl2 is required for the stability of VAMP1 as well as for the maintenance of the stable microtubule architecture of the oocyte. Vangl2 interacts with atypical PKC, and both the acetylated microtubule cytoskeleton and the Vangl2-VAMP1 distribution are dependent on the presence of aPKC. aPKC and Vangl2 are required for the cell membrane asymmetry that is established during oocyte maturation, and for the asymmetrical distribution of maternal transcripts for the germ layer and dorsal/ventral determinants VegT and Wnt11. This study demonstrates the interaction and interdependence of Vangl2, VAMP1, aPKC and the stable microtubule cytoskeleton in the oocyte, shows that maternal Vangl2 and aPKC are required for specific oocyte asymmetries and vertebrate embryonic patterning, and points to the usefulness of the oocyte as a model to study the polarity problem (Cha, 2011).
A key feature of early vertebrate development is the formation of superficial, epithelial cells that overlie non-epithelial deep cells. In Xenopus, deep and superficial cells show a range of differences, including a different competence for primary neurogenesis. The two cell populations are generated during the blastula stages by perpendicularly oriented divisions. These take place during several cell divisions, in a variable pattern, but at a percentage that varies little between embryos and from one division to the next. The orientation of division correlates with cell shape, suggesting that simple geometric rules may control the orientation of division in this system. Dividing cells are molecularly polarized such that aPKC is localized to the external, apical, membrane. Membrane localised aPKC can be seen as early as the one-cell stage and during the blastula divisions, it is preferentially inherited by superficial cells. Finally, it has been shown that when 64-cell stage isolated blastomeres divide perpendicularly and the daughters are cultured separately, only the progeny of the cells that inherit the apical membrane turn on the bHLH hairy/enhancer of split related gene ESR6e. It is concluded that oriented cell divisions generate the superficial and deep cells and establish cell fate diversity between them (Chalmers, 2003).
During early C. elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3. Together with the fact that aPKC and a mammalian PAR-3 homolog, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells, this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. The overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. aPKC associates not only with ASIP/PAR-3, but also with a mammalian homolog of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and that aPKC plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells (Suzuki, 2001).
To clairify the molecular basis underlying aPKC function in mammalian epithelial cells, the localization of aPKC and PAR-3 was examined during the cell repolarization process accompanied by wound healing of MTD1-A epithelial cells. Immunofluorescence analysis revealed that PAR-3 and aPKClambda translocate to cell-cell contact regions later than the formation of the primordial spot-like adherens junctions (AJs) containing E-cadherin and ZO-1. Comparison with three tight junction (TJ) membrane proteins, JAM, occludin and claudin-1, further indicates that aPKClambda is one of the last TJ components to be recruited. Consistently, the expression of a dominant-negative mutant of aPKClambda (aPKClambdakn) in wound healing cells does not inhibit the formation of the spot-like AJs; rather, it blocks their development into belt-like AJs. These persistent spot-like AJs in aPKClambda-expressing cells contain all TJ membrane proteins and PAR-3, indicating that aPKC kinase activity is not required for their translocation to these premature junctional complexes but is indispensable for their further differentiation into belt-like AJs and TJs. Cortical bundle formation is also blocked at the intermediate step where fine actin bundles emanating from premature cortical bundles link the persistent spot-like AJs at apical tips of columnar cells. These results suggest that aPKC contributes to the establishment of epithelial cell polarity by promoting the transition of fibroblastic junctional structures into epithelia-specific asymmetric ones (Suzuki, 2002).
Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts -- claudins, occludin, and junctional adhesion molecule (JAM) -- plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca2+ depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays have confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs , but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells (Gao, 2002).
A model is proposed in which Par6 exists in an equilibrium between an inactive and an active state. The active state binds to the PDZ1 domain of Par3 and may block the interaction of Par3 with junctional adhesion molecule (JAM), thereby preventing TJ assembly. The Par3-JAM interaction is proposed to be reversible, but recruitment of further components stabilizes the TJ, so that junctions will eventually form even in the presence of inhibitory Par6. Activation is induced by binding either Cdc42-GTP or the regulatory domain of aPKC. Activation can be artificially induced by removing the N-terminal domain of Par6 (which binds aPKC). This model accounts for the inhibitory effects of the overexpression of wild-type Par6 (which increases the concentration of the active form), of the DeltaN mutant (constitutively active), and of the PKCzeta (1-126) fragment (which will bind to and activate endogenous Par6) and for the reduced potency of Par6BdeltaPro136 (defective in Cdc42 binding). It also accounts for the inhibitory effects of Cdc42 (Q61L) (which will activate endogenous Par6) and of the N-terminal region of Par3 (which will compete with endogenous Par3 for JAM binding). Whether this model is indeed correct will require further investigation (Gao, 2002).
aPKC (atypical protein kinase C), PAR-3 and PAR-6 interact with each other to form a ternary complex and thus mutually regulate their functionality and localization. The biochemical nature of the aPKC-PAR-3 interaction has been investigated in detail to clarify its functional importance in cell polarity. The highly conserved 26 amino acid sequence 816-841, in PAR-3 was found to be necessary and sufficient for the tight association with aPKC. Among several conserved serine/threonine residues within the region, aPKC preferentially phosphorylates serine-827 in vitro, and this phosphorylation reduces the stability of the PAR-3-aPKC interaction. Several analyses using a phospho-serine 827 specific antibody have established that this phosphorylation by aPKC occurs in vivo. Over-expression of a point mutant of PAR-3 (S827A), which is predicted to form a stable complex with aPKC, causes defects in the cell-cell contact-induced cell polarization of epithelial MDCK cells, similarly to a dominant negative mutant of aPKC. These results imply that serine 827 in the aPKC binding site of PAR-3 is a target of aPKC and that the regulated interaction between a protein kinase, aPKC, and its substrate, PAR-3, plays an essential role in the establishment of cell polarity (Nagai-Tamai, 2002).
The MEK5-extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. The aPKCs are shown to interact in an EGF-inducible manner with MEK5; this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, zetaPKC and lambda/iotaPKC are shown to activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, it is concluded that MEK5 is a critical target of the aPKCs during mitogenic signaling (Diaz-Meco, 2001).
Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Many of these PKCs participate in synaptic plasticity in the CA1 region of the hippocampus. Multiple isoforms are transiently activated in the induction phase of long-term potentiation (LTP). In contrast, a single species, zeta, is persistently activated during the maintenance phase of LTP through the formation of an independent, constitutively active catalytic domain, protein kinase Mzeta (PKMzeta). Immunoblot and immunocytochemical techniques with isoform-specific antisera have been used to examine the distribution of the complete family of PKC isozymes and PKMzeta in rat brain. Each form of PKC shows a widespread distribution in the brain with a distinct regional pattern of high and low levels of expression. PKMzeta, the predominant form of PKM in brain, has high levels in hippocampus, frontal and occipital cortex, striatum, and hypothalamus. In the hippocampus, each isoform is expressed in a characteristic pattern, with zeta prominent in the CA1 stratum radiatum. These results suggest that the compartmentalization of PKC isoforms in neurons may contribute to their function, with the location of PKMzeta prominent in areas notable for long-term synaptic plasticity (Naik, 2000).
aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. In contrast, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. PAR-1b is shown to be essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. In contrast, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity (Suzuki, 2004).
In the C. elegans one-cell embryo as well as the Drosophila late oocyte, the complex segregate along the A-P axis: the aPKC/PAR-3/PAR-6 complex then localizes at the anterior cortex, whereas PAR-1 is at the posterior cortex. In Drosophila and mammalian epithelial cells, the complex segregates along the apicobasal axis: PAR-1 localizes at the basolateral membrane in contrast with the apical localization of the aPKC/PAR-3/PAR-6 complex. These observations raise questions whether the functional hierarchy of the aPKC/PAR-3/PAR-6 complex and PAR-1 is conserved evolutionarily. The functional relationship between aPKC, PAR-1b, and 14-3-3/PAR-5 suggested in this study is different from that suggested for Drosophila epithelial cells. In Drosophila follicle cells, PAR-1 inhibits the lateral invasion of aPKC, and the phospho-motif binding site of 14-3-3 binds to BAZ. These differences suggest the possibility that mammals and flies independently evolved similar but distinct mechanisms that regulate epithelial cell polarity using aPKC/PAR proteins. However, it is also possible that mutual regulations between PAR-1 and aPKC occur in both organisms, because most of the results in each study are not completely exclusive. For example, although the current study observed that PAR-1b depletion from MDCK cells did not induce the lateral leakage of aPKC and PAR-3, the possibility cannot be excluded that other mammalian PAR-1 proteins compensate for the PAR-1b function in these cells. To address this issue, perfectly corresponding experiments should be performed in each organism (Suzuki, 2004).
The establishment and maintenance of cellular polarity are essential biological processes that must be maintained throughout the lifetime of eukaryotic organisms. The Par-1 protein kinases are key polarity determinants that have been conserved throughout evolution. Par-1 directs anterior-posterior asymmetry in the one-cell C. elegans embryo and the Drosophila oocyte. In mammalian cells, Par-1 may regulate epithelial cell polarity. Relevant substrates of Par-1 in these pathways are just being identified, but it is not yet known how Par-1 itself is regulated. Human Par-1b (hPar-1b) has been shown to interacts with and is negatively regulated by atypical PKC. hPar-1b is phosphorylated by aPKC on threonine 595, a residue conserved in Par-1 orthologs in mammals, worms, and flies. The equivalent site in hPar-1a, T564, is phosphorylated in vivo and by aPKC in vitro. Importantly, phosphorylation of hPar-1b on T595 negatively regulates the kinase activity and plasma membrane localization of hPar-1b in vivo. This study establishes a novel functional link between two central determinants of cellular polarity, aPKC and Par-1, and suggests a model by which aPKC may regulate Par-1 in polarized cells. These findings suggest that one mechanism by which atypical PKC exerts its effect on the generation and/or maintenance of polarity may be to actively exclude Par-1 from particular subcellular compartments. Presumably the localization of Par-1 to different cellular domains would also allow it to interact with a different set of relevant effectors. Thus, phosphorylation of Par-1 by aPKC may enforce the mutual exclusion of Par-1 from the anterior cortex of C. elegans embryos and from epithelial cell tight junctions where Par-3/Par-6/aPKC complexes reside (Hurov, 2004).
The atypical protein kinase C is a key regulator of polarity and cell fate in lower organisms. However, whether mammalian aPKCs control stem cells and fate in vivo is not known. This study shows that loss of aPKCλ in a self-renewing epithelium, the epidermis, disturbs tissue homeostasis, differentiation, and stem cell dynamics, causing progressive changes in this tissue. This was accompanied by a gradual loss of quiescent hair follicle bulge stem cells and a temporary increase in proliferating progenitors. Lineage tracing analysis showed that loss of aPKCλ alters the fate of lower bulge/hair germ stem cells. This ultimately leads to loss of proliferative potential, stem cell exhaustion, alopecia, and premature aging. Inactivation of aPKCλ produces more asymmetric divisions in different compartments, including the bulge. Thus, aPKClambda is crucial for homeostasis of self-renewing stratifying epithelia, and for the regulation of cell fate, differentiation, and maintenance of epidermal bulge stem cells likely through its role in balancing symmetric and asymmetric division (Niessen, 2013).
An increasing number of independent studies indicate that the atypical protein kinase C (PKC) isoforms (aPKCs) are critically involved in the control of cell proliferation and survival. The aPKCs are targets of important lipid mediators such as ceramide and the products of the PI 3-kinase. In addition, the aPKCs have been shown to interact with Ras and with two novel proteins, LIP (lambda-interacting protein; a selective activator of lambda/iotaPKC) and the product of par-4 (a gene induced during apoptosis), which is an inhibitor of both lambda/iotaPKC and zetaPKC. LIP and Par-4 interact with the zinc finger domain of the aPKCs where the lipid mediators have been shown to bind. p62, a previously described phosphotyrosine-independent p56(lck) SH2-interacting protein, interacts potently with the V1 domain of lambda/iotaPKC and, albeit with lower affinity, with zetaPKC. Ectopically expressed p62 colocalizes perfectly with both lambda/iotaPKC and zetaPKC. Interestingly, the endogenous p62, like the ectopically expressed protein, displays a punctate vesicular pattern and clearly colocalizes with endogenous lambda/iotaPKC and endogenous zetaPKC. P62 colocalizes with Rab7 and partially with lamp-1 and limp-II as well as with the epidermal growth factor (EGF) receptor in activated cells, but not with Rab5 or the transferrin receptor. Of functional relevance, expression of dominant negative lambda/iotaPKC, but not of the wild-type enzyme, severely impairs the endocytic membrane transport of the EGF receptor with no effect on the transferrin receptor. These findings strongly suggest that the aPKCs are anchored by p62 in the lysosome-targeted endosomal compartment; this seems critical for the control of growth factor receptor trafficking. This is particularly relevant in light of the role played by the aPKCs in mitogenic cell signaling events (Sanchez, 1998).
The two members of the atypical protein kinase C (aPKC) subfamily of isozymes (zetaPKC and lambda/iotaPKC) are involved in the control of NF-kappaB through IKKbeta activation. The previously described aPKC-binding protein, p62, selectively interacts with RIP but not with TRAF2 in vitro and in vivo. p62 bridges the aPKCs to RIP, whereas the aPKCs link IKKbeta to p62. In this way, a signaling cascade of interactions is established from the TNF-R1 involving TRADD/RIP/p62/aPKCs/IKKbeta. These observations define a novel pathway for the activation of NF-kappaB involving the aPKCs and p62. Consistent with this model, the expression of a dominant-negative mutant lambda/iotaPKC impairs RIP-stimulated NF-kappaB activation. In addition, the expression of either an N-terminal aPKC-binding domain of p62, or its C-terminal RIP-binding region are sufficient to block NF-kappaB activation. Furthermore, transfection of an antisense construct of p62 severely abrogates NF-kappaB activation. Together, these results demonstrate that the interaction of p62 with RIP serves to link the atypical PKCs to the activation of NF-kappaB by the TNFalpha signaling pathway (Sanz, 1999).
The atypical protein kinase C (aPKC)-interacting protein, p62, interacts with RIP, linking these kinases to NF-kappaB activation by tumor necrosis factor alpha (TNFalpha). The aPKCs have been implicated in the activation of IKKbeta in TNFalpha-stimulated cells and have been shown to be activated in response to interleukin-1 (IL-1). The inhibition of the aPKCs or the down-regulation of p62 severely abrogates NF-kappaB activation by IL-1 and TRAF6, suggesting that both proteins are critical intermediaries in this pathway. Consistent with this, p62 is shown to selectively interact with the TRAF domain of TRAF6 but not that of TRAF5 or TRAF2 in co-transfection experiments. The binding of endogenous p62 to TRAF6 is stimulus dependent, reinforcing the notion that this is a physiologically relevant interaction. Furthermore, the N-terminal domain of TRAF6, which is required for signaling, interacts with zetaPKC in a dimerization-dependent manner. Together, these results indicate that p62 is an important intermediary not only in TNFalpha but also in IL-1 signaling to NF-kappaB through the specific adapters RIP and TRAF6 (Sanz, 2000).
Atypical protein kinase Cs zeta and lambda/iota play a functional role in the regulation of NGF-induced differentiation and survival of pheochromocytoma, PC12 cells. An NGF-dependent interaction of aPKC with its binding protein, ZIP/p62, has been demonstrated. Although, ZIP/p62 is not a PKC-iota substrate, the formation of a ZIP/p62-aPKC complex in PC12 cells by NGF occurs post activation of PKC-iota and is regulated by the tyrosine phosphorylation state of aPKC. Furthermore, NGF-dependent localization of ZIP/p62 is observed within vesicular structures, identified as late endosomes by colocalization with a Rab7 antibody. Both ZIP/p62 as well as PKC-iota colocalize with Rab7 upon NGF stimulation. Inhibition of the tyrosine phosphorylation state of PKC-iota does not prevent movement of ZIP/p62 to the endosomal compartment. These observations indicate that the subcellular localization of ZIP/p62 does not depend entirely upon activation of aPKC itself. Of functional importance, transfection of an antisense p62 construct into PC12 cells significantly diminishes NGF-induced neurite outgrowth. Collectively, these findings demonstrate that ZIP/p62 acts as a shuttling protein involved in routing activated aPKC to an endosomal compartment and is required for mediating NGF's biological properties (Samuels, 2001).
Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor kappaB (NF-kappaB). The atypical protein kinase C-interacting protein, p62, that binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. The formation is demonstrated of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, p62 serves as a scaffold for activation of the NF-kappaB pathway, which mediates NGF survival and differentiation responses (Wooten, 2001).
It has been reported that prostate apoptosis response-4 (PAR-4) binds to and inhibits protein kinase Czeta (PKCzeta) which phosphorylates IkappaB kinase beta (IKKbeta) for nuclear factor kappaB (NFkappaB) activation, while p62 binds to and recruits PKCzeta to the NFkappaB signaling complex. Thus, a mechanism to coordinate the two binding proteins for the regulation of PKCzeta is expected to exist. The present data show that p62 and PAR-4 do not compete for PKCzeta binding but directly interact with one another and form a ternary complex with PKCzeta. Furthermore, p62 not only enhances the catalytic activity of PKCzeta but also reactivates catalytically inactive PAR-4-bound PKCzeta. As the result, over-expression of p62 protects cells from PAR-4-mediated inactivation of NFkappaB and apoptotic death. Thus, the regulatory role of p62 for free and PAR-4-bound PKCzeta is important in activation of NFkappaB (Chang, 2002).
Maximal activation of NADPH oxidase requires formation of a complex between the p40phox and p67phox subunits via association of their PB1 domains. The crystal structure has been determined of the p40phox/p67phox PB1 heterodimer; the structure reveals that both domains have a β grasp topology and that they bind in a front-to-back arrangement through conserved electrostatic interactions between an acidic OPCA motif [the short sequence motif present in some PB1 domains, that previously has been referred to as the octicosapeptide repeat (OPR), PC motif (phox and cdc24p), and the AID motif (atypical protein kinase C-interaction domain)] on p40phox and basic residues in p67phox. The structure enabled the identification of residues critical for heterodimerization among other members of the PB1 domain family, including the atypical protein kinase Cζ (PKCζ) and its partners Par6 and p62 (ZIP, sequestosome). Both Par6 and p62 use their basic 'back' to interact with the OPCA motif on the 'front' of the PKCζ. Besides heterodimeric interactions, some PB1 domains, like the p62 PB1, can make homotypic front-to-back arrays (Wilson, 2003).
In order to resolve which interfaces p62, Par6, and PKCζ actually use in formation of heterodimeric complexes, site-specific mutants of these proteins were constructed. Using GST pull-down binding assays, it was found that PKCζ interacts with both Par6 and p62 only when it has a wild-type OPCA motif on its front. Mutation D62A/D66A in the OPCA motif of PKCζ abolishes binding to both Par6 and p62 PB1 domains. The same mutation affects PKCζ function in vivo. In contrast, a point mutation of a basic residue in the PKCζ 'back' (equivalent to Lys 355p67) has no influence on binding to wild-type PB1 domains from p62 and Par6. This suggests that PKCζ uses its acidic front to interact with the basic back of Par6 and p62. Consistent with this notion, mutation of a single basic residue at the back of either Par6 or p62 PB1 domains eliminates interaction with wild-type PKCζ, whereas mutations of the acidic cluster at the front of these adaptors have no impact on binding to PKCζ. These results suggest that binding of the adaptor proteins p62 and Par6 to PKCζ is mutually exclusive. Indeed, this is confirmed in direct competition experiments (Wilson, 2003).
Epithelial cells have apicobasal polarity and an asymmetric junctional complex that provides the bases for development and tissue maintenance. In both vertebrates and invertebrates, the evolutionarily conserved protein complex, PAR-6/aPKC/PAR-3, localizes to the subapical region and plays critical roles in the establishment of a junctional complex and cell polarity. In Drosophila, another set of proteins called tumor suppressors, such as Lgl, which localize separately to the basolateral membrane domain but genetically interact with the subapical proteins, also contribute to the establishment of cell polarity. However, how physically separated proteins interact remains to be clarified. Mammalian Lgl is shown to compete for PAR-3 in forming an independent complex with PAR-6/aPKC. During cell polarization, mLgl initially colocalizes with PAR-6/aPKC at the cell-cell contact region and is phosphorylated by aPKC, followed by segregation from apical PAR-6/aPKC to the basolateral membrane after cells are polarized. Overexpression studies establish that increased amounts of the mLgl/PAR-6/aPKC complex suppress the formation of epithelial junctions; this contrasts with a previous observation that the complex containing PAR-3 promotes it.These results indicate that PAR-6/aPKC selectively interacts with either mLgl or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity (Yamanaka, 2003).
Thus evidence is presented showing that the PAR-6β/aPKCλ complex interacts with either mLgl or PAR-3 in a mutually exclusive manner, forming two independent protein complexes. Notably, overexpression of mLgl-2 inhibits TJ formation; this finding is in direct contrast with the data found for PAR-3, whose overexpression, but not that of its mutant lacking the aPKC binding region, promotes TJ formation. This suggests that the two independent complexes have distinct functions in the establishment of epithelial cell polarity. This is consistent with the results of genetic studies of Drosophila in which Lgl is required for formation of the basolateral membrane domain through the inhibition of the formation of apical identity, whereas subapical Bazooka (PAR-3) is required for the formation of the apical membrane domain (Yamanaka, 2003).
In polarized epithelial cells, mLgl localizes to the lateral region, in contrast to the PAR-6β/aPKCλ/PAR-3 complex that localizes to the apical end of the lateral domain. Interestingly, mLgl-2 transiently codistributes with PAR-6β and aPKCλ during the initial phase of epithelial cell polarization, whereas PAR-3 stably codistributes with them at the apical end of the cell-cell contact region; this finding indicates that the balance between the two independent complexes changes during the initial phase of epithelial cell polarization. Further, overexpression of aPKCλ kn (kinase deficient aPKC) results in the abnormal codistribution of PAR-6β and mLgl-2 at the cell periphery; this finding suggests that aPKCλ activity is required for the segregation of PAR-6β and mLgl-2 localization during this process. Thus, the present results, as well as previous findings, led to the following working model. The cell-cell contact initially stimulates the localization of the protein complex containing PAR-6β, aPKCλ, and mLgl at the cell-cell contact region. The complex is 'inactive' for TJ formation. Once aPKCλ is activated, mLgl segregates from the PAR-6β/aPKCλ complex. This triggers the formation of the 'active' PAR-6β/aPKCλ/PAR-3 complex that promotes the formation of the epithelial junctional complex. Segregated mLgl remains in the lateral region and seems to contribute to the establishment of the basolateral membrane identity, because mLgl-1 has been reported to interact with syntaxin-4, a component of the basolateral exocytic machinery. Although the mechanism for activation of aPKCλ remains to be clarified, Cdc42 and/or Rac1 are strong candidates as activators of aPKC in MDCK cells, since the GTP-bound form of Cdc42 activates aPKCλ kinase activity through PAR-6 in vitro and cell-cell adhesion activates Cdc42 and Rac1 in epithelial cells (Yamanaka, 2003).
mLgl is phosphorylated by aPKCλ and this phosphorylation increases in response to cell-cell adhesion-mediated cell polarization. Further, a phosphomimicking mutant of mLgl-2 (3SE) fails to bind to aPKCλ. These results imply that aPKCλ-dependent phosphorylation of mLgl is involved in the regulation of its interaction with the PAR-6β/aPKCλ complex. In contrast, no difference could be detected between mLgl-2 wild-type and its 3SE mutant in their interactions with the PDZ domain of PAR-6β. In addition, overexpression of mLgl-2 mutants (3SA or 3SE) affects TJ formation similarly to that of wild-type. These results suggest the existence of another mechanism regulating the interaction of mLgl-2 with PAR-6β. Mammalian Crumbs/Stardust (Pals1) has been shown to interact with the PDZ domain of PAR-6β and this interaction is enhanced by activated Cdc42. Taken together with the present results, this suggests that the Crumbs/Pals1 complex might also be involved in the regulation of the interaction between mLgl and the PAR-6β/aPKCλ complex; the PAR-6β/aPKCλ complex, together with PAR-3, may involve the Crumbs/Pals1 complex to promote TJ formation. Thus, the dissociation of mLgl from the PAR-6β/aPKCλ complex likely triggers the interaction of the PAR-6β/aPKCλ complex with the Crumbs/Pals1 complex in addition to its interaction with PAR-3. The functional interactions proposed by this model are consistent with the results of recent genetic studies of Drosophila in which Lgl and Crumbs compete with each other to define respective membrane identity (Yamanaka, 2003).
The findings in this study suggest a notable analogy in the mechanism regulating epithelial polarity between Drosophila and mammals. This notion is supported by a recent observation in Drosophila that Lgl interacts with PAR-6 and aPKC and regulates the apicobasal polarity of Drosophila neuroblasts. In mammalian fibroblasts, mLgl-1 has been reported to form a protein complex with PAR-6α (also called PAR-6C) and aPKC and is involved in the polarized migration of wounded MEF cells. Further studies will further an understanding of the molecular mechanism underlying the establishment of cell polarity in a variety of biological contexts (Yamanaka, 2003).
Thus mammalian Lgl competes for PAR-3 in forming an independent protein complex with PAR-6 and aPKC in epithelial cells. During epithelial cell polarization, mLgl transiently colocalizes with PAR-6 and aPKC at the cell-cell contact region, and increased localization of mLgl and PAR-6 to the cell-cell contact region suppresses TJ formation. This finding contrasts with the data found for PAR-3, which promotes TJ formation and thus indicates that the balance between the two independent protein complexes regulates the establishment of epithelial cell polarity. It is also suggested that aPKC activity-mediated phosphorylation of mLgl is involved in the regulation of mLgl's interaction with PAR-6/aPKC. These findings provide new insight into the mechanism underlying the establishment of epithelial cell polarity (Yamanaka, 2003).
In early vertebrate development, apicobasally polarised blastomeres divide to produce inner non-polarised cells and outer polarised cells that follow different fates. How the polarity of these early blastomeres is established is not known. The role of Crumbs3, Lgl2 and the apical aPKC in the polarisation of frog blastomeres was examined. Lgl2 localises to the basolateral membrane of blastomeres, while Crumbs3 localises to the apical and basolateral membranes. Overexpression aPKC and Crumbs3 expands the apical domain at the expense of the basolateral and repositions tight junctions in the new apical-basolateral interface. Loss of aPKC function causes loss of apical markers and redirects basolateral markers ectopically to the apical membrane. Cell polarity and tight junctions, but not cell adhesion, are lost and outer polarised cells become inner-like apolar cells. Overexpression of Xenopus Lgl2 phenocopies the aPKC knockout, suggesting that Lgl2 and aPKC act antagonistically. This was confirmed by showing that aPKC and Lgl2 can inhibit the localisation of each other and that Lgl2 rescues the apicalisation caused by aPKC. It is concluded that an instrumental antagonistic interaction between aPKC and Lgl2 defines apicobasal polarity in early vertebrate development (Chalmers, 2005).
Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell-cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E-cadherin in stratified epithelium of the epidermis, its gene was conditionally inactivated in mice. Loss of E-cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, the results indicate that E-cadherin is specifically required for tight junction (but not desmosome) formation and this appears to involve signalling rather than cell contact formation (Tunggal, 2005).
Mammalian polarity proteins have been studied predominantly in cell culture systems, and little is known about their functions in vivo. To address this issue, a shRNA lentiviral system was used to manipulate gene expression in mouse mammary stem/progenitor cells. Transplantation of Par3-depleted stem/progenitor cells into the mammary fat pad severely disrupted mammary development, and glands were characterized by ductal hyperplasia, luminal filling, and highly disorganized end bud structures that were unable to remodel into normal ductal structures. Unexpectedly, Par3-depleted mammary glands also had an expanded progenitor population. A novel function was identified for the atypical protein kinase C (aPKC)-binding domain of Par3 in restricting Par3 and aPKC to the apical region in mammary epithelia in vivo; it was found that mammary morphogenesis is dependent on the ability of Par3 to directly bind aPKC. These results reveal a new function for Par3 in the regulation of progenitor differentiation and epithelial morphogenesis in vivo and demonstrate for the first time an essential requirement for the Par3-aPKC interaction (McCaffrey, 2009).
Regulation of cell polarity is an important biological event that governs diverse cell functions such as localization of embryonic determinants and establishment of tissue and organ architecture. The Rho family GTPases and the polarity complex Par6/Par3/atypical protein kinase C (PKC) play a key role in the signaling pathway, but the molecules that regulate upstream signaling are still not known. The guanine nucleotide exchange factor ECT2 (Drosophila homolog: Peeble) has been identified as an activator of the polarity complex. ECT2 interacts with Par6 as well as Par3 and PKCzeta. Coexpression of Par6 and ECT2 efficiently activates Cdc42 in vivo. Overexpression of ECT2 also stimulates the PKCzeta activity, whereas dominant-negative ECT2 inhibits the increase in PKCzeta activity stimulated by Par6. ECT2 localization was detected at sites of cell-cell contact as well as in the nucleus of MDCK cells. The expression and localization of ECT2 are regulated by calcium, which is a critical regulator of cell-cell adhesion. Together, these results suggest that ECT2 regulates the polarity complex Par6/Par3/PKCzeta and possibly plays a role in epithelial cell polarity (Liu, 2004).
During mouse pre-implantation development, extra-embryonic primitive endoderm (PrE) and pluripotent epiblast precursors are specified in the inner cell mass (ICM) of the early blastocyst in a 'salt and pepper' manner, and are subsequently sorted into two distinct layers. Positional cues provided by the blastocyst cavity are thought to be instrumental for cell sorting; however, the sequence of events and the mechanisms that control this segregation remain unknown. This study shows that atypical protein kinase C (aPKC), a protein associated with apicobasal polarity, is specifically enriched in PrE precursors in the ICM prior to cell sorting and prior to overt signs of cell polarisation. aPKC adopts a polarised localisation in PrE cells only after they reach the blastocyst cavity and form a mature epithelium, in a process that is dependent on FGF signalling. To assess the role of aPKC in PrE formation, its activity was interfered with using either chemical inhibition or RNAi knockdown. Inhibition of aPKC from the mid blastocyst stage not only prevents sorting of PrE precursors into a polarised monolayer but concomitantly affects the maturation of PrE precursors. These results suggest that the processes of PrE and epiblast segregation, and cell fate progression are interdependent, and place aPKC as a central player in the segregation of epiblast and PrE progenitors in the mouse blastocyst (Saiz, 2013).
Formation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, misorientation creates multiple lumens. This study shows that silencing of the polarity protein Par3 causes spindle misorientation in Madin-Darby canine kidney cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3 or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects. It is concluded that the Par3 and aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation (Hao, 2010).
Phosphorylation of the polarity protein Par-3 by the serine/threonine kinases aPKCzeta/iota and Par-1 (EMK1/MARK2) regulates various aspects of epithelial cell polarity, but little is known about the mechanisms by which these posttranslational modifications are reversed. This study finds that the serine/threonine protein phosphatase PP1 (predominantly the alpha isoform) binds Par-3, which localizes to tight junctions in MDCKII cells. PP1alpha can associate with multiple sites on Par-3 while retaining its phosphatase activity. By using a quantitative mass spectrometry-based technique, multiple reaction monitoring, it was shown that PP1alpha specifically dephosphorylates Ser-144 and Ser-824 of mouse Par-3, as well as a peptide encompassing Ser-885. Consistent with these observations, PP1alpha regulates the binding of 14-3-3 proteins and the atypical protein kinase C (aPKC) zeta to Par-3. Furthermore, the induced expression of a catalytically inactive mutant of PP1alpha severely delays the formation of functional tight junctions in MDCKII cells. Collectively, these results show that Par-3 functions as a scaffold, coordinating both serine/threonine kinases and the PP1alpha phosphatase, thereby providing dynamic control of the phosphorylation events that regulate the Par-3/aPKC complex (Traweger, 2008).
Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, evidence is provided of involvement of the atypical zeta protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous zeta protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of zeta protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks zeta but not alpha protein kinase C plasma membrane translocation. Synthetic peptides from zeta protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of zeta but not alpha protein kinase C. These data suggest the involvement of zeta protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation (Laudanna, 1998).
Long-term potentiation in the CA1 region of the hippocampus, a model for memory formation in the brain, is divided into two phases. A transient process (induction) is initiated, which then generates a persistent mechanism (maintenance) for enhancing synaptic strength. Protein kinase C (PKC), a gene family of multiple isozymes, may play a role in both induction and maintenance. In region CA1 from rat hippocampal slices, most of the isozymes of PKC translocated to the particulate fraction 15 sec after a tetanus. The increase of PKC in the particulate fraction does not persist into the maintenance phase of long-term potentiation. In contrast, a constitutively active kinase, PKM, a form specific to a single isozyme (zeta), increases in the cytosol during the maintenance phase. The transition from translocation of PKC to formation of PKM may help to explain the molecular mechanisms of induction and maintenance of long-term potentiation (Sacktor, 1993).
The maintenance of long-term potentiation (LTP) in the CA1 region of the hippocampus has been reported to require both a persistent increase in phosphorylation and the synthesis of new proteins. The increased activity of protein kinase C (PKC) during the maintenance phase of LTP may result from the formation of PKMzeta, the constitutively active fragment of a specific PKC isozyme. To define the relationship among PKMzeta, long-term EPSP responses, and the requirement for new protein synthesis, the regulation of PKMzeta was examined after sub-threshold stimulation that produced short-term potentiation (STP) and after suprathreshold stimulation by single and multiple tetanic trains that produced LTP. Although no persistent increase in PKMzeta follows STP, the degree of long-term EPSP potentiation is linearly correlated with the increase of PKMzeta. The increase is first observed 10 min after a tetanus that induces LTP and lasts for at least 2 hr, in parallel with the persistence of EPSP enhancement. Both the maintenance of LTP and the long-term increase in PKMzeta++ are blocked by the protein synthesis inhibitors anisomycin and cycloheximide. These results suggest that PKMzeta is a component of a protein synthesis-dependent mechanism for persistent phosphorylation in LTP (Osten, 1996).
The possibility that protein kinase C (PKC) is involved in the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) at CA1 synapses in the hippocampus has been the subject of considerable investigation. However, many of the conclusions have been drawn from the use of relatively nonspecific PKC inhibitors. The role of PKC in tetanus-induced LTP of AMPA receptor-mediated synaptic transmission has been examined in hippocampal slices obtained from adult rats. In particular, the possible role of PKC was investigated in a molecular switch process that is triggered by the synaptic activation of metabotropic glutamate receptors and regulates the induction of LTP. The three PKC inhibitors examined, chelerythrine, Ro-31-8220 and Go 6983, all block the setting of the molecular switch at concentrations consistent with inhibition of PKC. In contrast, these inhibitors are without affect on the induction of LTP, even when applied in very much higher concentrations. A PKA inhibitor, Rp-cAMPS, has no effect on either process. It is suggested that neither PKC nor PKA is required to induce LTP at this synapse. However, PKC is involved in the regulation of LTP induction, via the molecular switch process (Bortolotto, 2000).
Persistent dephosphorylation has been implicated in the molecular mechanisms of long-term depression (LTD). Dephosphorylation may be due to either a persistent increase in phosphatase activity or a persistent decrease in kinase activity. Protein kinase Mzeta (PKMzeta), the autonomously active form of the atypical PKCzeta isozyme that increases in long-term potentiation (LTP), decreases in LTD. This is consistent with the hypothesis that decreased levels of phosphorylation by PKC are important in LTD. Recently, however, increased phosphorylation by PKC has also been implicated in LTD. These contradictory results might be explained, in part, by the multiple isoforms of PKC, which may be independently regulated during the different phases of LTD. It has been found that 45 s after low-frequency (3 Hz) stimulation that induces LTD in the CA1 region of hippocampal slices, conventional Ca(2+)/lipid-dependent PKC isoforms translocate from the cytosol to the membrane. This translocation is transient, lasting less than 15 min. In contrast, PKMzeta is persistently decreased through 2 h of LTD maintenance. Therefore, the activation and downregulation of distinct PKC isoforms may participate in the induction and maintenance mechanisms of LTD (Hrabetova, 2001).
Long-term potentiation (LTP), a persistent synaptic enhancement thought to be a substrate for memory, can be divided into two phases: induction, triggering potentiation, and maintenance, sustaining it over time. Many postsynaptic events are implicated in induction, including N-methyl-D-aspartate receptor (NMDAR) activation, calcium increases and stimulation of several protein kinases; in contrast, the mechanism maintaining LTP is not yet characterized1. The constitutively active form of an atypical protein kinase C (PKC) isozyme, protein kinase M zeta (PKMzeta), is necessary and sufficient for LTP maintenance (Ling, 2002).
Protein kinase M zeta (PKM zeta) is a newly described form of PKC that is necessary and sufficient for the maintenance of hippocampal long term potentiation (LTP) and the persistence of memory in Drosophila. PKM zeta is the independent catalytic domain of the atypical PKC zeta isoform and produces long term effects at synapses because it is persistently active, lacking autoinhibition from the regulatory domain of PKC zeta. PKM has been thought of as a proteolytic fragment of PKC. Brain PKM zeta is a new PKC isoform, synthesized from a PKM zeta mRNA encoding a PKC zeta catalytic domain without a regulatory domain. Multiple zeta-specific antisera show that PKM zeta is expressed in rat forebrain as the major form of zeta in the near absence of full-length PKC zeta. A PKC zeta knockout mouse, in which the regulatory domain was disrupted and catalytic domain spared, still expresses brain PKM zeta, indicating that this form of PKM is not a PKC zeta proteolytic fragment. Furthermore, the distribution of brain PKM zeta does not correlate with PKC zeta mRNA but instead with an alternate zeta RNA transcript thought incapable of producing protein. In vitro translation of this RNA, however, generates PKM zeta of the same molecular weight as that in brain. Metabolic labeling of hippocampal slices shows increased de novo synthesis of PKM zeta in LTP. Because PKM zeta is a kinase synthesized in an autonomously active form and is necessary and sufficient for maintaining LTP, it serves as an example of a link coupling gene expression directly to synaptic plasticity (Hernandez, 2004).
Protein kinase Mzeta (PKMzeta) is an atypical protein kinase C isoform that has been implicated in the protein synthesis-dependent maintenance of long term potentiation and memory storage in the brain. Synapse-associated kinases are uniquely positioned to promote enduring consolidation of structural and functional modifications at the synapse, provided that kinase mRNA is available on site for local input-specific translation. This study reporta that the mRNA encoding PKMzeta is rapidly transported and specifically localized to synaptodendritic neuronal domains. Transport of PKMzeta mRNA is specified by two cis-acting dendritic targeting elements (Mzeta DTEs). Mzeta DTE1, located at the interface of the 5'-untranslated region and the open reading frame, directs somato-dendritic export of the mRNA. Mzeta DTE2, in contrast, is located in the 3'-untranslated region and is required for delivery of the mRNA to distal dendritic segments. Colocalization with translational repressor BC1 RNA in hippocampal dendrites suggests that PKMzeta mRNA may be subject to translational control in local domains. Dendritic localization of PKMzeta mRNA provides a molecular basis for the functional integration of synaptic signal transduction and translational control pathways (Muslimov, 2004).
Little is known about the neuronal mechanisms that subserve long-term memory persistence in the brain. The components of the remodeled synaptic machinery, and how they sustain the new synaptic or cellwide configuration over time, are yet to be elucidated. In the rat cortex, long-term associative memories vanished rapidly after local application of an inhibitor of the protein kinase C isoform, protein kinase M zeta (PKMzeta). The effect was observed for at least several weeks after encoding and may be irreversible. In the neocortex, which is assumed to be the repository of multiple types of long-term memory, persistence of memory is thus dependent on ongoing activity of a protein kinase long after that memory is considered to have consolidated into a long-term stable form (Shema, 2007).
Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral (BL) surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. It is concluded that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation (Martin-Belmonte, 2007).
Formation of the apical surface and lumen is a central problem in understanding how epithelial tissues arrange themselves into tubes and other hollow structures, such as cysts. This study has uncovered a molecular mechanism of AP surface and lumen formation. PTEN is needed for segregation of PtdIns(4,5)p2 to the apical plasma membrane (PM) and PtdIns(3,4,5)p3 to the BL PM. Apical PtdIns(4,5)p2 recruits Anx2, which in turn recruits Cdc42 to the apical PM, causing the organization of the sub-apical actin cytoskeleton and formation of the apical surface and lumen. Cdc42 binds and localizes the Par6/aPKC complex to the apical PM to promote establishment of polarity (Martin-Belmonte, 2007).
PtdIns(4,5)p2 is thus a key determinant of the apical surface. Similarly, PtdIns(3,4,5)p3 is a key determinant of the BL surface (Gassama-Diagne, 2006). Together, PtdIns(4,5)p2 and PtdIns(3,4,5)p3 play complementary roles in epithelial polarity. More generally, phosphoinositides have emerged as general determinants of membrane identity. An advantage of epithelia is that exogenous phosphoinositides can be inserted into limited ectopic locations. These gain-of-function experiments provide a direct test of the role of the lipid in specifying domain identity (Martin-Belmonte, 2007).
To exert its effects, PtdIns(4,5)p2 interacts with Anx2, which clusters this lipid with high affinity and specificity. Ectopic PtdIns(4,5)p2 in the BL surface recruits Anx2 to the BL PM. Although loss of Anx2 by RNAi prevents lumen formation, RNAi of Anx2 did not produce as strong a phenotype as expression of the DN Anx2CM or RNAi of PTEN or Cdc42. One explanation could be the existence of >20 annexin family members, which might have redundancy with each other. Indeed, Anx2 knockout (KO) mice are viable. Alternatively, even low levels of Anx2 might suffice to exert its function (Martin-Belmonte, 2007).
Cdc42 interacts with Anx2 in a GTP-dependent manner. Cdc42 is activated during cystogenesis. Most activated Cdc42 relocalizes from cell-cell contacts to the apical pole as lumens form. RNAi of Cdc42 caused malformation of the central lumen in cysts but did not affect polarization of MDCK cells in 2D monolayers. This effect of Cdc42 depletion in cysts highlights the importance of using 3D models in analysis of lumen formation. Interestingly, an intracellular accumulation of Anx2 was seen in the cells with reduced Cdc42, suggesting a potential positive feedback loop whereby Cdc42 and Anx2 each promote the localization of the other at the lumen of mature cysts. Anx2 might work by recruiting Cdc42 or a GEF for Cdc42, and this GEF may in turn activate Cdc42 at this location. In contrast, active Cdc42 might promote the exocytosis of Anx2 and other apical proteins (Martin-Belmonte, 2007).
Formation of the apical surface and lumen has been suggested to be mediated by exocytosis of large intracellular vacuoles, termed vacuolar apical compartment (VAC). Formation of endothelial lumens occurs by vacuolar exocytosis. Cdc42 is needed for the exocytosis of secretory vesicles from neuroendocrine cells, apparently via rearrangement of the actin cytoskeleton, and this may be analogous to the fusion of VACs or smaller vesicles with the apical surface. Accumulation of apparent VACs was seen when Cdc42 was depleted. Similarly, DN Cdc42 blocks capillary lumen formation. Perhaps during normal MDCK cyst lumen formation smaller vesicles are rapidly exocytosed to form the lumen. Inhibition of this by Cdc42 depletion may cause the accumulation of larger, more easily detected VACs. Indeed, this may be the defect underlying the phenotypes observed with loss of function of PTEN, Anx2, Cdc42, or aPKC. Cdc42 is also needed for exit of apical and BL proteins from the trans-Golgi network (TGN), so Cdc42 may act at multiple levels in the formation of the apical surface (Martin-Belmonte, 2007).
Localized active Cdc42 may promote formation of the apical surface and lumen by additional mechanisms. Active Cdc42 binds to Par6, a member of the Par3/Par6/aPKC complex that regulates TJ and polarity formation. In Drosophila, Par6/aPKC functions at the apical PM independently of Par3, which is associated with the junctional complex. Indeed, Par6/aPKC localizes at the apical PM of MDCK cysts independently of Par3, and the disruption of aPKC function inhibits normal lumen formation. Mutation of zebrafish aPKCλ causes defects in lumen formation in the intestine. These data suggest the existence of two distinct Par complexes for the establishment of epithelial polarity: a complex of Par6/aPKC localized to the apical PM and involved in the formation of this domain; and a complex that also includes Par3, localized at the TJs and required for their formation (Martin-Belmonte, 2007).
It has been reported that inhibition of Rac1 or β1-integrin in cysts leads to inversion of polarity orientation and abnormal organization of laminin. Rac1, β1-integrin, and laminin may be part of a pathway that determines orientation of the axis of polarity, while PTEN, PtdIns(4,5)p2, Anx2, Cdc42, and Par6/aPKC are part of a pathway that controls formation of the apical surface and lumen. The Rac1/β1-integrin/laminin pathway might be upstream and/or parallel to the PTEN/PtdIns(4,5)p2/Anx2/Cdc42 pathway, and it determines the location of the apical surface. Because activation of Rac1 at the primordial adhesions of epithelial cells controls the association and activation of the Par3/Par6/aPKC complex to induce TJ biogenesis and cell polarity, one potential connection between these pathways might be the targeting of PTEN to the apical domain through its interaction with TJs. PTEN localizes to the adherens junctions in fly epithelium through its interaction with Bazooka/Par3. This study shows that PTEN is needed for apical PM and lumen formation during cyst development and that Par3 localizes specifically to the TJs in MDCK cyst. This observation is consistent with previous studies showing that the expression of DN Par-3 cells disrupted MDCK cyst morphogenesis, causing the lack of a central lumen (Martin-Belmonte, 2007).
Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4beta-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produces a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment is independent of protein synthesis, while it is disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level (Manseau, 1999).
Using a balanced conditioned place preference (CPP) paradigm, the role of protein kinases A (PKA) and C (PKC) on the acquisition, consolidation and expression of cocaine place conditioning was studied. H7, a non-selective inhibitor of protein kinases, was administered intracerebroventricularly at 1 and 10 micrograms/10 microliters. The higher dose significantly reduces the time spent by rats in the cocaine compartment when given immediately after each conditioning session (consolidation), whereas it had no effect when administered before cocaine during the training phase (acquisition) or before testing for place preference in the absence of cocaine (expression). The same effect was found on administering immediately after each training session 3 micrograms/10 microliters chelerythrine, a selective PKC inhibitor, or 10 micrograms/10 microliters H89, a selective PKA inhibitor, suggesting that both kinases contribute to the consolidation of stimulus-reward association which determines rats' behavior in the cocaine CPP. Changes in the activity of PKA and PKC may thus be part of the cascade of events that contribute to enhancing synaptic responses in the consolidation phase of cocaine CPP and determine rats' behavior associated with the memory of the rewarding effect of cocaine during cocaine CPP expression. These findings may have implications for the study of cocaine 'craving' and relapse (Cervo, 1997).
Migrating cells extend protrusions to establish new adhesion sites at their leading edges. One of the driving forces for cell migration is the directional trafficking of cell-adhesion molecules such as integrins. The endocytic adaptor protein Numb is an important component of the machinery for directional integrin trafficking in migrating cells. In cultured mammalian cells, Numb binds to integrin-βs and localizes to Clathrin-coated structures (CCSs) at the substratum-facing surface of the leading edge. Numb inhibition by RNAi impairs both integrin endocytosis and cell migration toward integrin substrates. Numb is regulated by phosphorylation since the protein is released from CCSs and no longer binds integrins when phosphorylated by atypical protein kinase C (aPKC). Because Numb interacts with the aPKC binding partner PAR-3, a model is proposed in which polarized Numb phosphorylation contributes to cell migration by directing integrin endocytosis to the leading edge (Nishimura, 2007).
Numb localizes at a part of CCSs and functions in integrin endocytosis as a cargo-selective adaptor. Integrin is thought to be recycled from the tail to the front of migrating cells by endocytosis. However, many focal adhesions or focal complexes formed at the cell front disassemble behind the F-actin-rich lamellipodia. Numb mainly accumulated behind lamellipodia, although a certain population of Numb still remained and colocalized with integrin at the trailing edge. In addition, localization of Numb among CCSs correlated with the position of integrin adhesions, supporting the role of Numb in integrin endocytosis. Talin is a key molecule that tethers integrin to components of focal adhesions and actin stress fibers and is critical for focal-adhesion disassembly. Mutation of a conserved tyrosine residue within the integrin-β3 intracellular domain abolished the binding of both talin and Numb, suggesting that talin and Numb cannot bind to integrin simultaneously. Consistent with these observations, interaction of Numb with talin could not be detected. In addition, the binding of Numb to integrin does not activate the integrin extracellular domain, whereas the binding of talin does. The overexpression or knockdown of Numb does not directly affect cell adhesion. Thus, it appears that Numb does not actively promote focal-adhesion disassembly, but rather recruits free integrins without the components of focal adhesions to the AP-2 complex for internalization. Preferential localization of Numb around focal adhesions at the substratum-facing surface would facilitate recruitment of integrin during focal adhesion disassembly (Nishimura, 2007).
Recent genetic screening isolated Numb as a mutant defective for peripheral glia migration along axons in Drosophila (Edenfeld, 2007). Migration defects of postmitotic neurons have been described in Numb-knockout mice, indicating that Numb regulates particular cell migration in vivo. However, the defects of Numb knockdown on integrin endocytosis and cell migration are less marked than those of AP-2 and clathrin knockdown, suggesting that another adaptor molecule(s) may function in integrin endocytosis. A good candidate is disabled-2 (Dab2), which has a similar domain structure as Numb and binds to both components of clathrin-mediated endocytosis and to integrin-β. Dab2 is expressed in HeLa cells and positively controls cell adhesion and spreading. In contrast to Numb, Dab2 appears to preferentially localize to the apical surface. Thus, Numb and Dab2 may coordinately function in integrin endocytosis in different subcellular compartments for cell motility (Nishimura, 2007).
How does Numb localize at the substratum-facing surface and polarize toward the leading edge? Integrin adhesions could activate several intracellular signaling events and promote protein transport to adhesion sites by targeting microtubules and linking actin stress fibers. The actin cytoskeleton and/or adhesion itself are important for the preferential localization of Numb around adhesions. However, Numb still localized at the substratum-facing surface in the presence of cytochalasin-D, indicating that an additional mechanism may exist. Observations indicate that direct phosphorylation by aPKC may be a part of the regulatory mechanism underlying Numb localization at the substratum-facing surface. In addition, polarized localization of Numb toward the leading edge was lost upon aPKC knockdown. In support of these observations, asymmetric localization of Numb in Drosophila has been shown to be dependent on cortical actomyosin and the polarized localization/function of aPKC and PAR-3. Conclusive evidence will require isolation of the responsible motor(s) and anchor protein(s) for specific Numb localization (Nishimura, 2007).
Numb-full-3A, mutated at three phosphorylation sites, did not function as a constitutively active form that promotes integrin endocytosis and cell migration, but rather inhibited these processes. Similarly, both the phospho-mimic and nonphosphorylated form of μ2-adaptin, which is phosphorylated by AAK1, inhibit transferrin endocytosis, suggesting that clathrin-mediated endocytosis is tightly controlled by cycles of phosphorylation and dephosphorylation. Additional phosphorylation during endocytosis may be required for the dissociation of Numb from the binding proteins, integrin-β, and α-adaptin. Numb is indeed phosphorylated by several PKCs and CaMKs, and phosphatase inhibitor dramatically increases the phosphorylation level of Numb. Thus, local phosphorylation and dephosphorylation seems to allow Numb to localize defined CCSs around adhesion sites (Nishimura, 2007).
Trafficking of internalized integrin is regulated by growth factors and the extracellular matrix through several adaptors/kinases, including PI3-kinase, PKB/Akt, GSK3β, and PKCs. Several growth factors and adhesions indeed promote the recycling of integrins, leading to the upregulation of cell-surface expression, whereas treatment of cells with PDGF does not affect the internalization rate of integrin. The degree of colocalization and interaction of Numb with integrin-β1 was not significantly altered in HeLa cells before and after wounding. These data suggest that Numb functions constitutively in integrin endocytosis, although it is possibile that polarized migration promotes the internalization rate and amount of integrin endocytosis. It might be difficult to detect the changes in the interaction of Numb and integrin during migration due to the nature of rapid cycling of endocytosis and exocytosis and possibly due to the transient interaction. It has been reported that the inhibition of directional membrane trafficking causes membrane extension in all directions. Membrane trafficking controls the directionality of migrating cells. Taking into account the fact that Numb localization becomes polarized coincidently with directional migration, the subcellular region at which integrin is internalized and the subsequent coupling with the recycling processes could be important for efficient cell migration suitable for the particular environment (Nishimura, 2007).
The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. This study shows that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, it was further shown that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity (Zhang, 2008).
Non-muscle myosin IIA (NMII-A) and the tumor suppressor Lgl1 play a central role in the polarization of migrating cells. Mammalian Lgl1 interacts directly with NMII-A, inhibiting its ability to assemble into filaments in vitro. Lgl1 also regulates the cellular localization of NMII-A, the maturation of focal adhesions and cell migration. In Drosophila, phosphorylation of Lgl affects its association with the cytoskeleton. This study shows that phosphorylation of mammalian Lgl1 by aPKCζ prevents its interaction with NMII-A both in vitro and in vivo, and affects its inhibition on NMII-A filament assembly. Phosphorylation of Lgl1 affects its cellular localization and is important for the cellular organization of the acto-NMII cytoskeleton. It was further shown that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6α-aPKCζ, and that the complexes formation is affected by the phosphorylation state of Lgl1. The complex Lgl1-Par6α-aPKCζ resides in the leading edge of the cell. Finally, it was shown that aPKCzeta and NMII-A compete to bind directly to Lgl1 via the same domain. These results provide new insights into the mechanism regulating the interaction between Lgl1, NMII-A, Par6α, and aPKCζ in polarized migrating cells (Dahan, 2013).
A model is proposed for the role of Lgl1-NMIIA and Lgl1-Par6α-aPKCζ in establishing front-rear polarization in migrating cells. In migrating polarized cells Lgl1 resides at the cell’s leading edge in a complex with Par6α-aPKCζ, and it is this complex which defines the leading edge of the cell. In the lamellipodium Lgl1 binds to NMII-A but not to aPKCζ, inhibiting NMII-A filament assembly. These events allow the cell to polymerize F-actin to move the cell forward. According to this model Lgl1 is absent from the rear part of the cell, allowing NMII-A to assemble into filaments to enable cell retraction (Dahan, 2013).
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