PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1

A polarity complex of PAR-3, PAR-6 and atypical protein kinase C (aPKC) functions in various cell-polarization events, including neuron specification. The small GTPase Cdc42 binds to PAR-6 and regulates cell polarity. However, little is known about the downstream signals of the Cdc42-PAR protein complex. Here, we found that PAR-3 directly interacted with STEF/Tiam1, which are Rac-specific guanine nucleotide-exchange factors, and that STEF formed a complex with PAR-3-aPKC-PAR-6-Cdc42-GTP. Cdc42 induces lamellipodia in a Rac-dependent manner in N1E-115 neuroblastoma cells. Disruption of Cdc42-PAR-6 or PAR-3-STEF binding inhibited Cdc42-induced lamellipodia but not filopodia. The isolated STEF-binding PAR-3 fragment was sufficient to induce lamellipodia independently of Cdc42 and PAR-6. PAR-3 is required for Cdc42-induced Rac activation, but is not essential for lamellipodia formation itself. In cultured hippocampal neurons, STEF accumulated at the tip of the growing axon and colocalized with PAR-3. The spatio-temporal activation and signalling of Cdc42-PAR-6-PAR-3-STEF/Tiam1-Rac seem to be involved in neurite growth and axon specification. We propose that the PAR-6-PAR-3 complex mediates Cdc42-induced Rac activation by means of STEF/Tiam1, and that this process seems to be required for the establishment of neuronal polarity.     

APC and GSK-3beta are involved in mPar3 targeting to the nascent axon and establishment of neuronal polarity

In developing hippocampal neurons in culture, the evolutionarily conserved polarity complex mPar3/mPar6/aPKC selectively accumulates at the tip of one, and only one, of the immature neurites of a neuron and thus specifies the axon and generates neuronal polarity. How mPar3/mPar6 is enriched at the tip of the nascent axon, but not the dendrites, is not fully understood. Here, we report that mPar3 forms a complex with adenomatous polyposis coli (APC) and kinesin superfamily (KIF) 3A, proteins that move along microtubules. In polarizing hippocampal neurons, APC selectively accumulates at the nascent axon tip and colocalizes with mPar3. Expression of dominant-negative C terminus deletion mutants of APC or ectopic expression of APC leads to dislocalization of mPar3 and defects in axon specification and neuronal polarity. In addition to spatial polarization of APC, the selective inactivation of the GSK-3beta activity at the nascent axon tip is required for mPar3 targeting and polarization and establishing neuronal polarity. These results suggest that mPar3 is polarized in developing neurons through APC- and kinesin-mediated transport to the plus ends of rapidly growing microtubules at the nascent axon tip, a process that involves a spatially regulated GSK-3beta activity.     

Hippocampal neuronal polarity specified by spatially localized mPar3/mPar6 and PI 3-kinase activity

How a neuron becomes polarized remains an outstanding question. Here, we report that selection of the future axon among neurites of a cultured hippocampal neuron requires the activity of growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), as well as atypical protein kinase C (aPKC). The PI 3-kinase activity, highly localized to the tip of the newly specified axon of stage 3 neurons, is essential for the proper subcellular localization of mPar3, the mammalian homolog of C. elegans polarity protein Par3. Polarized distribution of not only mPar3 but also mPar6 is important for axon formation; ectopic expression of mPar6 or mPar3, or just the N terminus of mPar3, leaves neurons with no axon specified. Thus, neuronal polarity is likely to be controlled by the mPar3/mPar6/aPKC complex and the PI 3-kinase signaling pathway, both serving evolutionarily conserved roles in specifying cell polarity.     

A conserved oligomerization domain in drosophila Bazooka/PAR-3 is important for apical localization and epithelial polarity

The PAR-3/PAR-6/aPKC complex is required to establish polarity in many different cell types, including the C. elegans zygote and epithelial and neuronal cells in Drosophila and mammals. In each context, the components of this complex display a mutually dependent asymmetric cortical localization. PAR-6 is a direct effector of Rho family GTPases and binds to and regulates aPKC. Mammalian PAR-3 (mPar3) can associate with transmembrane proteins and may link the complex to the membrane, but this can account for only part of the requirement for this protein in the complex. Here we investigate the function of a novel conserved domain, CR1, of PAR-3 using computational, biochemical, and genetic approaches. Sequence-structure comparison by FUGUE predicts that CR1 has the same structural fold as a bacterial oligomerization domain. We show that CR1 of the Drosophila homolog, Bazooka (BAZ), mediates oligomerization in vitro and in vivo. Furthermore, deletion of CR1 disrupts BAZ localization in both epithelial cells and the germline and strongly impairs BAZ function in epithelial polarity. These results indicate that this domain is important for the localization and activity of the PAR-3/PAR6/aPKC complex and define a new role for PAR-3 in assembling higher order protein complexes.     

Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures

We have previously shown that during early Caenorhabditis 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 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607--3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95--106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that 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. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue 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 plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.     

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.     

aPKC phosphorylates JAM-A at Ser285 to promote cell contact maturation and tight junction formation

The PAR-3-atypical protein kinase C (aPKC)-PAR-6 complex has been implicated in the development of apicobasal polarity and the formation of tight junctions (TJs) in vertebrate epithelial cells. It is recruited by junctional adhesion molecule A (JAM-A) to primordial junctions where aPKC is activated by Rho family small guanosine triphosphatases. In this paper, we show that aPKC can interact directly with JAM-A in a PAR-3-independent manner. Upon recruitment to primordial junctions, aPKC phosphorylates JAM-A at S285 to promote the maturation of immature cell-cell contacts. In fully polarized cells, S285-phosphorylated JAM-A is localized exclusively at the TJs, and S285 phosphorylation of JAM-A is required for the development of a functional epithelial barrier. Protein phosphatase 2A dephosphorylates JAM-A at S285, suggesting that it antagonizes the activity of aPKC. Expression of nonphosphorylatable JAM-A/S285A interferes with single lumen specification during cyst development in three-dimensional culture. Our data suggest that aPKC phosphorylates JAM-A at S285 to regulate cell-cell contact maturation, TJ formation, and single lumen specification.     

Mammalian Lgl forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity

BACKGROUND: 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. RESULTS: We show that mammalian Lgl competes 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 the previous observation that the complex containing PAR-3 promotes it. CONCLUSIONS: 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.     

A distinct PAR complex associates physically with VE-cadherin in vertebrate endothelial cells

A cell polarity complex consisting of partitioning defective 3 (PAR-3), atypical protein kinase C (aPKC) and PAR-6 has a central role in the development of cell polarity in epithelial cells. In vertebrate epithelial cells, this complex localizes to tight junctions. Here, we provide evidence for the existence of a distinct PAR protein complex in endothelial cells. Both PAR-3 and PAR-6 associate directly with the adherens junction protein vascular endothelial cadherin (VE-cadherin). This association is direct and mediated through non-overlapping domains in VE-cadherin. PAR-3 and PAR-6 are recruited independently to cell-cell contacts. Surprisingly, the VE-cadherin-associated PAR protein complex lacks aPKC. Ectopic expression of VE-cadherin in epithelial cells affects tight junction formation. Our findings suggest that in endothelial cells, another PAR protein complex exists that localizes to adherens junctions and does not promote cellular polarization through aPKC activity. They also point to a direct role of a cadherin in the regulation of cell polarity in vertebrates.     

Drosophila PAR-1 and 14-3-3 inhibit Bazooka/PAR-3 to establish complementary cortical domains in polarized cells

PAR-1 kinases are required for polarity in diverse cell types, such as epithelial cells, where they localize laterally. PAR-1 activity is believed to be transduced by binding of 14-3-3 proteins to its phosphorylated substrates, but the relevant targets are unknown. We show that PAR-1 phosphorylates Bazooka/PAR-3 on two conserved serines to generate 14-3-3 binding sites. This inhibits formation of the Bazooka/PAR-6/aPKC complex by blocking Bazooka oligomerization and binding to aPKC. In epithelia, this complex localizes apically and defines the apical membrane, whereas Bazooka lacking PAR-1 phosphorylation/14-3-3 binding sites forms ectopic lateral complexes. Lateral exclusion by PAR-1/14-3-3 cooperates with apical anchoring by Crumbs/Stardust to restrict Bazooka localization, and loss of both pathways disrupts epithelial polarity. PAR-1 also excludes Bazooka from the posterior of the oocyte, and disruption of this regulation causes anterior-posterior polarity defects. Thus, antagonism of Bazooka by PAR-1/14-3-3 may represent a general mechanism for establishing complementary cortical domains in polarized cells.     

The interaction of mPar3 with the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

The Par polarity complex consisting of the evolutionarily conserved proteins mPar3, mPar6, and aPKC regulates cell polarity in many cell types including neurons. Here we show that mPar3 is required for the establishment of neuronal polarity and links the Smurf2 to Kinesin-2. The HECT domain E3 ubiquitin ligase Smurf2 ensures that neurons extend only a single axon by initiating the degradation of inactive Rap1B through the ubiquitin/proteasome system. Its interaction with mPar3 is required to localize Smurf2 to growth cones and restrict Rap1B to the axon. Interfering with the binding of mPar3 to Kinesin-2 or Smurf2 to mPar3 and knockdown of mPar3 by RNAi disrupt the establishment of neuronal polarity through the failure to restrict Rap1B to a single neurite.     

The PAR-6 polarity protein regulates dendritic spine morphogenesis through p190 RhoGAP and the Rho GTPase

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. Here we show 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, we further show 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.     

Loss of aPKCλ in Differentiated Neurons Disrupts the Polarity Complex but Does Not Induce Obvious Neuronal Loss or Disorientation in Mouse Brains

Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6β, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6β and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation.     

aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity

BACKGROUND: 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. On the other hand, 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. RESULTS: We demonstrate that PAR-1b is 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. On the other hand, 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. CONCLUSIONS: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.     

Binding to PKC-3, but not to PAR-3 or to a conventional PDZ domain ligand, is required for PAR-6 function in C. elegans

PAR-6 is a conserved protein important for establishment and maintenance of cell polarity in a variety of metazoans. PAR-6 proteins function together with PAR-3, aPKC and CDC-42. Mechanistic details of their interactions, however, are not fully understood. We studied the biochemical interactions between C. elegans PAR-6 and its binding partners and tested the requirements of these interactions in living worms. We show that PB1 domain-mediated binding of PAR-6 to PKC-3 is necessary for polarity establishment and PAR-6 cortical localization in C. elegans embryos. We also show that binding of PAR-6 and PAR-3 is mediated in vitro by a novel type of PDZ-PDZ interaction; the βC strand of PAR-6 PDZ binds the βD strand of PAR-3 PDZ1. However, this interaction is dispensable in vivo for PAR-6 function throughout the life of C. elegans. Mutations that specifically abolish conventional ligand binding to the PAR-6 PDZ domain also failed to affect PAR-6 function in vivo. We conclude that PAR-6 binding to PKC-3, but not to PAR-3 nor to a conventional PDZ ligand, is required for PAR-6 cortical localization and function in C. elegans.     

Similar requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in diverse cell types

During animal development, a complex of Par3, Par6 and atypical protein kinase C (aPKC) plays a central role in cell polarisation. The small G protein Cdc42 also functions in cell polarity and has been shown in some cases to act by regulating the Par3 complex. However, it is not yet known whether Cdc42 and the Par3 complex widely function together in development or whether they have independent functions. For example, many studies have implicated Cdc42 in cell migrations, but the Par3 complex has only been little studied, with conflicting results. Here we examine the requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in a range of different developmental events. We found similar requirements in all tissues examined, including polarised growth of vulval precursors and seam cells, migrations of neuroblasts and axons, and the development of the somatic gonad. We also propose a novel role for primordial germ cells in mediating coalescence of the Caenorhabditis elegans gonad. These results indicate that CDC-42 and the PAR-3/PAR-6/aPKC complex function together in diverse cell types.     

Microtubules induce self-organization of polarized PAR domains in Caenorhabditis elegans zygotes

A hallmark of polarized cells is the segregation of the PAR polarity regulators into asymmetric domains at the cell cortex. Antagonistic interactions involving two conserved kinases, atypical protein kinase C (aPKC) and PAR-1, have been implicated in polarity maintenance, but the mechanisms that initiate the formation of asymmetric PAR domains are not understood. Here, we describe one pathway used by the sperm-donated centrosome to polarize the PAR proteins in Caenorhabditis elegans zygotes. Before polarization, cortical aPKC excludes PAR-1 kinase and its binding partner PAR-2 by phosphorylation. During symmetry breaking, microtubules nucleated by the centrosome locally protect PAR-2 from phosphorylation by aPKC, allowing PAR-2 and PAR-1 to access the cortex nearest the centrosome. Cortical PAR-1 phosphorylates PAR-3, causing the PAR-3-aPKC complex to leave the cortex. Our findings illustrate how microtubules, independently of actin dynamics, stimulate the self-organization of PAR proteins by providing local protection against a global barrier imposed by aPKC.     

The Drosophila PAR-1 spacer domain is required for lateral membrane association and for polarization of follicular epithelial cells

The Ser/Thr kinases of the PAR-1/MARK/Kin1 family are conserved regulators of polarity in epithelial and non-epithelial cells . Drosophila PAR-1 localizes laterally in the follicular epithelium of the ovary , where it has been shown to function at two distinct levels: It stabilizes the cytoskeleton and it regulates apical-basal polarity by directly inhibiting lateral assembly of the apical aPKC/Bazooka/PAR-6 complex . However, it has been unclear how lateral localization of Drosophila PAR-1 is achieved and whether this localization contributes to epithelial polarity in vivo. Here we show that, through its spacer domain, Drosophila PAR-1 accumulates on the lateral plasma membrane (PM) in cells of the follicular epithelium (FE). Rescue experiments indicate that in FE cells PAR-1 kinase activity is essential for all the described functions of PAR-1. In contrast, the spacer domain of PAR-1 is required for apical-basal polarity and growth control but is dispensable for microtubule (MT) stabilization. Our data indicate that the spacer domain of PAR-1 is required for lateral PM localization of PAR-1 kinase and for development of a polarized FE.     

Meiotic maturation induces animal-vegetal asymmetric distribution of aPKC and ASIP/PAR-3 in Xenopus oocytes

The asymmetric distribution of cellular components is an important clue for understanding cell fate decision during embryonic patterning and cell functioning after differentiation. In C. elegans embryos, PAR-3 and aPKC form a complex that colocalizes to the anterior periphery of the one-cell embryo, and are indispensable for anterior-posterior polarity that is formed prior to asymmetric cell division. In mammals, ASIP (PAR-3 homologue) and aPKCgamma form a complex and colocalize to the epithelial tight junctions, which play critical roles in epithelial cell polarity. Although the mechanism by which PAR-3/ASIP and aPKC regulate cell polarization remains to be clarified, evolutionary conservation of the PAR-3/ASIP-aPKC complex suggests their general role in cell polarity organization. Here, we show the presence of the protein complex in Xenopus laevis. In epithelial cells, XASIP and XaPKC colocalize to the cell-cell contact region. To our surprise, they also colocalize to the animal hemisphere of mature oocytes, whereas they localize uniformly in immature oocytes. Moreover, hormonal stimulation of immature oocytes results in a change in the distribution of XaPKC 2-3 hours after the completion of germinal vesicle breakdown, which requires the kinase activity of aPKC. These results suggest that meiotic maturation induces the animal-vegetal asymmetry of aPKC.     

Dishevelled promotes axon differentiation by regulating atypical protein kinase C

The atypical protein kinase C (aPKC) in complex with PAR3 and PAR6 is required for axon-dendrite differentiation, but the upstream factors responsible for regulating its activity are largely unknown. Here, we report that in cultured hippocampal neurons aPKC is directly regulated by Dishevelled (Dvl), an immediate downstream effector of Wnt. We found that downregulation of Dvl abrogated axon differentiation, whereas Dvl overexpression resulted in multiple axon formation. Interestingly, Dvl was associated with aPKC and this interaction resulted in aPKC stabilization and activation. Furthermore, the multiple axon formation resulting from Dvl overexpression was attenuated by expressing a dominant-negative aPKC in these neurons and overexpression of aPKC prevented the loss of axon caused by Dvl downregulation. Finally, Wnt5a, a noncanonical Wnt, activated aPKC and promoted axon differentiation. The Wnt5a effect on axon differentiation was attenuated by downregulating Dvl or inhibiting aPKC. Thus, Dvl-aPKC interaction can promote axon differentiation mediated by the PAR3-PAR6-aPKC complex.