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

Amino acid alignment of murPKCζII with murPKMζ, murPKCζ (accession number M94632). The amino acid sequences of PKCζ and PKCζII were predicted from the nucleic acid sequence in Figure 1. The PKMζ 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 PKCζ these are PB1, C1, and kinase domains.

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 μm). The right panels show confocal X–Y sections through an apical plane (bars=50 μM). (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) PKCζ activity is not altered by suppression of PKCζII expression. PKCζ was immunoisolated from cells as described in the Materials and methods section. PKCζ protein and activity were determined and no change in activity was observed. Quantitation of four observations is shown under the representative western (PKCζ) and autoradiograph (³²P-myelin basic protein).

Characterisation of PKCζII localisation and protein interactions. (A) Localisation of PKCζII, PKCζIIA119E, PKCζ, and Par6c by live-cell confocal microscopy. YFP-tagged plasmids encoding PKCζII (top left), PKCζIIA119E (bottom left), PKMζ (bottom right) and Par6c (top right) were transfected into Cos7 cells and visualised (pseudocolour red) by confocal microscopy (bar=50 μM). (B) PKCζII and PKMζ colocalise in the cytoplasm of coexpressing cells. Cos7 cells (phase, top right) expressing YFP-PKMζ (red) and CFP-PKCξII (green) were observed by live-cell confocal microscopy (merge, bottom right) (bar=50 μM). (C) PKCζII and Par6c colocalise in the cytoplasm of coexpressing cells. Cos7 cells (phase, top right) expressing YFP-PKCζII (red) and CFP-Par6c (green) were visualised by live-cell confocal microscopy (merge, bottom right) (bar=30 μM). (D) Interaction with PKMζ stabilises PKCζII in the cytoplasm. Cos7 cells (Cont) expressing mycPKCζII (mycζII), PKMζ (PKMζ) or coexpressing mycPKCζII and PKMζ (mycζII+PKMζ) 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. PKCλ/ι is endogenously expressed in Cos7 cells. (E) The intensity of the indicated bands (PKCλ/ι, PKMζ, or mycζII) 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) PKCζII forms a complex with Par6c or PKMζ that is not dependent on the pseudosubstrate site. Cos7 cells (lane 1) expressing PKMζ (lane 2), flagPar6c (lane 3), mycPKCζIIA119E (lane 4), mycPKCζIIA119E+PKMζ (lane 5), mycPKCζIIA119E+flagPar6c (lane 6) mycPKCζII (lane 7), mycPKCζII+PKMζ (lane 8) or mycPKCζII+flagPar6c (lane 9) were lysed and the detergent-soluble fraction was incubated with Protein G Sepharose-coupled 9E10 (αmyc) 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 (PKMζ), anti-flag (flagPar6c), or anti-myc (mycPKCζII/mycζIIA119E) primary antibodies.

PKCζII inhibits formation of tight junctions in HC11 cells. (A) PKCζII RNAi specifically reduces PKCζII, but not PKCζ RNA. RT–PCR detection of PKCζII, PKCζ, and β-actin message in control HC11 cells (pSR) and PKCζII 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 β-casein mRNA expression (not shown). DNA contamination was minimal as detected by the lack of amplification of β-actin in the absence of reverse transcriptase using the specified primers (β-actin – RT). (B) Western blot detection of PKCζ and PKCζII in HC11 pSR (control) and HC11 pSRII (PKCζII 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 PKCζ or the C-terminus of atypical PKCs. PKCζII is recognised by the N-terminal but not C-terminal antibody and is decreased in HC11 pSRII (PKCζII 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 (PKCζII 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 β-catenin, β-actin, and ZO-1. Single confocal sections (0.5 μm) are shown for the two cell lines (bar=50 μm). (D) PKCζII 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 μm) of stained cells are shown above confocal images in the X–Y plane (bottom, bar=50 μM).

The gene encoding PKCζII is on mouse chromosome number 7 and is transcribed and translated in various tissues and cell lines. (A) Chromosome localisation of the PKCζII gene. FISH was performed with a specific probe recognising the PKCζII 3′UTR and chromosomal paints recognising chromosome 7. Hybridising regions of the PKCζII probe are indicated with red arrows and correlate with the highlighted chromosome 7s (right panel). (A′) Chromosome localisation of the PKCζ gene. FISH was performed with a specific probe recognising an intron sequence from the murine PKCζ gene and chromosomal paints recognising chromosome 4. Fluorescent-tagged PKCζ 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 β-actin or PKCζII product in the absence of reverse transcriptase (not shown). The specific primers used to detect PKCζ, PKMζ, and PKCζII are indicated in Materials and methods. (C) In vitro transcription/translation of isolated PKCζII genomic clone and detection of a 45 kDa protein. Genomic DNA encoding PKCζII 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—PKCζII no ATG, lane 2—PKCζII no 5′-UTR, lane 3—PKCζII with 5′ and 3′ UTR, lane 4—PKCζII 3′UTR only, lane 5—luciferase control. (D) myc-PKCξ or PKCξII 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 PKCζII 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 (λ/ι/ζ) (top panel) or an antibody directed against the N-terminus to detect PKCζII (bottom panel) (see Materials and methods for specificity). Lysate from Cos7 cells expressing a PKCζII cDNA plasmid (Cos7 (PKCζII)) is included as a migration control.