Structure, Expression, and Properties of an Atypical Protein Kinase C (PKC3) from Caenorhabditis elegans
PKC3 IS REQUIRED FOR THE NORMAL PROGRESSION OF EMBRYOGENESIS AND VIABILITY OF THE ORGANISM*

Shi-Lan WuDagger , Jeff Staudinger§, Eric N. Olson, and Charles S. RubinDagger par

From the Dagger  Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461, the § Molecular Endocrinology Department, Glaxo Wellcome, Research Triangle Park, North Carolina, and the  Department of Molecular Biology and Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Little is known about differential expression, functions, regulation, and targeting of "atypical" protein kinase C (aPKC) isoenzymes in vivo. We have cloned and characterized a novel cDNA 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 ~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 revealed 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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The "atypical" subclass of protein kinase C (PKC)1 isoforms is composed of PKCzeta and PKClambda /iota (1-5). Distinct genes encode mammalian atypical PKCs (aPKCs), but the proteins are similar in size (Mr ~68,000) and amino acid sequence (72% identity) (3-5). aPKCs are included in the PKC family because of the following three characteristic features: (a) catalytic domains of aPKCs and other PKCs are homologous; (b) like other PKC isoforms, PKCs zeta  and lambda /iota contain a Cys-rich region that is preceded by a pseudosubstrate site; (c) aPKCs and other PKC isoforms require phosphatidylserine (PS) as a co-factor for expression of maximal phosphotransferase activity (1-5). In contrast to the classical (alpha , beta I, beta II, and gamma ) and novel (delta , epsilon , eta , and theta ) PKCs (cPKCs and nPKCs, respectively), the zeta  and lambda /iota isoforms lack binding/regulatory sites for diacylglycerol (DAG), phorbol esters, and Ca2+ (3-6).

PKCs zeta  and lambda /iota have been tentatively linked with the regulation of mitogenesis, inflammation, growth factor controlled gene transcription and inhibition of apoptosis (7-13). However, knowledge of biochemical pathways by which PKCs zeta  and lambda /iota control aspects of cell physiology is limited. Activation of platelet-derived growth factor or epidermal growth factor receptor tyrosine kinases in cells that overexpress aPKCs promotes autophosphorylation of atypical kinases (on Thr/Ser) and transcription of an AP-1-controlled reporter gene (10). Both effects depend upon activation of phosphatidylinositol 3-kinase. Activated PKCs zeta  and lambda /iota apparently promote mitogenic signaling via the Ras-MEK-mitogen-activated protein kinase pathway through interactions downstream from Ras and upstream from MEK (7-10, 14). The precise mechanism(s) and number of steps that couple phosphatidylinositol 3,4,5-trisphosphate synthesis with aPKC activation and mitogenesis are unknown.

Tumor necrosis factor-alpha and interleukin-1 stimulate sphingomyelinase-catalyzed synthesis of ceramide (15). Ceramide activates PKCzeta in vitro and apparently promotes phosphorylation of Ikappa B by sequential activation of PKCzeta and an uncharacterized, 50-kDa protein kinase in cells (11, 12). Phosphorylated Ikappa B dissociates from its' cytoplasmic ligand NFkappa B and is rapidly proteolyzed. NFkappa B then translocates to the nucleus and stimulates gene transcription (16). However, the ability of PKCzeta (and the 50-kDa effector kinase) to regulate activity of NFkappa B or the cytokine mediator p85 Ikappa B kinase (17) in a normal physiological context remains to be established.

An 80-kDa protein named LIP binds PKClambda /iota in vitro and in serum-stimulated Cos cells that overexpress both LIP and PKClambda /iota (18). PKC lambda /iota ·LIP complex formation correlates with increased kinase activity and NFkappa B-stimulated transcription of a reporter gene. Both aPKCs are inhibited by a 35-kDa protein named Par-4, which accumulates during apoptosis (13, 19). Formation of Par-4·PKCzeta complexes in doubly transfected NIH-3T3 cells correlates with inhibition of AP-1-mediated transcription and appearance of biochemical and morphological characteristics of apoptosis (13).

Conclusions regarding functions and regulation of aPKCs are derived principally from studies on overexpressed wild-type and mutant PKC lambda /iota and zeta  transgenes in immortalized or transformed cells that also overexpress receptor tyrosine kinases, protein modulators (Par-4, LIP), constitutively activated or inhibited Ras or MEK, etc. (7-14, 18). High level co-expression of regulatory proteins in transfected cells enables characterization of ordered interactions among members of a signaling cascade. However, such experiments provide no information on whether observed interactions are of major or minor importance in normal cells that contain low levels of endogenous signaling molecules. There are also major gaps in our basic knowledge of (a) patterns and levels of aPKC gene expression, (b) the nature and quantitative significance of upstream regulators of aPKC catalytic activity, (c) intracellular anchoring/targeting of aPKCs, and (d) the number and identity of downstream substrate/effector proteins controlled by aPKCs in specific cells in intact organisms. Furthermore, aPKC expression, activity, location, and functions may be regulated during and after cell differentiation and tissue development.

The nematode Caenorhabditis elegans provides a system for investigating properties and functions of aPKCs in intact cells in vivo. Adult C. elegans are composed of 959 somatic cells that are organized into tissues that constitute digestive, reproductive, muscular, hypodermal, and nervous systems (20-22). The cellular and developmental biology of C. elegans have been characterized in exceptional detail (20-22). C. elegans development and homeostasis are regulated by signal transduction systems that are the same as those operative in mammals (23, 24). Methods for producing mutant, transgenic, and "knock-out" strains of C. elegans enable analysis of gene promoter activities and gene functions in individual cells in situ (25-29). Immunofluorescence microscopy (30) permits detection of specific proteins in individual cells of intact C. elegans at all stages of development.

We have characterized a cDNA that encodes a novel Ca2+, DAG-independent C. elegans PKC (PKC3). PKC3 is partially homologous with mammalian aPKCs, and its catalytic properties provide a functional basis for its assignment to the atypical subclass of PKCs. A 647-bp promoter/enhancer that precedes the pkc-3 gene governs expression of PKC3 mRNA and protein in cells that are involved in feeding, digestion, excretion, and reproduction. A high proportion of PKC3 is anchored/targeted in the vicinity of cortical actin cytoskeleton at all stages of development. A candidate PKC3 anchoring protein has been identified. Antisense RNA-mediated depletion of PKC3 disrupted embryogenesis and compromised the viability of C. elegans. Accumulation of PKC3 in terminally differentiated muscle and epithelial cells indicates that the kinase can regulate functions that are independent of cell cycle progression and apoptosis.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of cDNAs Encoding PKC3-- Three oligonucleotides (5'-CTCAATAGACTCACAAATGGAACTTGATGAAGCAGTTCGATG-3', 5'GAATATGGATTCAGTGTTGATTGGTGGGCATTGGGAGTGTTAATG-3', and 5'-TTTGAATACGTGAATCCTCTACAAATGAGTCGGGAAGATTCAGTC-3'), whose sequences correspond to portions of C. elegans expressed sequence tags (GenBank) that are homologous with human PKCzeta (31), were synthesized and end-labeled with 32P as described in Hu and Rubin (32). Labeled oligonucleotides (107 cpm/ml) were used to screen 450,000 plaques from a C. elegans cDNA library in bacteriophage lambda ZAPII (Stratagene). Nitrocellulose filter lifts were hybridized and washed as described previously (32). Positive plaques were purified to homogeneity, and recombinant pBluescript SK phagemids that contain hybridizing cDNA inserts were excised from the phage in Escherichia coli (33) and purified. Two positive cDNA clones were isolated. The larger cDNA (2.4 kbp) contains the complete coding sequence and 3'-untranslated region for PKC3 mRNA; the sequence of the smaller cDNA (1 kbp) is identical with a segment of the larger insert.

DNA Sequence Analysis-- cDNA inserts were sequenced by the dideoxynucleotide chain termination procedure of Sanger et al. (34) as described previously (32).

Computer Analysis-- Analysis of sequence data, sequence comparisons, and data base searches were performed using PCGENE-IntelliGenetics software (IntelliGenetics, Mountainview, CA) and BLAST programs (35, 36) provided by the NCBI Server at the National Institutes of Health.

Preparation of Genomic DNA and Southern Gel Analysis-- C. elegans genomic DNA was isolated by using a QIAamp Kit (Qiagen) according to the manufacturer's instructions. Fragments of DNA were generated by digestion with restriction endonucleases, fractionated by electrophoresis in a 1% agarose gel, and transferred to a Nytran membrane as described previously (32). The Southern blot was probed with 32P-labeled PKC3 cDNA (2 × 106 cpm/ml) that was prepared as reported previously (32). A 2.4-kbp PKC3 cDNA insert (see Fig. 1, below), which was released from pBluescript SK by cleaving with PstI and XhoI, served as a template for synthesis of the radiolabeled probe. Conditions for hybridization, as well as high and low stringency washing of the membrane, and autoradiography are given in Hu and Rubin (32).

Expression and Purification of a PKC3 Fusion Protein-- A segment of cDNA encoding amino acid residues 477-597 of PKC3 (see Fig. 1) was synthesized via polymerase chain reaction. The 5' primer contained an NdeI restriction site followed by nucleotides 1429-1451 of PKC3 cDNA (Fig. 1); the 3' primer consisted of the inverse complement of nucleotides 1770-1794 in PKC3 cDNA preceded by a BglII restriction site. The cDNA fragment was amplified by the polymerase chain reaction as described previously (32). Product DNA was digested with NdeI and BglII and cloned into the bacterial expression plasmid pET-14b (Novagen) that was cleaved with NdeI and BamHI. The cDNA insert is preceded by vector DNA that encodes a 20-residue N-terminal peptide. The N-terminal fusion peptide includes six consecutive His residues, which constitute a Ni2+-binding site. Transcription of the fusion gene is driven by the isopropyl-1-thio-beta -D-galactopyranoside-inducible T7 RNA polymerase. E. coli BL21 (DE3) transformed with recombinant expression plasmid was grown and induced with isopropyl-1-thio-beta -D-galactopyranoside as described previously (37). Bacteria were harvested, disrupted, and separated into soluble and particulate fractions as described in previous studies (37). PKC3 fusion protein, which was recovered in the pellet fraction, was dissolved in 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 8 M urea, 2 µg/ml pepstatin, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 1 mM EDTA, 10 mM benzamidine HCl. Fusion protein was purified to near-homogeneity by nickel-chelate affinity chromatography (in the presence of M urea) as described previously (38). The purified partial PKC3 protein remained soluble when urea was removed by dialysis against 10 mM sodium phosphate, pH 7.4, 0.15 M NaCl (phosphate-buffered saline, PBS). Approximately 2.5 mg of highly purified PKC3 fusion protein was isolated from a 1-liter culture of E. coli.

Production of Antiserum Directed against PKC3-- Samples of the PKC3 fusion protein were injected into rabbits (0.4-mg initial injection; 0.2 mg for each of five booster injections) at Covance Laboratories (Vienna, VA) at 3-week intervals. Antiserum was collected at 3-week intervals after the first injection.

Affinity Purification of Anti-PKC3 Immunoglobulins-- Purified PKC3 fusion protein was coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) (39) to yield a final concentration of 1.5 mg of protein bound per ml of resin. Serum (2 ml) was incubated with 2 ml of PKC3 fusion protein-Sepharose 4B for 2 h at 20 °C. Next, resin containing anti-PKC3 IgGs was packed into a column and washed with 20 mM Tris-HCl, pH 7.6, 0.5 M NaCl until the flow-through reached an A280 of 0. The column was successively eluted with 5 ml of 0.5% acetic acid, pH 2.5, 0.15 M NaCl, and 5 ml of 0.1 M diethylamine, pH 11.8, to release IgGs. Sufficient 1 M Na2 HPO4 or 0.5 M Hepes was added to column fractions to adjust the pH to ~7.5. IgG concentration was estimated from the absorbance at 280 nm. IgGs were dialyzed against PBS containing 50% glycerol and stored at -20 °C.

Growth and Synchronization of C. elegans-- The Bristol N2 strain of C. elegans was grown as described previously (32). Embryos, prepared by the method of Sulston and Hodgkin (40), were hatched in the absence of nutrients. Hatched nematodes were transferred to plates containing Azotobacter vinelandii as a food source, whereupon the animals develop synchronously (41). L1 larvae were harvested 6 h after feeding, L2 larvae at 20 h, L3 larvae at 29 h, L4 larvae at 40 h, young adult C. elegans at 52 h, and egg-laying adult animals were collected at 78 h. Pelleted C. elegans were suspended in an equal volume of 0.1 M NaCl and frozen at -196 °C. Frozen C. elegans were pulverized in a mortar cooled with liquid N2, and the resulting powder was stored at -75 °C.

Electrophoresis of Proteins and Western Immunoblot Assays-- Cytosolic and particulate fractions of C. elegans and hamster AV-12 cell homogenates were isolated as described previously (32, 42). Proteins were fractionated by electrophoresis in a 10% polyacrylamide gel containing 0.1% SDS (39). Phosphorylase (Mr = 97,000), transferrin (77,000), albumin (67,000), ovalbumin (43,000), and carbonic anhydrase (29,000) were used as standards for estimation of Mr values.

Western blots of proteins from C. elegans and AV-12 cells were prepared, incubated with anti-PKC3 IgGs (1:2000, relative to serum), and washed as previously reported (43). Antigen-antibody complexes were visualized by using an enhanced chemiluminescence procedure (43).

Immunofluorescence Analysis of the Cellular and Intracellular Distribution of PKC3-- Post-embryonic C. elegans were fixed, washed, incubated sequentially with affinity purified anti-PKC3 IgGs (1:100, relative to serum) and fluorescein isothiocyanate-tagged goat IgGs directed against rabbit immunoglobulins as described previously (37). Actin was visualized by serially incubating the specimens with a mouse monoclonal IgG (1:100 dilution) (clone C4, ICN Inc.) directed against actin and rhodamine-coupled sheep antibodies directed against mouse IgGs (Amersham Corp.).

Embryos were fixed in 0.1 M sodium phosphate, pH 7.2, containing 4% paraformadehyde for 10 min. This step was repeated once. After two rinses with 0.1 M sodium phosphate, pH 7.2, embryos were immersed in methanol at -20 °C overnight. Next, embryos were rehydrated in 10% incremental steps using TTBS (0.1% (w/v) Tween 20, 0.15 M NaCl, 1% albumin, 0.1 M Tris-HCl, pH 7.5) as the diluent. Samples were then incubated with affinity purified PKC3 antibodies (1:100, relative to serum) for 2 h at 20 °C. After 3 washes with TTBS, goat anti-rabbit IgG antibody conjugated with fluorescein was added, and the incubation was continued for 1 h. Secondary antibody was preabsorbed with fixed embryos that did not receive anti-PKC3 antibody. Following three washes with TTBS and a final rinse with PBS, embryos were incubated for 30 min at 37 °C in PBS containing 10 µg/ml RNase. After three additional washes with PBS, stained embryos (3 µl) were mixed with 3 µl of 33 mM Tris-HCl, pH 9.5, containing 30 µM propidium iodide, 2% n-propylgallate, and 70% glycerol and were mounted on a 2% agarose pad. Fluorescence signals corresponding to PKC3-IgG complexes were obtained and recorded with a Bio-Rad MRC 600 laser scanning confocal microscope system (Image Analysis Facility, Albert Einstein College of Medicine) as described previously (37).

Cell Culture-- A cell line derived from a hamster subcutaneous tumor (AV-12) was obtained from the American Type Culture Collection. Cells were grown as described previously (42).

Expression of PKC3 in AV-12 Cells-- Complementary DNA that encodes the full-length PKC3 polypeptide was excised from the recombinant pBluescript plasmid (see above) by digestion with XbaI. The cDNA insert was cloned into the mammalian expression vector pCis2 (44), which was cleaved with XbaI and dephosphorylated. This placed PKC3 cDNA downstream from a powerful, constitutively active cytomegalovirus promoter. AV12 cells were transfected with recombinant pCis2 plasmid, as described previously (42). Since (a) pCis2 contains a constitutively active dihydrofolate reductase gene (44) and (b) AV12 cells are killed by methotrexate (45), stable transfectants can be selected by adding 0.25 µM methotrexate to the culture medium.

Purification of PKC3 from Transfected AV-12 Cells-- AV-12 cells that contain the PKC3 transgene were harvested in buffer A (20 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 10 mM EGTA, 2 mM EDTA, 1 mM DTT, 50 µg/ml leupeptin, 25 µg/ml aprotinin, 25 µg/ml pepstatin, 10 µg/ml soybean trypsin inhibitor, 10 mM benzamidine HCl, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.2% (v/v) Triton X-100) and disrupted with 20 strokes of a motor-driven Teflon glass homogenizer. All operations were performed at 4 °C. The homogenate was centrifuged at 40,000 × g for 10 min, and the supernatant solution was collected. Next, the sample was centrifuged at 150,000 × g for 45 min. Conductivity of the supernatant solution was adjusted to match the conductivity of buffer B (20 mM Tris-HCl, pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM DTT) supplemented with 0.1 M NaCl. The sample was applied to a column (1.5 × 10 cm) of DEAE-Sepharose that was equilibrated with buffer B containing 0.1 M NaCl. After washing with 60 ml of starting buffer, elution was performed with a linear gradient (360 ml) of NaCl (0.1-0.5 M) in buffer B at a flow rate of 1 ml/min. Fractions (3 ml) enriched in PKC3 were identified by Western immunoblot analysis and phosphotransferase assays (see below) and were pooled. Pooled fractions were diluted with an equal volume of buffer B and applied to a column (1.5 × 10 cm) of heparin-Sepharose 4B that was equilibrated with buffer B containing 0.1 M NaCl. The column was washed with 40 ml of buffer C (buffer B plus 10% glycerol) containing 0.1 M NaCl. Elution was performed with a 180 ml of linear gradient of NaCl (0.1-1.0 M) in buffer C at a flow rate of 0.5 ml/min. Fractions (1.5 ml) were collected and assayed as described above. Fractions containing peak levels of PKC3 were pooled, dialyzed against buffer D (20 mM potassium phosphate, pH 7.4, 1 mM DTT, 10% glycerol), and applied to a column (1 × 10 cm) of hydroxylapatite that was equilibrated with buffer D. After washing with 40 ml of equilibration buffer the column was eluted with a 60-ml linear gradient of potassium phosphate buffer (20-300 mM, pH 7.4) at a flow rate of 0.2 ml/min. PKC3 was eluted at ~ 0.1 M potassium phosphate. Fractions (1 ml) containing the highest levels of PKC3 polypeptide and kinase activity were pooled and stored at -20 °C. Overall, the enzyme (15 µg of total protein) was purified 1,750-fold and had a specific activity of 0.2 µmol/min/mg protein.

Assay for PKC3 Activity-- Catalytic activity of PKC3 was assayed by measuring the incorporation of 32P radioactivity from [gamma -32P]ATP into the synthetic peptide YRRGSRRWKKIY (General Biotechnologies, Inc.), which is a specific substrate. The reaction mixture (30 µl) contained 25 mM Tris-HCl, pH 7.4, 30 µM peptide substrate, 50 µM [gamma -32P]ATP (100-200 cpm/pmol), 5 mM MgCl2, 0.5 mM EGTA, 1 mM DTT and enzyme, unless otherwise indicated. Phospholipid, diacylglycerol, ceramide, fatty acids, CaCl2, or inhibitors were added as indicated in "Results." Reaction mixtures were incubated at 30 °C for 10 min. Assays were terminated by addition of 5 µl of 0.2 M EDTA, pH 8.0, containing 60 mM NaF. Reaction mixtures were then applied to P81 filter papers (Whatman). After filters were washed and dried (46), 32P radioactivity incorporated into substrate peptide was determined in a scintillation counter. One unit of protein kinase activity is defined as the amount of kinase that incorporates 1 nmol of phosphate into peptide substrate per min.

Protein Determination-- Protein concentrations were determined by using the Coomassie Plus Protein Assay Reagent (Pierce) with bovine serum albumin as a standard.

Preparation of Transgenic C. elegans-- The cosmid F09E5, which contains the pkc-3 gene and 5'-flanking DNA (see "Results"), was obtained from Dr. Alan Coulson, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. A 4.4-kbp fragment of genomic DNA was excised from the cosmid by digestion with NdeI and XmaIII and was cloned into the plasmid pGEM5Z (Promega), which was cleaved with NotI and NdeI. Next, recombinant pGEM5Z plasmid was digested with SphI and SmaI, and the excised 3.9-kbp genomic DNA insert was subcloned into the multiple cloning site of a C. elegans expression vector (pPD 90.23) (25, 26). The pPD90.23 plasmid was digested with SphI and BalI to generate a cloning site compatible with the staggered and blunt ends of the insert. The DNA insert from the pkc-3 gene contains 3.2 kbp of 5'-flanking DNA followed by exon I, intron I, and 78 codons of exon II. Codon 78 of exon II is fused in-frame with DNA encoding a nuclear localization signal and the E. coli lacZ reporter gene (25, 26). The lacZ coding region is followed by translation termination and poly(A) addition signals. Thus, the 5' promoter/enhancer region of the pkc-3 gene will drive expression of a fusion beta -galactosidase reporter enzyme that accumulates in nuclei.

A second fusion reporter gene was created by digesting the recombinant pPD90.23 plasmid (described above) with NcoI and StuI, filling in with Klenow DNA polymerase, and then re-ligating the blunt ends. This manipulation deletes nucleotides 1-2,549 from the 5' end of the C. elegans genomic DNA insert. Thus, the re-ligated vector contains only 647 bp of contiguous 5'-flanking DNA (putative promoter-enhancer region) upstream from pkc-3 exon I. Exon I, intron I, and 78 codons of exon II of the pkc-3 gene precede the lacZ reporter gene, as described above.

C. elegans were transformed by microinjecting recombinant reporter plasmid DNA (20 µg/ml) and a plasmid containing the dominant selectable marker gene rol-6 into the gonadal syncytium of young adult C. elegans as described previously (28, 47). Transgenic C. elegans were selected and maintained as indicated in previous studies (47). Transgenic C. elegans were fixed and stained for beta -galactosidase activity as described in Freedman et al. (47).

Ablation of PKC3 Function via Injection of Antisense RNA-- Capped sense and antisense PKC3 RNAs were synthesized, purified, and precipitated by using the mCAPTM RNA synthesis and capping kit (Stratagene) according to the manufacturer's instructions. The recombinant pBluescript SK plasmid that contains the 2.4-kbp cDNA shown in Fig. 1 served as a template for RNA synthesis. Synthesis of antisense PKC3 RNA was catalyzed by bacteriophage T7 RNA polymerase after recombinant plasmid was linearized by cleavage with PstI; T3 RNA polymerase synthesized sense RNA (mRNA) from the same template after linearization via XhoI digestion. Sense and antisense PKC3 RNAs (50 µg/ml) were microinjected into the gonadal syncytium of young adult C. elegans. The procedure is identical with that used for the injection of C. elegans expression plasmids during the creation of transgenic animals (see above). Individual injected nematodes were placed on separate culture plates and were allowed to lay eggs (containing developing embryos) for 24 h. Subsequently, parental C. elegans were removed and embryo morphology and ability of embryos to hatch were assessed after an additional 24-h period.

Assay for Binding of PKC3 with a Candidate Anchor Protein-- The TNTTM coupled reticulocyte lysate system (Promega Corp.) was programmed with 1 µg of recombinant pBluescript that contains full-length PKC3 cDNA. Incubations (50 µl) were performed at 30 °C for 90 min under conditions recommended by the manufacturer. This enabled phage T3 RNA polymerase to catalyze synthesis of PKC3 mRNA, which was immediately translated into the cognate protein in the presence of [35S]Met and [35S]Cys by ribosomes, t-RNAs, and enzymes in the reticulocyte lysate.

Complementary DNA encoding the C. elegans homolog (PIKC1CE) of mammalian PICK1 (48) was cloned into the expression plasmid pGEX-3X (Pharmacia Biotech Inc.). This enabled inducible high level synthesis in E. coli of full-length PICK1CE (445 amino acids)2 fused to the C terminus of glutathione S-transferase (GST). GST-PICK1CE fusion protein was purified to near-homogeneity by affinity chromatography on GSH-Sepharose 4B as described previously (37) and dialyzed against PBS containing 50% glycerol. GST-PICK1CE (2 µg) was mixed with 10% of the translation mixture (5 µl) containing 35S-labeled PKC3 or control protein in 20 mM Tris-HCl, pH 7.5, containing 0.2 mM DTT, 0.15 M NaCl, 1 mM EDTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 10 µg/ml soybean trypsin inhibitor, 10 mM benzamidine HCl, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 0.05% (w/v) Triton X-100. Samples (final volume = 25 µl) were incubated at 20 °C for 45 min and then at 4 °C for an additional 45 min. Next, 20 µl of GSH-Sepharose 4B beads (50% suspension in binding buffer) was added, and the incubation was continued for 1 h at 4 °C. Subsequently, 400 µl of wash buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.3 M NaCl, 5 mM EDTA, 0.1% Triton X-100) was added, and the beads were pelleted by centrifugation at 3,000 × g. The supernatant solution was removed and then the beads were washed 4 additional times by resuspension in 500 µl of wash buffer and centrifugation at 3,000 × g. Finally, bound proteins were released from the GSH-Sepharose 4B beads by boiling in 20 µl of SDS gel loading buffer. Proteins were subjected to denaturing electrophoresis as described above. Gels were impregnated with ENHANCE (Amersham Corp.), dried, and exposed to x-ray film to generate fluorograms. Autoradiography produced similar results.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning and Sequence Analysis of cDNA Encoding C. elegans PKC3-- A cDNA that encodes a novel C. elegans PKC (designated PKC3) was retrieved from a bacteriophage library as described under "Experimental Procedures." The 2,351-bp sequence of the cloned cDNA is presented in Fig. 1. Conceptual translation of the DNA sequence revealed an open reading frame that begins with a Met codon (nucleotides 1-3) in a C. elegans consensus context for translation initiation ((A/G)NNATGT) and terminates with a translation stop codon at nucleotides 1792-1794. The 3'-untranslated region is composed of 537 nucleotides and precedes a polyadenylate tail. C. elegans mRNAs contain classical (AATAAA) or variant poly(A) addition signals 12-17 nucleotides upstream from the target site for poly(A) polymerase (49). Three nested copies of a variant hexanucleotide signal, AAAAAA (49), occupy this region (nucleotides 2314-2322, Fig. 1) in PKC3 mRNA, thereby providing a recognition site(s) for 3' end processing enzymes.


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Fig. 1.   Sequences of PKC3 cDNA and protein. The nucleotide sequence of PKC3 cDNA is shown. The derived amino acid sequence is presented below the corresponding codons.

Since only three nucleotides in the 5'-untranslated region of PKC3 cDNA were determined by direct sequencing (Fig. 1), it was necessary to verify the assignment of the initiator ATG by further analysis. This task was facilitated by the recent determination of the sequence of a 45-kbp genomic DNA insert from cosmid F09E5 (GenBank accession number U37429) by the C. elegans genome project consortium (50). The 3' end of this genomic DNA segment includes the entire pkc-33 gene, as well as upstream and downstream flanking DNA. An in-frame translation stop codon lies 18 bp upstream from the consensus translation start site. No alternative Met codons are present in the intervening 18 bp. Further evidence supporting translation initiation at the indicated ATG codon was obtained by in vitro transcription and translation of the cDNA shown in Fig. 1. In vitro synthesis yielded a 35S-labeled protein that is identical in size (apparent Mr = 75,000) with PKC3 obtained from C. elegans (e.g. see Fig. 3C, below). To exclude the possibility that translation is initiated at the second, in-frame Met codon (nucleotides 211-213, Fig. 1), PKC3 cDNA was truncated by deletion of nucleotides -4 to 200. The mutant cDNA programmed the in vitro synthesis of a polypeptide that is substantially smaller (apparent Mr = 67,000) than authentic C. elegans PKC3 (data not shown).

Copy Number and Chromosomal Localization of the PKC3 Gene-- C. elegans DNA was digested with restriction enzymes that cut infrequently, and fragments were characterized by Southern gel analysis (data not shown). Identical, simple patterns of hybridizing DNA fragments were observed when Southern blots were probed with full-length, 32P-labeled PKC3 cDNA and then washed at either high or low stringency. Thus, PKC3 appears to be encoded by a single copy gene. Determination of DNA sequences that flank the 5' and 3' ends of the pkc-3 gene by the C. elegans genome project places the pkc-3 locus slightly to the left of center on chromosome II, where it is flanked by genes named clr-1 and vhp-1.

Structure/Function Relationships in C. elegans PKC3-- The cDNA sequence in Fig. 1 encodes a novel protein kinase (PKC3) that is composed of 597 amino acids and has a Mr of 68,017. Functional roles for conserved amino acids in S/T protein kinases have been established by a combination of biochemical analysis, comparisons of sequences of hundreds of phosphotransferases, mutagenesis, and determination of three-dimensional structures for several prototypic S/T protein kinases (51-54). Application of this knowledge to the amino acid sequence of PKC3 enables tentative identification of functional domains in the nematode enzyme. Amino acids 250-515 (Figs. 1 and 2) constitute the catalytic domain of C. elegans PKC3. A segment of PKC3 that includes amino acids 260-282 is a variant version (GXGX2AX16K) of the classical GXGX2GX16K motif found in most S/T protein kinases. Mammalian aPKCs also contain Ala at the corresponding position (Fig. 2), whereas all other PKCs have Gly. This domain probably provides hydrogen bonds and charged side chains that anchor the alpha  and beta  phosphates of ATP in the catalytic cleft. Lys282 is essential for catalysis. Asp395 is also involved in binding Mg-ATP. Glu422, which is part of a conserved APE tripeptide motif, as well as Asp431 and Arg502 subserve stabilization of the catalytic core region (52, 53). The RDLKLDN segment (residues 376-382) of PKC3 constitutes a S/T protein kinase "signature" sequence (51) and is homologous with a portion of the catalytic loop in protein kinase A (52, 53).


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Fig. 2.   Comparison of C. elegans PKC3 with mammalian aPKCs. The derived amino acid sequence of PKC3 is aligned with sequences of human PKClambda /iota (4) and human PKCzeta (31). Amino acids conserved in all three kinases are marked with asterisks.

C. elegans PKC3 contains a domain (residues 128-177) in which six Cys and two His residues are arranged in perfect register with a pattern conserved in all previously characterized PKC isoforms: HX12CX2CX13CX2CX4HX2CX7C (1, 2, 56). A predicted pseudosubstrate site is evident in a basic region of the PKC3 polypeptide (DTVYRRGARRWKK, residues 109-121, Figs. 1 and 2) that precedes the Cys-rich domain. Pseudosubstrate domains mimic structural features of PKC phosphorylation sites but lack Ser and Thr and are thought to form intramolecular inhibitor complexes with the catalytic site (55).

C. elegans PKC3 shares a substantial degree of overall homology (~56% identity) with the mammalian lambda /iota and zeta  isoforms (Table I, Fig. 2). In contrast, the primary structure of PKC3 has diverged markedly (only 35% overall identity) from sequences of C. elegans PKC 1B (an nPKC) and PKC 2A (a cPKC) (37, 56). No other protein kinases share >30% identity with PKC3. Sequence identity between PKC3 and aPKC isoforms varies widely among discrete domains (Table I).

                              
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Table I
Sequence identities among the functional domains of C. elegans PKC3, mammalian aPKCs, and C. elegans PKC1B
Functional domains corresponding to the indicated segments of the PKC3 sequence were aligned with the corresponding regions of human PKC lambda /iota (4), human PKCzeta (31), and C. elegans PKC1B (37), a representative nPKC.

Organization of the C. elegans pkc-3 Gene-- During the course of our investigations the C. elegans genome project (50) deposited DNA sequences for chromosome II in the GenBank data base. A search of the data base with the cDNA sequence for PKC3 (Fig. 1) disclosed that cosmid F09E5 (accession number U37429) contained the pkc-3 gene. (No previous studies have addressed experimentally any aspects of PKC3 gene transcription/regulation or properties and functions of the PKC3 polypeptide.) Alignment of PKC3 cDNA with the cosmid DNA sequence revealed the intron/exon organization of the cognate gene (Table II). The compact C. elegans pkc-3 structural gene contains 9 exons, but spans only 3.6 kbp of DNA because of the small to moderate sizes of the introns (Table II).

                              
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Table II
Organization of the C. elegans pkc-3 gene

Preparation and Specificity of Anti-PKC3 Immunoglobulins-- Antiserum directed against a C-terminal segment (amino acids 477-597) of PKC3 was produced in rabbits. Affinity purified anti-PKC3 IgGs (see "Experimental Procedures") bound a 75-kDa protein in homogenates of C. elegans (Fig. 3A, lane 1). The antibodies also complexed a protein of the same size in cytosol derived from hamster AV-12 cells that were stably transfected with a PKC3 transgene (Fig. 3B, lane 1). The target antigen was not present among cytosolic proteins isolated from non-transfected AV-12 cells (Fig. 3B, lane 4). Moreover, the 75-kDa antigen was not detected when Western blots of proteins from C. elegans and transfected AV-12 cells were probed with preimmune IgGs or anti-PKC3 IgGs that were preincubated with excess partial PKC3 fusion protein (Fig. 3, A and B, lanes 2 and 3). The apparent Mr of the nematode kinase was confirmed by in vitro translation. PKC3 mRNA (generated from the cDNA template shown in Fig. 1), programmed synthesis of a 35S-labeled polypeptide that exhibited a Mr of 75,000 in a denaturing polyacrylamide gel (Fig. 3C, lane 3). Thus, affinity purified IgGs selectively bind C. elegans PKC3. The discrepancy between the apparent Mr (75,000) and calculated Mr (68,000) values for PKC3 is presumably due to a decreased concentration of SDS binding sites in the PKC3 polypeptide chain relative to standard Mr marker proteins. A similar disparity between apparent and calculated Mr values is observed for mammalian aPKCs (4, 5, 57).


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Fig. 3.   IgGs directed against a C-terminal fragment of PKC3 specifically bind recombinant and endogenous protein kinase C3. A, a Western blot was prepared and developed as described under "Experimental Procedures." Each lane received 30 µg of total C. elegans proteins. Lanes 1 and 3 were incubated with affinity purified IgGs (25 ng/ml, 1:2000 relative to serum) directed against PKC3. Lane 2 was incubated with preimmune serum (1:1000); excess purified antigen (3 µg) was present when lane 3 was probed with affinity purified antibodies. Chemiluminescence signals were recorded on x-ray film. B, a Western immunoblot is shown. Lanes 1-3 received cytosolic proteins (35 µg) isolated from AV-12 cells that were stably transfected with a PKC3 transgene. Lane 4 contained 35 µg of cytosolic proteins obtained from control AV-12 cells. Lanes 1, 3, and 4 were probed with affinity purified IgGs (25 ng/ml) directed against PKC3. Excess, purified PKC3 antigen (3 µg) was included when lane 3 was incubated with the IgGs. Lane 2 was incubated with preimmune serum. C, an in vitro transcription-translation system (see "Experimental Procedures") was programmed with a cDNA template encoding firefly luciferase (a 61-kDa control polypeptide) (lane 1), cDNA encoding PKC3 (lane 3), or buffer lacking cDNA (lane 2). The translation mixture contained 40 µCi of [35S]Met and [35S]Cys. Radiolabeled polypeptides were fractionated by SDS-polyacrylamide gel electrophoresis and detected by autoradiography on x-ray film as described under "Experimental Procedures." Each lane received 5 µl of the translation reaction mixture. The lower bands in lane 3 apparently correspond to proteolytic fragments of PKC3 or truncated PKC3 proteins generated by the use of internal Met codons. Only the relevant portions of the gels are shown. No other bands were observed.

Expression, Purification, and Properties of C. elegans PKC3-- To obtain a preparation of C. elegans PKC3 that is free from other PKC isoforms and unrelated but potentially interfering protein kinases, the nematode enzyme was stably overexpressed in the cytoplasm of hamster AV-12 cells. Cytosol was isolated from 100 15-cm plates of cells and PKC3 was purified 1,750-fold by three serial chromatographic procedures (see "Experimental Procedures"). Elution of PKC3 was monitored by Western immunoblot analysis and phosphotransferase assays that employed RRGSRRWKKIY (a modified pseudosubstrate peptide in which Ser is substituted for Ala) as a specific substrate. Purified PKC3 had a specific activity of 0.2 µmol/min/mg protein in the absence of activators. Despite the high basal activity, PS or arachidonic acid increased PKC3 phosphotransferase activity 2.5-4-fold (Table III). Both compounds activate PKCs zeta , lambda /iota , and certain other PKC isoforms (1, 2). A substrate peptide based on the autoinhibitory sequence of C. elegans PKC1B (an nPKC) was phosphorylated at only 25% the rate of the PKC3-derived peptide. PKC3 also displayed a high degree of selectivity for protein substrates. The C. elegans kinase catalyzed the PS-stimulated (~4-fold) phosphorylation of myelin basic protein, whereas histones and casein were not substrates. Comparison of the specific activity of partially purified PKC3 with values obtained for homogeneous aPKCs (57, 58) suggests that the C. elegans enzyme is 10-20% pure. More importantly, immunoprecipitation with anti-PKC3 IgGs eliminated nearly all of the PS-activated kinase activity (Table III). Thus, the assays report the properties of PKC3 and are not compromised by contaminating kinases.

                              
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Table III
Effects of potential activators on the activity of C. elegans PKC3
Activity of purified PKC3 (100 ng) was measured in the presence and absence of the indicated concentrations of potential activators as described under "Experimental Procedures." For immunodepletion, a sample of PKC3 was immunoprecipitated with 0.5 µg of affinity purified IgGs bound to protein A-Sepharose. Activity remaining in the supernatant solution was then determined. Assays were repeated 4 times. Typical results are shown.

PKC3 activity was not affected by physiological stimulators of cPKC and nPKC isoforms: DAG and Ca2+ (Table III). Ceramide, a second messenger that activates mammalian aPKCs, did not alter PKC3 activity (Table III). The same activators also failed to increase PKC3 activity in the presence of PS (data not shown). PKC3 has apparent Km values for ATP (10 µM), peptide substrate (28 µM), and Mg2+ (1.2 mM) that are similar to Km values measured for aPKCs (57).

Susceptibility of PKC3 to inhibitors of cPKCs and nPKCs was assessed by determining dose-response curves over a 1,000-fold concentration range for several drugs. IC50 values derived from these analyses are presented in Table IV. PKC3 was not inhibited by two compounds (calphostin C and a DAG analog) that bind with the Cys-rich regulatory regions of c- and nPKCs and suppress DAG-stimulated enzyme activity. In comparison with c- and nPKC isoforms, the C. elegans kinase was 6-40-fold more resistant to inhibition by H-7, rottlerin, and GF-109203X, three compounds that interact with the ATP binding region of the catalytic domain (Table IV) (58-61). PKC3 is strikingly insensitive (300-fold increase in IC50) to staurosporine, a potent competitive inhibitor of ATP binding in nPKCs and cPKCs. PKCzeta also has lower affinities for ATP binding site-directed inhibitors than c- and nPKCs (58). The preceding results are consistent with the high level of sequence conservation among the catalytic domains of PKC3, PKC lambda /iota , and PKCzeta and the divergence in sequences of the corresponding regions in C. elegans PKC1B (Table I) and all other PKC isoforms (<= 50% identity). Together, structural and enzymatic properties of PKC3 demonstrate that the C. elegans kinase is a member of the aPKC subclass of PKC isoforms.

                              
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Table IV
Inhibition of PKC3 activity by drugs that bind with either the catalytic domain or a Cys-rich regulatory region
The IC50 values for inhibition of C. elegans PKC3 were determined from dose-response curves as described in the text. Typical results are shown. Representative IC50 values for nPKCs and cPKCs were taken from Refs. 58-61.

Regulation of Expression and Localization of PKC3 during Development-- Cytosolic and total particulate proteins were prepared from C. elegans at each stage of development. Western immunoblot analysis revealed that PKC3 is expressed throughout the lifespan of C. elegans (Fig. 4). However, both the content and intracellular distribution of PKC3 vary with development. For example, total PKC3 content in L1 larvae is increased ~7-fold relative to the minimal level observed in L3 animals. Embryos and L4 larvae are also 3- to 4-fold enriched in PKC3. A very high proportion (75-100%) of PKC3 partitions with the insoluble fraction of homogenates from embryos, L2-L4 larvae, and young adult C. elegans (Fig. 4). L1 larvae have the highest level of particulate PKC3 but also contain a similar amount of the kinase in cytosol. PKC3 synthesized in AV-12 cells accumulates predominantly in cytosol and can be purified as a soluble protein (see "Results" above). Association of PKC3 with the organelles and cytoskeleton of C. elegans at six consecutive stages of development suggests that the enzyme may be targeted to specific intracellular locations via interactions with anchoring proteins and/or lipids.


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Fig. 4.   Developmental regulation of PKC3 expression and localization. Particulate (P) and soluble cytosolic (S) proteins were isolated from C. elegans embryos (E), L1-L4 larvae, young adults (A), and egg-laying adults (A/E) as described under "Experimental Procedures." Samples of these proteins (30 µg) were assayed for PKC expression by Western immunoblot analysis as described under "Experimental Procedures." Only the relevant portion of the blot is shown.

Accumulation of PKC3 in cytosol is developmentally regulated. The atypical kinase is principally a cytosolic enzyme (of moderate abundance) only in adult egg-laying nematodes (Fig. 4). However, the highest level of soluble PKC3 is detected in L1 larvae, a stage at which C. elegans begins to respond to external stimuli after emerging from the eggshell-encased embryonic environment.

The pkc-3 Gene Promoter Is Differentially Activated in Cells Involved in Feeding, Digestion, Excretion, and Egg Laying in Vivo-- Lines of transgenic C. elegans that carry a chimeric reporter gene were created to assay pkc-3 gene promoter activity in individual cells of intact animals. A 3.2-kbp DNA fragment that flanks the 5' end of the pkc-3 gene in cosmid F09E5 (see above) was inserted upstream from a beta -galactosidase reporter gene (lacZ) in a C. elegans expression plasmid (26). An octapeptide nuclear localization signal is appended to the N terminus of the reporter enzyme to direct accumulation of beta -galactosidase in the nucleus. Promoter activity is visualized by histochemical staining for beta -galactosidase; cells with an active pkc-3 promoter were identified by microscopy and reference to a well established anatomical data base. The fusion gene containing the pkc-3 promoter was designated pkc3P:lacZ.

Patterns of beta -galactosidase expression were similar in multiple transgenic lines, and typical results are presented in Fig. 5. pkc-3 gene promoter activity is evident in nuclei of cells comprising the anterior and posterior bulbs of the pharynx (Fig. 5A). Maximal levels of pkc3P:lacZ fusion gene expression were consistently detected in pharyngeal muscle (and epithelial) cells and cells that constitute the pharyngeal-intestinal valve (Fig. 5A). High level promoter activity was also noted in hypodermal cells located near the tip of the head of the animal. Coordinated actions of the pharyngeal muscle cells govern the ingestion of food (bacteria) into the anterior region of the pharynx; the disruption of bacteria on the hardened, cuticular surfaces of muscle cells that exert a grinding motion, at the posterior portion of the pharynx; and the pumping of the macerated bacteria into the intestine via the valve structure that links the pharynx to the gut. Muscle cells are usually anchored to hypodermal cells to provide support for contractile activity.


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Fig. 5.   Cell-specific expression of pkc-3 gene promoter activity in C. elegans. Lines of transgenic C. elegans carrying the pkc3P:lacZ reporter gene were created as described under "Experimental Procedures" and the text of "Results." The histochemical stain for beta -galactosidase produces an insoluble product in nuclei transcribing the pkc-3 gene. A shows accumulation of the reporter gene product in hypodermal cells near the tip of the head (a), muscle cells in the anterior (b), and posterior (c) bulbs of the pharynx, and cells comprising the pharyngeal-intestinal valve (d). B, reveals PKC3 promoter activity in the large nuclei of intestinal cells (e). C documents high level pkc-3 gene transcription in a complex of cells corresponding to the intestinal-rectal valve and the anal depressor and sphincter muscles (f). Lower level activity is detected in nuclei of four hypodermal cells (g) that provide the cuticle and muscle anchorage in the tail region. D demonstrates transcriptional activity of the pkc-3 gene in vulval cells, which are symmetrically arranged on either side of the vulval opening (h) and somatic cells of the spermatheca (i). C. elegans were photographed using Nomarski optics (magnification = 1000 ×) as described previously (37, 47).

The pkc3P:lacZ gene is also expressed in the large nuclei of cells that constitute the intestine of C. elegans (Fig. 5B). At the posterior end of the intestine, pkc-3 gene promoter/enhancer DNA drives transcription of lacZ in the anal sphincter and anal depressor muscle cells, as well as cells that are components of the intestinal-rectal valve (Fig. 5C). This small subset of cells controls expulsion of the nutrient-depleted suspension of bacterial remnants from the intestine through the anal opening. Modest levels of pkc-3 promoter activity are apparent in four hypodermal cells that provide cuticle and muscle anchorage in the tail of the nematode. Overall, pkc-3 gene transcription is selectively activated in cells involved in the physiologically linked phenomena of food uptake, digestion, nutrient uptake, and excretion of non-absorbed waste.

Further assessment of expression of the pkc3P:lacZ fusion gene revealed robust transcriptional activity in vulval epithelial and muscle cells and the somatic cells of the spermatheca (Fig. 5D). Thus, PKC3 may also be involved in the control of egg laying and the maintenance/organization of a tissue that concentrates sperm and mediates fertilization of oocytes. No beta -galactosidase activity was observed when transgenic C. elegans carrying a promoterless reporter gene or lacZ downstream from a metal-inducible promoter (47) were assayed under similar conditions. Activation of pkc-3 gene transcription is a tightly regulated process that is detected in only ~85-90 cells. Thus, the gene is either expressed at a very low level, or is silent, in >90% of the somatic cells of C. elegans. Cell-specific patterns and levels of lacZ expression described above were replicated in transgenic C. elegans when the promoter/enhancer DNA was truncated to a 647-bp fragment that is immediately adjacent to the 5' end of the pkc-3 structural gene. Thus, a relatively small segment of 5'-flanking DNA contains all of the cis regulatory elements and transcription factor binding sites that are crucial for cell-specific, developmentally controlled expression of a C. elegans aPKC gene.

Expression and Distribution of PKC3 in Vivo-- Accumulation and distribution of the PKC3 protein were determined by confocal immunofluorescence microscopy. Intense fluorescence signals corresponding to PKC3-IgG complexes disclosed that the atypical C. elegans kinase is abundantly expressed in the anterior and posterior pharyngeal bulbs, the pharyngeal-intestinal valve, vulval cells, and a region at the posterior end of the intestine that corresponds to site of co-assembly of the intestinal-rectal valve and anal sphincter and depressor muscles (Fig. 6, A-C). In addition, affinity purified anti-PKC3 IgGs selectively decorated the apical surfaces of all intestinal cells, thereby outlining the lumen of the gut (Fig. 6D). The highly polarized concentration of the kinase near the apical plasma membrane and the absence of antigen in cytoplasm and all other membranes/organelles of the large intestinal cells strongly suggest that PKC3 is targeted and anchored in situ (Fig. 6D). When immunochemical analysis was extended to the level of electron microscopy, PKC3 molecules were readily detected at the apical surface (villi) of C. elegans intestinal and pharyngeal cells,4 in proximity with the cortical actin cytoskeleton that lies under the plasma membrane. PKC3 also accumulates in discrete patches and spots in vulval cells (Fig. 6C). The cell/tissue-specific patterns of pkc-3 gene transcription and PKC3 polypeptide accumulation in post-embryonic C. elegans are in excellent agreement.


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Fig. 6.   In situ distribution of PKC3 in post-embryonic C. elegans. The location of PKC3 polypeptides in post-embryonic C. elegans was determined by confocal immunofluorescence microscopy. A shows an overview of accumulation of PKC3 in cells that constitute the pharynx and intestine of an L2 larva. The region marked tail includes the intestinal-rectal valve and anal sphincter and depressor muscle cells. Intense fluorescence signals in B demonstrate the high level expression of the nematode aPKC in cells of the anterior (a) and posterior (p) bulbs of pharynx of an L4 nematode. C, depicts the asymmetric distribution of PKC3 protein in vulval cells that abut two embryos in route to expulsion to the external environment. Selective and targeted accumulation of PKC3 at the apical surfaces of intestinal cells (in an L1 larva) is evident in D. PKC3-derived immunofluorescence outlines the lumen of the intestine. The robust signal observed at the top left of D originates from the posterior bulb of the pharynx and the pharyngeal-intestinal valve (magnification = 600 ×)

C. elegans embryos also contain a substantial amount of PKC3 (Fig. 4). Immunofluorescence microscopy revealed that PKC3 is present at the earliest stages of development (e.g. in 2-cell embryos, Fig. 7A). Expression of the kinase is sustained in various cells as embryos progress through middle and late developmental stages that culminate in the hatching of an L1 larva (558 cells) ~14 h after fertilization (Fig. 7, B-D). During early embryogenesis (1-~200 cells, 0-3 h post-fertilization) PKC3 accumulates in numerous cells, and a substantial portion of the kinase is typically clustered and enriched in the vicinity of the cell periphery and cell-cell junctions (Fig. 7, A, B, and D). Double immunostaining with IgGs directed against PKC3 and actin (Fig. 7, D and E) suggests that PKC3 is (in part) co-localized with the cortical actin cytoskeleton. When the pharynx and intestine are assembled during mid- to late embryogenesis (e.g. Fig. 7C), generalized PKC3 expression ceases and the kinase selectively accumulates in pharyngeal muscle, gut cells, and the pharyngeal-intestinal and intestinal-rectal valve regions. Thus, the nematode aPKC appears to be differentially targeted, but available to mediate signaling at all stages of embryogenesis.


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Fig. 7.   In situ accumulation of PKC3 in C. elegans embryos. The disposition of PKC3 polypeptides in C. elegans embryos was determined by immunofluorescence analysis as described under "Experimental Procedures." Fluorescence signals that correspond to IgG-PKC3 complexes were obtained from C. elegans in early (2 cells, A; 32 cells, D), middle (~150 cells, B), and late (C) stages of embryogenesis. In the late embryo (C), PKC3 is differentially enriched in cells of the newly assembled digestive system (pharynx and intestine), whereas PKC3 content is diminished in most other embryonic tissues. D and E show the partially overlapping distribution of PKC3 (fluorescein signal) and F-actin (rhodamine signal), respectively, in a doubly stained embryo. Representative embryos are shown. Multiple examples of each staining pattern were reproducibly observed in independent immunostaining experiments.

PKC3 Is Critical for the Progression of Embryogenesis and Viability of C. elegans-- During the first 7 h after fertilization C. elegans embryos engage in intensive protein synthesis, mitogenesis, and cycles of re-organization of the cytoskeleton that together generate a spheroidal organism with 558 cells. The succeeding 7 h are devoted to elongation and further morphogenesis that transforms the spheroidal embryo into a cylindrical nematode. The apparent targeting and sustained expression of PKC3 throughout embryogenesis raise the possibility that signaling via this protein kinase plays an important role in early development. To test this proposition, we ablated PKC3 by injecting capped, antisense PKC3 RNA into the gonadal syncytium of young adult nematodes. This enables distribution of the antisense RNA throughout a common cytoplasm shared by many oocyte nuclei. As oogenesis proceeds individual plasma membranes are formed, thereby sequestering injected RNA molecules in individual oocytes that are destined for fertilization. Injected animals were placed on individual plates and allowed to lay eggs (i.e. developing embryos) for 24 h. Subsequently parental animals were removed, and embryo morphology and the ability to hatch were monitored. More than 90% (338 of 367) of embryos containing antisense PKC3 mRNA failed to hatch. Examination by interference microscopy revealed that arrested embryos contain ~100-200 highly disorganized cells (Fig. 8A). The few animals that hatched exhibited a normal L1 phenotype, indicating that they had received neither the phenotypic marker gene rol-6 that produces easily detected "roller" C. elegans nor co-injected antisense RNA (see "Experimental Procedures"). In contrast, 98% of 781 eggs produced by C. elegans injected with the same concentration of capped, PKC3 mRNA (sense RNA) had a normal morphology. These eggs hatched and produced viable, reproductive nematodes (Fig. 8, B and C). A high percentage of these animals had the roller phenotype (Fig. 8C). Thus, it appears that the atypical PKC3 of C. elegans is essential for the normal progression of early to mid-embryogenesis and the viability of the intact organism.


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Fig. 8.   PKC3 antisense RNA selectively perturbs embryogenesis and generates inviable animals. C. elegans oocytes were injected with 50 µg/ml antisense or sense RNAs for PKC3 as described under "Experimental Procedures." Individual parental, hermaphrodite animals were placed on plates and allowed to lay eggs for 24 h. Subsequently, parental C. elegans were removed, and eggs were scored for morphology and ability to hatch 24 h later. A presents a typical embryo (~200 cells) produced by a nematode injected with PKC3 antisense RNA. These embryos do not progress beyond this stage, and the pattern of cellular organization is distinct and disorganized relative to wild-type embryos (B). Embryos with the phenotype shown in A were not viable. C. elegans embryos obtained from animals microinjected with PKC3 sense (messenger) RNA were indistinguishable from the wild-type embryos (B) in morphology. These embryos progressed through the remaining stages of development and hatched as normal, viable L1 larvae (C). Representative embryos are shown. However, hundreds of very similar embryos were observed in each repetition of the experiment. Typical data are presented in the inset.

PKC3 Is Bound by PICK-1CE, a Candidate Targeting Protein-- Expression of PKC3 in AV-12 cells yields a soluble kinase that exhibits ample phosphotransferase activity in the absence of activators (Fig. 3B and Table III). In contrast, a significant proportion of PKC3 is associated with cytoskeleton and/or organelles in vivo (Figs. 4, 6, and 7). Differential targeting/anchoring may regulate PKC3 activity in situ by (a) restricting access to substrates or lipid activators or (b) increasing its susceptibility to inhibition by protein modulators. A protein named PICK1 has an N-terminal PDZ domain (62),2 which binds a target motif (SXV) at the C terminus of protein kinase Calpha (48). Formation of PICK1-PKCalpha complexes may selectively direct PKCalpha to substrates clustered at sites adjacent to plasma membrane or cortical cytoskeleton. Other cPKCs and nPKCs are not bound by PICK1 (48). PKC3 contains an alternative PDZ binding site (DXV, Ref. 63) at its C terminus (Fig. 1). Moreover, a C. elegans PICK1 homolog (PIKC1CE) was recently discovered.2 Therefore, we tested the ability of a full-length C. elegans PICK1-GST fusion protein (designated GST-PICK1CE) to bind with 35S-labeled PKC3 generated by in vitro translation. GST-PICK1CE formed a stable complex with PKC3 that was isolated on GSH-Sepharose 4B beads; the complex was not dissociated by repeated washing with buffer containing 0.1% Triton X-100 and 0.3 M NaCl (Fig. 9, lane 2). Binding with PICK1CE is specific since neither GST alone nor a GST fused with a protein kinase A anchor protein interacted with PKC3 (Fig. 9, lanes 1 and 3).


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Fig. 9.   C. elegans PKC3 is bound by a candidate targeting/anchoring protein named PICK1CE. 35S-Labeled PKC3 was synthesized by coupled in vitro transcription/translation. Results of translation are shown in Fig. 3C. Radiolabeled PKC3 was incubated with 2 µg of GST-PICK1CE, 4 µg of GST, or 2 µg of GST fused with an A kinase anchor protein (R. Angelo and C. S. Rubin, unpublished results) as described under "Experimental Procedures." Subsequently, GSH-Sepharose 4B beads were added to sequester the GST fusion proteins and PKC3. After extensive washing, proteins on the beads were size-fractionated by denaturing electrophoresis (see "Experimental Procedures"). An autoradiogram obtained from the dried gel is shown. GST alone (lane 1) and the GST fused with the A kinase anchor protein (lane 3) failed to complex PKC3, thereby indicating the specificity of the assay. In contrast, GST-PIKC1CE bound (~50%) of added 75-kDa 35S-PKC3 (lane 2). The smaller protein bound by PICK1CE is apparently an N-terminally truncated PKC3 protein5 whose synthesis is initiated at an internal Met codon.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We characterized a cDNA that encodes a novel C. elegans protein kinase named PKC3. PKC3 is composed of 597 amino acids and contains catalytic, non-repeated Cys-rich pseudosubstrate and C-terminal domains that are homologous (~56% overall identity) with corresponding domains in mammalian PKC lambda /iota and PKCzeta . The latter enzymes comprise the atypical subclass of the superfamily of mammalian PKC isoforms. Enzymatic analysis demonstrated that PKC3 is a Ca2+- and DAG-independent phosphotransferase. However, the nematode kinase is stimulated ~3-4-fold by PS or arachidonic acid. These characteristic structural and enzymic properties (see Introduction) indicate that PKC3 is a new aPKC isoform. The nematode kinase is the first example of an invertebrate aPKC. Evidently, conserved features of aPKCs enable these enzymes to receive and propagate physiological signals in regulatory pathways shared by lower and higher eukaryotes.

Sequences of catalytic and putative regulatory domains of PKC3 share similar levels of homology with corresponding regions of both PKC lambda /iota and PKCzeta (Table I). Since neither lambda /iota nor zeta  sequences predominate, PKC3 may be a "composite" kinase that mediates functions in C. elegans that are collectively performed by two aPKCs in higher organisms. Low stringency Southern blotting and computer-based searches of the C. elegans genome data base (~80% sequenced) further suggest that PKC3 may be the only aPKC in the nematode. The PKC3 polypeptide contains two large modules (residues 1-89 and 178-249) with sequences that diverge sharply from sequences in mammalian aPKCs and other protein kinases (Table I and Fig. 2). These structural differences indicate that the C. elegans kinase is a distinct aPKC isoform. The unique N-terminal and central regions may constitute binding sites for cell- and/or species-specific substrate-effector proteins, activators, inhibitors, or targeting/anchoring proteins. The catalytic domain of PKC3 is >= 75% identical with the corresponding regions of PKCs lambda /iota and zeta  despite the evolutionary divergence of nematodes and mammals. A structural feature that distinguishes aPKCs from other PKC isoforms is the "insertion" of a 9- or 11-residue loop into a region of highly conserved sequence near the C terminus of the catalytic domain (e.g. residues 453-463 in PKC lambda /iota , Fig. 2) (5). This property is conserved in C. elegans PKC3, which has a decapeptide insert between residues 453 and 464 (Fig. 2). Crystal structures of several protein kinases (52, 53) suggest that these divergent inserts may play a role in determining the substrate specificity of aPKC isoforms (5, 53, 54).

Ten residues in the PKC3 pseudosubstrate region are identical with amino acids in aPKCs; the remaining three amino acids are conservative substitutions (Ile right-arrow Val; Ser right-arrow Thr; Arg right-arrow Lys). Conservation of the autoinhibitory sequence may reflect (a) the distinctive substrate specificity of aPKC isoforms (57) and (b) exertion of selection pressure favoring retention of an intramolecular ligand that binds with and regulates a conserved catalytic domain. The pseudosubstrate site sequence of PKC3 differs significantly from autoinhibitory regions in C. elegans PKC1B (see Table I) and PKC2A and mammalian cPKCs and nPKCs.

A Cys-His-rich motif provides a framework that mediates domain folding and sequestration of zinc in all PKC isoforms (1, 2, 64). However, sequences of polypeptide segments that connect conserved Cys and His residues in PKC3 and aPKCs diverge from sequences in regulatory domains of cPKCs and nPKCs (Table I). In PKCs lambda /iota and zeta  these differences apparently create novel surfaces that enable binding of isoform-specific regulatory proteins, while excluding interaction with DAG (5, 6, 13, 18, 57).

mRNAs encoding lambda /iota and zeta  PKC isoforms are expressed in many mammalian tissues (1-5). PKCzeta protein is detected in brain, lung, liver, and endocrine tissues (65). However, effects of development, hormones, growth factors, etc. on tissue-specific patterns and levels of expression and intracellular locations of PKCs lambda /iota and zeta  have not been systematically investigated. Likewise, little is known about roles for aPKCs in vivo. Activation of pkc-3 gene transcription and accumulation of PKC3 protein are tightly coordinated in a cell/tissue-specific manner in post-embryonic C. elegans. High levels of PKC3 are produced in muscle, epithelial, and hypodermal cells that are components of pharynx, pharyngeal-intestinal valve, intestine, intestinal-rectal valve, and the anal depressor/sphincter system. Thus, PKC3-mediated signaling may be involved in coordinating and regulating a series of highly related functions: ingestion and maceration of food, pumping food into the intestine, digestion and nutrient extraction, and excretion of nutrient-depleted waste. In addition, PKC3 is abundant in vulval cells and somatic cells of the spermatheca. Therefore, PKC3 may be involved in the regulation of egg laying and the maintenance/organization of the chamber in which sperm are concentrated and oocytes are fertilized. PKC3 is not observed in >90% of the somatic cells of C. elegans, including all motor and sensory neurons and body wall muscles. A common feature of PKC3-enriched tissues is that they undergo repeated physical stresses that result in cycles of expansion, deformation, and contraction during (a) pumping of E. coli into and out of intestine, (b) delivery of eggs to the external environment, and (c) constriction of oocytes as they enter and exit the smaller spermatheca. The possibility that physical stress is coupled to the regulation of PKC3 activity merits analysis in future studies.

Investigations on aPKC functions in model cell systems have focused principally on regulation of mitogenesis or apoptosis (7-14). The extensive data base on C. elegans development indicates that many cells that actively transcribe the pkc-3 gene and accumulate substantial levels of PKC3 protein in post-embryonic animals (Figs. 5 and 6) are incapable of further mitogenesis and will not undergo programmed cell death. Thus, PKC3 may participate in the regulation of distinct, highly differentiated functions in larval and adult tissues, instead of modulating cell death or division. PKC3 plays a different but centrally important role during embryogenesis. Elimination of PKC3 function by microinjection of PKC3 antisense RNA into oocytes results in arrested, disorganized, and non-viable early- to mid-stage embryos (Fig. 8). This striking result and the production of viable C. elegans from oocytes injected with sense PKC3 RNA (mRNA) indicate that the aPKC is essential for the normal progression of early development and viability of the nematode. Classical and novel PKC isoforms of C. elegans are expressed at low to undetectable levels in embryos (37, 56), thereby suggesting that only the aPKC isoform is specifically adapted for mediating critical aspects of signal transduction during early development.

Mammalian aPKCs are usually cytoplasmic enzymes that do not translocate to organelles in response to signals generated by hormones and growth factors (1, 2, 57, 66, 67). Exceptions to this generalization include reports of PKC lambda /iota in the nucleus and PKCzeta associated with plasma membrane in certain cultured cells (10, 68). In these instances hormonal activation elicited translocation of the kinases to cytoplasm. Analysis of the subcellular distribution of PKC3 yielded a sharply defined and unexpected pattern of aPKC localization. A high proportion of PKC3 is tightly bound to organelles/cytoskeleton in six of the seven developmental stages of C. elegans. In contrast, accumulation of PKC3 in cytoplasm is restricted to L1 larvae and adult nematodes (Fig. 4). Thus, the ability of the C. elegans aPKC to regulate cell functions in the cytoplasmic compartment may be developmentally regulated.

PKC3 is concentrated and anchored in a polarized fashion in vivo (Figs. 6D and 7). This is evident in the enrichment of PKC3 near the periphery of embryonic cells and the appearance of the kinase near the apical surfaces of cells comprising the intestine. The subcellular location of PKC3, co-immunostaining with actin in embryos, immunoelectron microscopy,4 and the resistance of PKC3 to solubilization with non-ionic detergents5 suggest that the kinase is associated with the cortical actin cytoskeleton. In intestine, only those portions of the cortical cytoskeleton/cell membrane complex that abut the lumen of the gut contain PKC3, and immunostaining outlines the entire length of C. elegans digestive system.

To begin an analysis of mechanisms for PKC3 targeting, we assessed the ability of C. elegans PICK1CE to bind with the kinase. Mammalian PICK1 differentially binds PKCalpha and presumably mediates translocation of the enzyme to sites near membranes/cytoskeleton that are enriched in target substrates (48). Binding is accomplished by the coupling of the consensus C-terminal SXV motif of PKCalpha with a PDZ domain in PICK1 (48, 62).2 PKC3, which contains a variant, but functional (66) C-terminal PDZ target site (DXV), forms a stable complex with the C. elegans homolog of PICK1 (PICK1CE). This suggests that an aPKC anchor/targeting protein could concentrate the kinase in the vicinity of inhibitory modulators. The subsequent generation of a lipid second messenger or protein activator could then produce phosphotransferase activity at a predetermined target site. An important caveat is that the PICK1CE·PKC3 model is currently hypothetical. It is not yet known whether the two proteins are co-expressed and form complexes in intact cells, nor do we know the exact nature of the binding domains in the partner proteins. However, these points can be directly addressed in future studies. Interactions reported herein document the feasibility of the model and suggest that careful evaluation of the targeting role for PICK1CE and a search for other potential anchor proteins are warranted. It is also possible that similar mechanisms govern the destination, activity, and functions of aPKCs in highly differentiated mammalian cells.

    ACKNOWLEDGEMENT

We thank Ann Marie Alba for expert secretarial assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DK44597 (to C. S. R.) and the Lucille P. Markey Charitable Trust (to C. S. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF025666.

par To whom correspondence should be addressed: Dept. of Molecular Pharmacology, F-229, Albert Einstein College of Medicine, Bronx, NY 10461. Tel.: 718-430-2505; Fax: 718-430-8922; E-mail: rubin{at}aecom.yu.edu.

1 The abbreviations used are: PKC, protein kinase C; PS, phosphatidylserine; DAG, diacylglycerol; bp, base pair(s); kbp, kilobase pairs; GST, glutathione S-transferase; cPKC, classical Ca2+, DAG-activated PKC; nPKC, novel Ca2+-independent, DAG-activated PKC, aPKC, atypical Ca2+, DAG-independent PKC; PBS, phosphate-buffered saline; DTT, dithiothreitol.

2 J. Staudinger, J. Lu, and E. N. Olson, manuscript submitted for publication.

3 In accord with standard C. elegans nomenclature, genes are named with three lowercase italic letters and a number (pkc-3); the same uppercase roman letters (PKC3) are used to designate protein encoded by the corresponding gene.

4 D. Hall, S.-L. Wu, and C. S. Rubin, unpublished results.

5 S.-L. Wu and C. S. Rubin, unpublished results.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
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