Article

• The EMBO Journal (2004) 23, 77 - 88
• doi:10.1038/sj.emboj.7600023

Published online: 18 December 2003

• Subject Categories:

  • Cell and Tissue Architecture | Signal Transduction

Identification of PKCII: an endogenous inhibitor of cell polarity

Scott J Parkinson^a, J Anne Le Good^b, Richard DH Whelan, Phil Whitehead and Peter J Parker

1. Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, London, UK

Correspondence to:

Peter J Parker, Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A3PX, UK. Tel.: 020 7242 0200; Fax: 020 7269 3094; E-mail: peter.parker@cancer.org.uk

^aCurrent address: Dana-Farber Cancer Institute, Division of Cancer Biology, Harvard Medical School, Boston 02115, USA. E-mail: Scott_Parkinson@dfci.harvard.edu

^bCurrent address: ISREC, Chemin des Boveresses 155, CH 1066 Epalinges, Switzerland

Received 13 June 2003; Accepted 13 November 2003

• Keywords:

  • cell polarity,
  • Par6,
  • PKC,
  • RNAi,
  • tight junction

Introduction

Cell polarisation is a fundamental property of every cell type, with symmetric and/or asymmetric distribution of cellular components being essential for the development, cell division, cell migration, and maintenance of specialised compartments within cells. Atypical protein kinase (aPKC) is implicated in the development of cellular asymmetry and contributes to astrocyte migration and epithelial cell polarity (Joberty et al, 2000; Lin et al, 2000; Etienne-Manneville and Hall, 2001; Hirose et al, 2002).

Epithelial cells are composed of distinct apical (lumenal) and basolateral domains separated by a specialised cellular structure known as the tight junction. The tight junction maintains a barrier that prevents diffusion of molecules across the epithelial cell layer (Dragsten et al, 1981). The protein components of the tight junction are beginning to be elucidated. The junctional adhesion molecules (JAMs) appear to be essential for tight junction assembly; monoclonal antibodies against JAMs inhibit junction formation following Ca²⁺ switch (Liu et al, 2000). These transmembrane proteins associate as dimers in trans through their extracellular domains at sites of cell–cell contact and recruit intracellular scaffolding components to the tight junction (Kostrewa et al, 2001). The intracellular domains of JAMs bind directly to the first PDZ domain of the cell polarity protein Par3/Asip/Bazooka (Ebnet et al, 2001). Par3 also associates with Par6, Cdc42, and aPKC (Joberty et al, 2000; Lin et al, 2000) all of which contribute to tight junction formation. The JAMs, as well as the tight-junction-localised proteins occludin and the claudins, also associate with the PDZ domains of the ZO family of scaffolding proteins (Furuse et al, 1993, 1998; Fanning et al, 1998; Haskins et al, 1998, Itoh et al, 1999).

aPKCs appear to be essential for the formation but not maintenance of tight junctions. MDCK cells expressing kinase-inactive aPKC mutants appear normal, however after Ca²⁺ depletion to disrupt cell–cell contacts, the localisation of the tight junction markers ZO-1, Par3, occludin, and claudin-1 is erratic following Ca²⁺ replacement (Suzuki et al, 2001). These and other observations led to the hypothesis that aPKCs catalyse the transition of premature cellular junctions into differentiated asymmetric structures (Suzuki et al, 2002).

The aPKCs are activated by protein–protein interactions within the regulatory domains of the proteins. This serves the two purposes of destabilising the pseudosubstrate site interaction with the catalytic domain permitting binding/phosphorylation of substrate and tethering the kinase activity to specific regions of the cell (Moscat and Diaz-Meco, 2000). PKC and PKC/ are homologous proteins that interact with Par6, a protein necessary for cellular asymmetry and asymmetric cell division. Interaction of aPKCs with Par6 restricts their localisation in the cell and activates their kinase activity (Joberty et al, 2000; Lin et al, 2000).

In addition to the full-length aPKCs, there is also a PKC isoform (PKM) synthesised following transcription initiation of the PKC gene from a brain-specific promoter located in an intron (Marshall et al, 2000). This results in translation of an aPKC catalytic protein lacking the regulatory domain. The PKM transcript is implicated in long-term potentiation, likely as a result of its constitutive kinase activity (Sacktor et al, 1993, Osten et al, 1996).

In this study, we describe the identification and characterisation of a distinct aPKC gene, designated PKCII. This gene has a unique chromosomal localisation and is 98% identical with the cDNA encoding PKC. An immunoreactive protein corresponding to PKCII is detected in cultured cell lines and RNA can be detected in many mouse tissues. Amongst the 29 bp differences with PKC, a deletion at nucleotide C1146 of PKCII shifts the reading frame and results in truncation of the protein at amino acid 376. Therefore, PKCII is composed of an aPKC regulatory domain without a functional catalytic domain. Recombinant PKCII is partitioned in the nucleus, however its location changes when it interacts with proteins intrinsic to development of tight junctions. Expression of endogenous PKCII in epithelial cells disrupts the assembly of tight junctions and promotes overgrowth of cells demonstrating its role in regulating aPKC-dependent functions.

Cloning and identification of PKCII

A stringent screen of a mouse genomic library (using the 5' end of the PKC cDNA as a probe) identified three hybridising clones. The three clones were identical by restriction analysis and Southern blotting. Clone 3, containing a 13 kb insert, was selected for further analysis. Sequencing revealed this clone to contain a DNA sequence homologous with PKC that was designated PKCII (the genomic sequence has been annotated recently by the NCBI Annotation Project and is designated locus XM_124895). The genomic sequence of PKCII was approximately 98% identical at the nucleotide level with the cDNA encoding 'authentic' murine PKC (Figure 1) and retained a 3'UTR containing AATAAA poly A addition signal and a poly A stretch (data not shown). The PKCII genomic sequence contains 29 nucleotide divergencies with respect to the PKC cDNA sequence including deletion of nucleotides C1146, G1943, and a region flanking the PKC stop codon (C1779–G1819).

Figure 1.

Nucleotide alignment of murine PKCII genomic DNA with murine PKC cDNA (accession number M94632). Predicted start codons are bold and overmarked with ^***. Differences in nucleotide sequences are red and underlined. Stop codons for murPKCII (green) and murPKC (blue) are also underlined.

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Translation of the putative coding region of PKCII demonstrates a high degree of identity at the protein level with PKC (Figure 2). The start codon of PKC is conserved in PKCII; however, the nucleotide deletion of C1146 in PKCII results in a shift of the reading frame and termination of the putative coding region at nt1152 (Figure 1). Following translation, PKCII would comprise a functional regulatory domain homologous with PKC. The differences between PKC and PKCII in the catalytic region, however, are significant. In addition to loss of 60% of the catalytic domain, the shift in the PKCII reading frame converts amino acid D356 to a threonine. This mutation ensures PKCII lacks all catalytic activity since D356 is the catalytic base of PKC responsible for stabilising the serine/threonine oxyanion of the substrate prior to transfer of the phosphate from ATP (Protein Kinase Resource: www.sdsc.edu/kinases/). The frame shift also terminates the coding sequence in subdomain VIB (the catalytic loop) of the kinase domain suggesting that there is no catalytic function for this putative protein (see below).

Figure 2.

Amino acid alignment of murPKCII with murPKM, murPKC (accession number M94632). The amino acid sequences of PKC and PKCII were predicted from the nucleic acid sequence in Figure 1. The PKM sequence was derived from sequenced RT–PCR products in Figure 3 (below). Amino acid differences are underlined. The three shadowed sequences define the three domains of the protein; in order from the amino-terminus of PKC these are PB1, C1, and kinase domains.

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Fluorescence in situ hybridisation (FISH) was performed to map the location of PKC and PKCII on mouse metaphase spreads stained with specific chromosome paints (Figure 3A, A'). Probes specific for PKCII (X7; see Materials and methods) hybridised strongly to chromosome 7(A_2–3 region). PKC-specific hybridising probes (pZ-X5; see Materials and methods) hybridised with chromosome 4(E₂ region). These locations were also confirmed by analysis of the Celera mouse genome database using the nucleotide sequences in Figure 1 (J Sgouros and S Parkinson, unpublished). Thus PKCII has a unique chromosomal location on chromosome 7.

Figure 3.

The gene encoding PKCII is on mouse chromosome number 7 and is transcribed and translated in various tissues and cell lines. (A) Chromosome localisation of the PKCII gene. FISH was performed with a specific probe recognising the PKCII 3'UTR and chromosomal paints recognising chromosome 7. Hybridising regions of the PKCII probe are indicated with red arrows and correlate with the highlighted chromosome 7s (right panel). (A') Chromosome localisation of the PKC gene. FISH was performed with a specific probe recognising an intron sequence from the murine PKC gene and chromosomal paints recognising chromosome 4. Fluorescent-tagged PKC hybridising regions are indicated with a red arrow and correlate with the presence of chromosome 4 in the right panel. (B) Transcription of aPKCs detected in mouse tissues by RT–PCR. Total RNA was isolated from the indicated Balb/C mouse tissues and treated with DNAse. All RNAs were free of DNA contamination as demonstrated by a lack of visible -actin or PKCII product in the absence of reverse transcriptase (not shown). The specific primers used to detect PKC, PKM, and PKCII are indicated in Materials and methods. (C) In vitro transcription/translation of isolated PKCII genomic clone and detection of a 45 kDa protein. Genomic DNA encoding PKCII was added to a rat reticulocyte lysate and transcribed and translated in vitro in the presence of ³⁵S methionine. The following templates were used: lane 1—PKCII no ATG, lane 2—PKCII no 5'-UTR, lane 3—PKCII with 5' and 3' UTR, lane 4—PKCII 3'UTR only, lane 5—luciferase control. (D) myc-PKC or PKCII were expressed in Cos7 cells and immunopurified. Activity was determined against myelin basic protein (MBP) as described in Materials and methods. (E) Detection of endogenous PKCII protein in various cell lines. The indicated cell lines were grown to confluence, trypsinised, counted, and lysed in sample buffer. Lysate from the equivalent of 10⁶ cells was loaded on 12.5% PAGE gel, transferred to PVDF and incubated with an antibody directed against the C-terminus to detect full-length aPKCs (//) (top panel) or an antibody directed against the N-terminus to detect PKCII (bottom panel) (see Materials and methods for specificity). Lysate from Cos7 cells expressing a PKCII cDNA plasmid (Cos7 (PKCII)) is included as a migration control.

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The data indicate that a PKC-like gene on chromosome 7, lacking any introns, encodes a protein with an atypical PKC regulatory domain. To determine if this PKC-homologous region is simply a nonexpressed pseudogene, Balb/c mouse RNA was extracted from various organs in order to assess PKCII transcription by RT–PCR (Figure 3B). Since PKCII genomic DNA contains no introns, extreme care was taken to ensure there was no DNA contamination of the extracted RNAs. Specific primers were designed that only amplified PKC, PKM, or PKCII, as determined in control reactions. RNA encoding PKC was detected primarily in muscle, heart, lung, kidney, and liver. PKM RNA was abundant in the brain and could not be detected in other tissues. RNA derived from PKCII was abundant in bone, lung, kidney, and liver, however it could be detected in most of the tissues tested. This demonstrates that PKCII is transcribed from its locus on chromosome 7.

Next, we sought to address whether the RNA detected (Figure 3B) could be translated, generating this putative novel aPKC protein. In vitro transcription/translation of the open reading frame of the PKCII genomic sequence identified the expected 45 kDa protein following autoradiography (Figure 3C). Detection was dependent on the ATG homologous to the PKC open reading frame and, as expected, the T7-dependent product was disrupted by the presence of the 5'UTR. No product was detected from the 3'UTR only, demonstrating that the identified protein product derives from translation of the predicted open reading frame identified in Figure 2. Expression and immunoprecipitation of PKCII or PKC as a positive control demonstrated conclusively that PKCII had no intrinsic catalytic activity (Figure 3D).

In order to determine whether PKCII was expressed in vivo, an antibody recognising the amino terminus of PKC (kindly provided by Dr T Sacktor) was used to determine the expression of PKCII in various cell lines. PKCII and PKC contain virtually identical N-termini. Therefore, detection of an immunoreactive 45 kDa band would indicate the presence of a PKCII-related protein (Figure 3E). Various cell lines were grown to confluence, the number of cells determined and lysates analysed by Western blot using the N-terminal PKC antibody or C-terminal aPKC antibody. A C-terminal antibody that does not distinguish between the aPKC subtypes (//) (71 kDa; Figure 3E, top panel) gave a signal following Western blot analysis of total cell lysates. NIH and U251 cells also gave a signal following longer exposure of the film. An N-terminal PKC-specific antibody detected a 45 kDa band that comigrated with that detected in lysates from Cos7 cells transfected with a plasmid encoding PKCII. Full-length PKC could also be detected by the N-terminal antibody in these epithelial cell lines. This data support the conclusion that the PKCII genomic locus on mouse chromosome 7A_2–3 encodes a new member of the aPKC family. An equivalent protein species is detected in human (293, Hela, MCF10A, MCF7), green monkey (Cos7), mouse (NIH3T3, HC11) and canine (MDCK)-derived cells suggesting PKCII is expressed across mammalian species.

PKCII interacts with Par6 and PKM

To determine the potential for PKCII to interact with candidate partners, we exploited the fact that ectopic expression of tagged PKCII in COS7 cells produced an accumulation of the fusion protein in nuclei, such that the influence of coexpressed proteins could be investigated. Recombinant PKCII and candidate binding partners were expressed in Cos7 cells tagged with the fluorescent proteins, CFP or YFP. YFP-PKCII (Figure 4A; top left) was found primarily in the nucleus and could also be observed at cell contacts (Figure 4A; top left, arrow). This was in contrast to YFP-PKM (Figure 4A; bottom right) and YFP-PKC (not shown), which were located almost exclusively in the cytoplasm.

Figure 4.

Characterisation of PKCII localisation and protein interactions. (A) Localisation of PKCII, PKCIIA119E, PKC, and Par6c by live-cell confocal microscopy. YFP-tagged plasmids encoding PKCII (top left), PKCIIA119E (bottom left), PKM (bottom right) and Par6c (top right) were transfected into Cos7 cells and visualised (pseudocolour red) by confocal microscopy (bar=50 M). (B) PKCII and PKM colocalise in the cytoplasm of coexpressing cells. Cos7 cells (phase, top right) expressing YFP-PKM (red) and CFP-PKCII (green) were observed by live-cell confocal microscopy (merge, bottom right) (bar=50 M). (C) PKCII and Par6c colocalise in the cytoplasm of coexpressing cells. Cos7 cells (phase, top right) expressing YFP-PKCII (red) and CFP-Par6c (green) were visualised by live-cell confocal microscopy (merge, bottom right) (bar=30 M). (D) Interaction with PKM stabilises PKCII in the cytoplasm. Cos7 cells (Cont) expressing mycPKCII (mycII), PKM (PKM) or coexpressing mycPKCII and PKM (mycII+PKM) were lysed and nuclear-rich fractions obtained as described in Materials and methods. Equivalent volumes of nuclear (N) or cytoplasmic (C) fractions in sample buffer were run on a 12.5% PAGE gel, transferred to PVDF and immunoblotted with 9E10 (anti-myc) or aPKC C-terminal (aPKC C-term) antibodies. PKC/ is endogenously expressed in Cos7 cells. (E) The intensity of the indicated bands (PKC/, PKM, or mycII) from Figure 4D were quantitated (NIH ImageQuant) from three independent experiments and the percentage of protein in the nuclear (open bars) or cytoplasmic (closed bars) fractions was quantified (s.d.). (F) PKCII forms a complex with Par6c or PKM that is not dependent on the pseudosubstrate site. Cos7 cells (lane 1) expressing PKM (lane 2), flagPar6c (lane 3), mycPKCIIA119E (lane 4), mycPKCIIA119E+PKM (lane 5), mycPKCIIA119E+flagPar6c (lane 6) mycPKCII (lane 7), mycPKCII+PKM (lane 8) or mycPKCII+flagPar6c (lane 9) were lysed and the detergent-soluble fraction was incubated with Protein G Sepharose-coupled 9E10 (myc) antibody. Detergent soluble (LOAD; bottom panels; 1:25 of total) and immunoprecipitates (mycIP; top panels) were run on 12.5% gels, transferred to PVDF and associated proteins were detected by probing with anti-aPKC C-terminal (PKM), anti-flag (flagPar6c), or anti-myc (mycPKCII/mycIIA119E) primary antibodies.

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PKCII retains the regulatory domains predicted to be required for interaction with the aPKC catalytic domain (PKM) and also with regulatory protein Par6.

Expressed alone, PKCII and PKM are located in nonoverlapping regions of the cell (Figure 4A). However, when CFP-PKCII and YFP-PKM were coexpressed in Cos7 cells, CFP-PKCII was observed in the cytoplasm in proportion to the relative PKM expression (Figure 4B). In high YFP-PKM-expressing cells (single arrowhead) CFP-PKCII was observed in the cytoplasm. In relatively low YFP-PKM-expressing cells (double arrowhead) CFP-PKCII still accumulated in the nucleus.

PKM and full-length PKC express identical catalytic domains. Despite the presence of its own regulatory domain, PKC could also stabilise PKCII in the cytoplasm although this required significantly higher expression levels for PKC than those for PKM (data not shown). These data suggest that the PKC catalytic domain can interact with PKCII.

Expressed alone, Par6c (Figure 4A; top right) could be detected in both the cytoplasm and nucleus of cells while, as noted above, PKCII (Figure 4A; top left) was primarily nuclear. In low expressing CFP-Par6c cells, YFP-PKCII retained its nuclear localisation (single arrowheads; Figure 4C), however in high CFP-Par6c-expressing cells YFP-PKCII resided in the cytoplasm (Figure 4C; double arrowhead) suggesting that Par6c interacts with PKCII in vivo.

The effects on cellular localisation of PKCII were also assessed by cell fractionation (Figure 4D, E). Crude nuclear and cytoplasmic fractions were isolated from Cos7 cells expressing mycPKCII, PKM or mycPKCII, and PKM and the distribution of the expressed proteins was evaluated in the two fractions. Endogenous PKC/ and exogenous PKM were primarily cytoplasmic in the presence or absence of mycPKCII. MycPKCII was detected primarily in the nucleus when expressed alone in Cos7 cells. However, it appeared mainly in the cytoplasmic fraction when coexpressed with PKM. This cell fractionation supports the fluorescent microscopy results presented in Figure 4B.

These data, exploiting the subcellular location observed on overexpression of PKCII, indicate that PKCII can interact with aPKCs and with aPKC regulatory proteins mutually regulating their cellular location. The pseudosubstrate site of PKCII is a potential regulatory site that mediates interaction with the catalytic domain of the aPKCs. In addition, Par6 interacts with the N-terminal 120 amino acids of the aPKCs that includes the pseudosubstrate site. Interestingly, mutation of the PKCII pseudosubstrate site (A119E) stabilises the protein in the cytoplasm (Figure 4A; bottom left). This indicates that the pseudosubstrate site determines the nuclear location of the overexpressed protein. To investigate this further, mycPKCII or mycPKCIIA119E was expressed in Cos7 cells with flagPar6c or PKM and their association was examined following immunoprecipitation with the anti-myc 9E10 antibody (Figure 4F). The flagPar6c and PKM detected in immunoprecipitates were specific for mycPKCII-expressing cells. Notably, mutation of the mycPKCII pseudosubstrate site (mycIIA119E) had no effect on the interaction between PKCII and PKM or flagPar6c (see Discussion). This indicates that while the pseudosubstrate site contributes to nuclear localisation it is not required for binding to PKC or Par6. It is likely however that PKCII binding to these proteins blocks recognition of its pseudosubstrate site, leading to inefficient nuclear import.

PKCII inhibits tight junction formation in mammary epithelial cells

PKCII protein interacts with the cell polarity protein Par6 (Figure 4). This suggests that PKCII could play a role in development of cellular junctions. It was previously reported that aPKC activity was required for the formation but not maintenance of tight junctions presumably via aPKC interaction with Par6 (Suzuki et al, 2001). PKCII lacks any catalytic activity and could compete with aPKCs for binding to Par6 (Figure 4) inhibiting tight junction formation. Therefore, the role of endogenous PKCII in the development of cell junctions was investigated using an RNAi approach. HC11 cells are a mouse mammary epithelial cell line endogenously expressing PKCII (Figure 3B). They are normally maintained in an undifferentiated state in the presence of EGF and insulin. However, they form tight junctions and become differentiated in response to lactogenic hormones and produce -casein and other differentiation markers (Stelwagen et al, 1999; Xie et al, 2002).

Preliminary experiments demonstrated that the selected RNAi sequence specifically inhibited expression of recombinant PKCII but not PKC in 293T cells (data not shown). HC11 cells were infected with an ecotropic retrovirus driving transcription of this RNAi molecule complementary and specific to PKCII. RNA was extracted from HC11 cells infected with the PKCII RNAi retrovirus (pSRII) or control virus (pSR), and the expression of PKC, PKCII, and -actin was assessed by RT–PCR (Figure 5A). HC11 pSRII cells showed a significant loss of PKCII message in all conditions tested while the detection of PKC was unaffected demonstrating the specificity of the targeting sequence. The levels of expressed proteins were also assessed in the two cell lines (Figure 5B). Western blot analysis revealed a loss of the 45 kDa immunoreactive band (PKCII) in pSRII cells while no significant difference in the 70 kDa band (PKC) was observed between pSR and pSRII HC11 lysates. These data demonstrate that the PKCII gene identified here is expressed and further that PKCII is translated from an mRNA distinct from PKC.

Figure 5.

PKCII inhibits formation of tight junctions in HC11 cells. (A) PKCII RNAi specifically reduces PKCII, but not PKC RNA. RT–PCR detection of PKCII, PKC, and -actin message in control HC11 cells (pSR) and PKCII RNAi-expressing HC11 cells (pSRII). Cells were grown to confluence on 6 cm² plates under standard growth conditions (EGF, INS, and SER). Following confluence, parallel cultures were switched to 2% serum for 24 h (serum) and then treated with prolactin/hydrocortisone (prolactin) for 72 h to induce differentiation (Xie et al, 2002). Cells grown under these different conditions were harvested, RNA extracted, DNAse treated and the indicated message amplified with specific primers. For clarity, the images shown are negatives. Differentiation was confirmed by -casein mRNA expression (not shown). DNA contamination was minimal as detected by the lack of amplification of -actin in the absence of reverse transcriptase using the specified primers (-actin – RT). (B) Western blot detection of PKC and PKCII in HC11 pSR (control) and HC11 pSRII (PKCII RNAi) cells. HC11 pSR and pSRII cells were grown for 72 h postconfluence and the proteins extracted in lysis buffer. Lysates were run on 10% PAGE gels, transferred to PVDF membrane and immunoblotted with rabbit antibodies against the N-terminus of PKC or the C-terminus of atypical PKCs. PKCII is recognised by the N-terminal but not C-terminal antibody and is decreased in HC11 pSRII (PKCII RNAi) versus pSR (control) cells. The asterisk denotes a nonspecific immunoreactive band. (C) Immunostaining of adherence and tight junction markers in HC11 pSR (control) and HC11 pSRII (PKCII RNAi) cells. Cells were seeded on glass coverslips and grown to confluence in EGF, insulin, and 10% serum. After 72 h, cells were fixed in MeOH and stained to detect -catenin, -actin, and ZO-1. Single confocal sections (0.5 m) are shown for the two cell lines (bar=50 m). (D) PKCII is necessary for the transformed phenotype of HC11 cells. HC11 pSR or pSRII cells were grown for 72 h postconfluence and stained with propidium iodide to identify nuclei (red) and an antibody against ZO-1 (green). Z stack profiles (top, bar=10 m) of stained cells are shown above confocal images in the X–Y plane (bottom, bar=50 M).

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Using the RNAi cell lines characterised in Figure 5A and B, we investigated the role of PKCII in the development of cell polarity.

HC11 pSR and HC11 pSRII cells were seeded on coverslips, allowed to grow to confluence, fixed and stained for various junction markers (Figure 5C). Loss of PKCII in pSRII cells correlated with the appearance of contiguous staining of ZO-1 in the apical plane. This contrasts with pSR cells where ZO-1 staining is punctate throughout the cytoplasm and disjointed at cell junctions. A similar pattern of behaviour was observed for the tight junction marker claudin-1 (data not shown). The -actin staining was consistent with the ZO-1 localisation. Bundled cortical actin could be observed in the apical plane of pSRII but not pSR cells. The adherens junction marker -catenin was located at cell–cell contacts in both cell lines suggesting that PKCII did not regulate adherens junction formation. These results suggest that PKCII specifically inhibits the development of tight junctions in HC11 cells.

In growth media containing EGF and insulin, HC11 cells adopt a transformed phenotype. They are characteristically rounded and grow in multiple layers losing contact inhibition (pSR cells, Figure 5D). However, in pSRII cells, loss of PKCII correlates with a monolayer phenotype as demonstrated in Z stacks of cells stained with propidium iodide to label nuclei. The loss of cell overgrowth (ie monolayer growth) can also be observed in orthogonal sections of cells stained with E-cadherin and -catenin (Figure 6). The adherens junction marker localises to areas of cell–cell contact and multiple layers of cells can be observed. Blocking PKCII expression repressed the transformed phenotype. HC11 pSRII cells stained with E-cadherin and -catenin were essentially monolayer and contact inhibited (Figure 6B). In pSRII cells, the tight junction marker ZO-1 is located apically to the adherens junction markers E-cadherin and -catenin and colocalises with the tight junction marker occludin (Figure 6B).

Figure 6.

Characterisation of cell–cell junctions in HC11 pSR (A) and pSRII knockdown (B) cells. HC11 pSR and HC11 pSRII cells were grown for 72 h postconfluence in the presence of EGF, insulin, and serum on glass coverslips, fixed in MeOH, and stained for various junctional markers as indicated. The left panels show composite Z stack profiles of stained cells (bars=20 m). The right panels show confocal X–Y sections through an apical plane (bars=50 M). (C) aPKC localisation is junctional in pSRII cells. aPKC was immunostained in pSR and pSRII cells as indicated. Examples of the junctional location of aPKC are highlighted by the arrows. (D) PKC activity is not altered by suppression of PKCII expression. PKC was immunoisolated from cells as described in the Materials and methods section. PKC protein and activity were determined and no change in activity was observed. Quantitation of four observations is shown under the representative western (PKC) and autoradiograph (³²P-myelin basic protein).

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The data presented suggest that PKCII interacts with Par6 and with the catalytic domain of aPKCs. Both interactions could potentially inhibit cell polarity and explain the formation of tight junctions in the absence of PKCII expression. aPKC localisation was also modified in the pSRII cells (Figure 6C; note the junctional accumulation of aPKC) while the total aPKC activity in immunoprecipitates was not stimulated following PKCII knockdown (Figure 6D). This data supports the hypothesis that PKCII inhibits tight junction formation by preventing aPKC accumulation and activation at tight junctions.

The data reported here introduce a new member of the murine aPKC family (PKCII) and implicate it in regulating aPKC-dependent cellular processes. The murine gene encoding this protein has a unique chromosome location and retains a high degree of identity with the PKC cDNA sequence. The synthesised protein has 98% amino acid identity to the regulatory domain of PKC but lacks any catalytic function. Despite lacking catalytic activity, domains for interaction with regulatory proteins are retained (Lin et al, 2000; Suzuki et al, 2001). It is shown that PKCII interacts with Par6c (Figure 4), an aPKC-binding protein required for tight junction formation, and so prevents tight junction formation (Figures 5, 6). Thus in vivo, PKCII functions to control aPKC signalling pathways contributing to cell polarity.

The knockdown of PKCII protein using an RNAi approach in HC11 epithelial cells demonstrates that this aPKC homolog inhibits tight junction formation. Previous studies have identified an essential role for aPKC activity in establishing tight junction formation (Suzuki et al, 2001, 2002). The N-terminus of Par6 contains an aPKC-binding motif that recognises the regulatory domain of the kinase. Cdc42 binds to Par6 through its CRIB and PDZ domains and activates aPKC kinase activity (Garrard et al, 2003). The Par6/aPKC complex also interacts with the scaffolding protein Par3 permitting its phosphorylation; an event correlated with development of tight junctions (Hirose et al, 2002). In the absence of any effect on aPKC activity it is concluded that PKCII competes for recruitment to cell junctions. This may be direct competition with Par6 or the Par6/PKCII complex may compete with Par6/aPKC for activated Cdc42 preventing phosphorylation of Par3 and/or recruitment of other complexes required for development of tight junctions. Other factors may also contribute to the phenotype observed in Figures 5 and 6 including the possibility that PKCII interacts with the catalytic domain of aPKCs (see Figure 4).

The data presented are consistent with a previous study that determined a PKC regulatory fragment inhibited tight junction formation in mammalian epithelial cells (Gao et al, 2002). In this study, a synthetic recombinant N-terminal fragment consisting of the first 126 amino acids of PKC lacking kinase activity delayed the development of mature tight junctions. This was dependent on its interaction with Par6 since mutation of the PKC Par6-binding site abrogated the delay in the recruitment of ZO-1 to tight junctions. This data support the idea that PKCII inhibits development of the Cdc42–Par6–aPKC complex although it is unclear whether the Par6-binding mutant used by Gao et al. (2002) still interacts with aPKC catalytic domains. In addition, a PKC mutation identified from a genetic screen in zebrafish was found to be responsible for the heart-and-soul (has) phenotype in which the animals demonstrate a loss of normal heart, kidney, and retinal patterning (Horne-Badovinac et al, 2001; Peterson et al, 2001). The has phenotype results from premature termination of the PKC coding sequence. The has mutants identified lack the C-terminal 69 and 73 amino acids and completely lack any catalytic activity.

The Ca²⁺ switch assay is routinely used to investigate formation of cellular junctions. Depletion of extracellular Ca²⁺ disrupts cell–cell contacts and addition of extracellular Ca²⁺ permits junctions to form. Using this assay, previous reports demonstrated that the formation of tight junctions is dependent on aPKC activity since kinase-dead mutants disrupt their formation (Suzuki et al, 2001). Supporting these findings, MDCK cells overexpressing PKCII over grow each other forming multiple layers and have disrupted ZO-1 localisation following Ca²⁺ switch (data not shown). This observation is consistent with the HC11 data, however PKCII is poorly expressed in transfected epithelial cells complicating analysis through ectopic expression.

The presence of the PKCII gene in mice likely represents a reverse transcriptase-dependent insertion of the PKC cDNA into the genome since the PKCII genomic sequence retains a high degree of homology but lacks any introns. Spontaneous mutation of the inserted sequence resulted in the deletion of nucleic acid 1146 and synthesis of a PKC regulatory domain lacking catalytic function. There are several reports of similar insertions occurring in the mammalian genome however most of these events result in nonfunctional genomic sequences referred to as pseudogenes. From the data presented it appears that PKCII was inserted into a region that permits transcription of its sequence resulting in a stable mRNA and 45 kDa protein.

Part of the analysis of the PKCII–Par6c interaction in vivo exploited the distinct subcellular compartmentation of the proteins. In Cos7 cells PKCII and a homologous fragment of PKC (not shown) are primarily localised in the nucleus of cells, suggesting the presence of a nuclear localisation signal (NLS) in the protein. These observations support previous studies identifying an NLS within the regulatory domain of full-length aPKCs (Perander et al, 2001). A pseudosubstrate site PKCII mutant is cytoplasmic suggesting this region plays a role in nuclear retention or targeting. In contrast, an homologous pseudosubstrate site mutation in PKC/ stabilises the protein in the nucleus (Perander et al, 2001). Differences in the nuclear trafficking of PKC/ and PKC have been reported previously. Significantly, identical mutants deleting the N-terminus up to and including the pseudosubstrate site had completely opposite cellular locations; the PKC mutant was cytoplasmic while the PKC mutant was nuclear (Perander et al, 2001). The location of the N-terminal mutant of PKC (Perander et al, 2001), PKM, PKCII, and PKCIIA119E (this study) are all consistent and demonstrate a contribution of the pseudosubstrate site to nuclear localisation of PKC. For PKC/, there may be an additional NLS located C-terminal to the pseudosubstrate site that is exposed upon pseudosubstrate site release from the catalytic domain.

The presence of PKCII in the cytoplasm following mutation of A119E correlates with the location of PKCII in vivo when it is in a complex with Par6c or PKM. Therefore, PKCII interaction with Par6c or PKM likely masks the NLS, of which the pseudosubstrate site appears to be a part. Furthermore, recombinant Par6c is also partially located in the nucleus when expressed on its own, however, in the presence of PKC or PKCII it is primarily cytoplasmic suggesting that the interaction either masks an NLS in Par6c or exposes a nuclear export signal somewhere in the complex. In differentiated epithelial cells, Par6 has only been detected at cell junctions. This may be due to its interaction with aPKC and other proteins that limit its localisation; the distribution of endogenous Par6 in other cell types and under various conditions may be varied. The observation that recombinant Par6c is entirely cytoplasmic when coexpressed with interacting proteins but only partially in their absence suggests this might be the case and needs to be investigated further.

Synthesis of truncated proteins that regulate cellular functions is a common theme in signal transduction. Translation of these inhibitory proteins can occur from alternate splicing of mRNAs or from distinct genes with unique chromosomal locations. Synthesis of the focal adhesion kinase (FAK)-related protein FRNK is an example of regulation by alternate splicing of a single mRNA (Schaller et al, 1993). Reminiscent of PKCII and PKC, FRNK is composed of the C-terminal regulatory domain of FAK but lacks any kinase activity. Another example is the soluble frizzled-related proteins (sFRPs). The sFRPs are located on distinct genes that are homologous to the frizzled receptors for the Wnts. These secreted proteins are homologous to the cysteine-rich domain (CRD) of the frizzled 7-transmembrane receptors and inhibit Wnt-dependent signalling pathways leading to stabilisation of -catenin in the cytoplasm (Rattner et al, 1997). Interestingly, it appears that PKC signalling pathways are also regulated by multiple strategies. PKM is synthesised from the PKC gene on mouse chromosome 4. It lacks most of the regulatory domain and likely constitutively phosphorylates substrates due to lack of an inherent pseudosubstrate site. PKCII, however, lacks all catalytic activity but can interact with regulatory proteins preventing aPKC-dependent signalling. All of the aPKC members likely contribute to form an integrated cellular response to each stimulus.

In conclusion, PKCII is identified as a new member of the aPKC family. It serves as an endogenous inhibitor of aPKC functions through interaction with known regulatory proteins. As a result of these properties, PKCII prevents tight junction formation in epithelial cells.

Reagents

Antibodies. The following antibodies were used: rabbit anti-aPKC C-terminal antibody (Santa Cruz) detects PKC/ and PKC (data not shown), rabbit anti-PKC N-terminal antibody (generously provided by Dr T Sacktor) is specific for PKC and does not cross-react with PKC/, mouse M2 anti-flag (Sigma), rabbit anti-ZO-1 (Zymed), mouse fitc-conjugated anti-occludin (Zymed), tritc-conjugated phalloidin (Molecular Probes), mouse fitc-conjugated E-cadherin (BD Transduction Labs), mouse anti--catenin (BD Transduction Labs), Cy3-conjugated anti-mouse (Jackson ImmunoResearch Laboratories), FITC-conjugated anti-rabbit (Dako).

Plasmids. PKCII was amplified from mouse brain DNA using the following primers; zetaII24(+) (5'-AAGCTTTGGCGGAGTGCGCCATG-3') and zetaII2133(-) (5'-GGTACCAGA-CATCATCGGTACAC-3'). The resulting 2 kb band was ligated into PCR Blunt (Invitrogen), excised with HindIII and KpnI and ligated into pDNA3.1(Hygro+) (Invitrogen). PcDNA3.1-PKC (rat) was made by subcloning PKC into pcDNA3.1-PKCII cut with NotI/XbaI. PcDNA3.1-PKM was made by subcloning in the RT–PCR product (see RT–PCR below) cut with SpeI/ClaI (500 bp fragment) into pcDNA3.1-PKC cut with NheI/ClaI. PEYFP-PKM was constructed by cutting pcDNA3.1-PKC with NdeI/PmeI, blunting the ends with Vent polymerase and ligating the 1.2 kb fragment into pEYFP-C1 (Clontech) cut with SmaI. The amino acid sequence of PKM was predicted from the sequence of RT–PCR products using PKM-specific primers and RNA from mouse brain (see below). PcDNA3.1-mycPKCII was generated by partial digestion of pcDNA3.1-PKCII with NheI/NgoMIV and ligation of oligos encoding the myc epitope. PEYFP-PKC was constructed by cutting pEYFP-PKM with BspE1/EcoRV and ligating in a fragment from pcDNA3.1-PKC cut with NgoMIV/EcoRV. PECFP-PKC was constructed by subcloning pEYFP-PKC into the pECFP-C1 vector (Clontech). PEYFP-PKCII was constructed by cutting pEYFP-PKC with BstEII/BamHI and ligating in the equivalent fragment from pcDNA3.1-PKCII. PECFP-PKCII was constructed by subcloning into the pECFP vector. Flag-tagged Par6c (pFlagPar6c) was a gift from Dr Dan Lin and Dr Tony Pawson. To create pECFP-Par6c and pEYFP-Par6c the pFlagPar6c cDNA was cut with EcoRI/PvuII and ligated into pECFP-C1 and pEYFP-C1 cut with EcoRI/SmaI. The PKCIIA119E mutation was generated by PCR using pcDNA3.1-PKCII as the template and the primers; 5'-CTACCGCCATGGAGAAAG-AAGATGGAGAAAGCCG-3' and zetaII2133(-) (described above). The product was cut with NcoI/ClaI and ligated into pcDNA3.1-mycPKCII to make pcDNA3.1-mycPKCIIA119E. All DNA constructs were confirmed by sequencing. The PKCII sequence obtained correlated with the sequence established by Celera for the mouse genome (J Sgouros and S Parkinson, unpublished).

Tissue culture. Cos7, MDCK, HELA, 293, NIH3T3, U251, and Caco2 cells were maintained in DMEM containing 10% FBS and penicillin/streptomycin. MCF10A cells were grown in DMEM containing Ham's F12, 5% horse serum, 10 g/ml insulin, 5 g/ml hydrocortisone, 20 ng/ml EGF, 100 ng/ml cholera toxin, and penicillin/streptomycin. HC11 cells were maintained in RPMI, 10% FBS, penicillin/streptomycin, 10 g/ml insulin, and 20 ng/ml EGF.

Virus production. Retrovirus expressing an RNAi against PKCII were developed using the pSuppressorRetro (pSR) system (Imgenex, San Diego). Complementary oligos (PKCII target sequence: 5'-aagcatgtatttgagcaggcg-3') were ligated into pSR, checked by sequencing, and cotransfected into 293T cells with the packaging vector pCL-Eco. Control virus was generated against PKC and a sealed pSR plasmid cotransfected with the packaging vector. Virus was collected 48 h post-transfection, filtered, added to HC11 cells and stable lines selected with neomycin. Under growth culture conditions (above), only the PKCII viral-infected lines demonstrated the change in phenotype; the other cell lines were indistinguishable from noninfected cells. However, in other culture conditions, virus targetting PKC demonstrated a significant phenotype distinct from PKCII. To avoid confusion concerning these cell lines, the 'vector only' line of pSR is shown as a control.

Cloning and chromosome localisation of murPKCII. Using a randomly labelled ([-³²P]-dCTP) 300 bp 5' fragment of PKC cDNA as a probe, a mouse genomic gt 10 phage library was screened. Three identical hybridising clones were isolated after primary screening and purified by two further rounds of selection. Clone 3, containing a 13 kb insert (flanking PKCII), was selected for further analysis. The chromosome localisation of PKCII was assessed by FISH using the following probes and chromosomal paints on mouse metaphase spreads, X7 (PKCII 3'UTR), pZ-X5 (PKC intron sequence).

Alignments. Alignments were performed using Clustal X software with the sequences encoding mouse PKCII and PKC (accession number M94632).

In vitro transcription/translation. In total, 1 g of genomic PKCII DNA composed of 5'UTR (2.5 kb), putative PKCII coding region (1.8 kb) and 3'UTR (8.7 kb) was transcribed in vitro and translated in rabbit reticulocyte lysates (Promega) according to manufacturers instructions at 30°C for 2 h in the presence of ³⁵S-methionine. The entire reaction was denatured in sample buffer, separated on a 10% acrylamide gel and analysed by autoradiography.

RT–PCR. Mouse tissue total RNA was extracted using Trizol reagent (Gibco) followed by extensive treatment of the RNA fraction with DNAse. Reverse transcription was carried out using the Access RT–PCR system (Promega) with the following changes to the manufacturers protocol. Reverse transciption was performed using random 9-mers as primers. RNA was ensured to be DNA-free if -actin was amplified using musBactI (5'-tgacggggtcacccacactgtgccc-atcta-3') and musBactII (5'-agtaacagtccgcctagaagcacttgcggt-3') in the presence but not absence of reverse transcriptase. The following primers were used for amplification by RT–PCR and were specific for their designated targets as confirmed by sequencing: PKC-(zeta199(+) 5'-tgggtggacagtgaaggtgac-3', zeta392(-) 5'-gttggctcggtacagcttc-3'), PKCII-(zeta199(+), zetaII390(-) 5'-attggctcggtac-ggcttt-3'), PKM-(psizeta(+) 5'-ggagacccacccagcccattg-3', zeta392(-)). reverse transcription: 48^oC—60 min, 95^oC—2 min. Add (+) and (-) primers and Tfl polymerase on ice. PCR: 95°C—2 min, 35 (95°C—1 min, 62°C—1 min, 68°C—1 min), 68°C—10 min.

Transfections. Cos7 cells (1.5 10⁵) were seeded into 3 cm dishes, left overnight, and transfected by CaPO₄ with 1 g of DNA. The following day the precipitate was removed, cells washed and left to recover overnight.

Immunoprecipitations. Cells (10 cm dish) were lysed in 1 ml buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5 mM EDTA, 5 mM MgCl₂, 2 mM DTT, 1 mM PMSF, and complete protease inhibitor cocktail tablet (Boehringer Mannheim). The lysate was collected by scraping and insoluble material removed by centrifugation (100 000 g, 30 min). Protein G-coupled antibody was added to the soluble lysate and rotated for 2–3 h at 4°C. Immunocomplexes were washed 4 with lysis buffer and solubilised for gel electrophoresis in 100 l of sample buffer.

Immunocomplex activity determination. Immunoprecipitates of PKC or myc-tagged PKC and PKCII were isolated essentially as above except in the presence of 1 M okadaic acid, 50 mM sodium fluoride, 10 mM p-nitrophenylphosphate, 10 mM sodium orthovanadate and in the absence of MgCl₂. Immunocomplexes on protein G-Sepharose beads were incubated with shaking at 22°C in the presence of 50 mM HEPES pH 7.5, 10 M [-³²P]ATP, 10 mM MgCl₂, 1 mg/ml myelin basic protein and 1 M okadaic acid. After 15 min the reaction was stopped by the addition of sample buffer. Radiolabelled myelin basic protein was separated on a 12.5% polyacrylamide gel, identified by autoradiography, and quantified by direct Cerenkov counting.

Fractionation. Cos7 cells were lysed with 1 ml (4°C) hypotonic buffer (10 ml Tris (pH 8.0), 10 mM NaCl, 3mM MgCl₂, 0.25% NP-40)/10 cm² dish. Cells were scraped into Eppendorf tubes and passed 4 through a 21 gauge needle. The lysate was spun for 6 s at 12 000 rpm at 4°C to isolate nuclei. The cytosolic fraction was removed and proteins precipitated with TCA. The nuclear- and cytosolic-rich fractions were resuspended in equal volumes of sample buffer and analysed by electrophoresis and Western blot.

Immunofluorescence and confocal microscopy. Live cell and fixed cell microscopy was performed on an inverted confocal laser scanning microscope (Carl Zeiss, Inc.) equipped with an 63X/1.4Plan-Apochromat oil-immersion objective and fitted with an environmental chamber. Each image represents a single two-dimensional projection. For live cell microscopy, cells were seeded on 35 mm glass bottom dishes (MatTek Corporation), and transfected with the indicated plamids the following day. Alternatively, cells were fixed on 1.5 mm coverslips with 100% MeOH (-20^oC) for 10 min, and blocked in Tris-buffered saline 0.1% Triton (TBST) with 3% BSA (30% stock, Sigma) for 30 min. Primary antibodies were added in TBST 3% BSA overnight at 4°C. Cells were washed, then fluorescent-conjugated secondary antibody (see reagents) added (1:200 in TBST) for 30 min. Cells were washed in TBST, TBS, and H₂O, then mounted in Mowiol Mount (Sigma) containing DABCO (Sigma).

We thank Dr T Sacktor, Dr T Pawson and Dr D Lin for generously providing reagents. Dr D Zicha, Dr J Moneypenny and Mr P Jordan in the Light Microscopy Laboratory at Cancer Research, UK for valuable help with confocal imaging. Dr J Williamson in the Human Cytogenetics Laboratory at Cancer Research, UK for the chromosomal localisation data and Dr J Sgouros for confirming the PKCII sequence in the Celera mouse genomic database. This work was funded by research grants from the Human Frontiers Science Program (SJP) and European Union (QLK3-CT-2000-01038).

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