From the 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
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ABSTRACT |
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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.
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INTRODUCTION |
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The "atypical" subclass of protein kinase C
(PKC)1 isoforms is composed
of PKC and PKC
/
(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
and
/
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 (
,
I,
II, and
) and novel (
,
,
,
and
) PKCs (cPKCs and nPKCs, respectively), the
and
/
isoforms lack binding/regulatory sites for diacylglycerol (DAG),
phorbol esters, and Ca2+ (3-6).
PKCs and
/
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
and
/
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
and
/
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- and interleukin-1 stimulate
sphingomyelinase-catalyzed synthesis of ceramide (15). Ceramide
activates PKC
in vitro and apparently promotes
phosphorylation of I
B by sequential activation of PKC
and an
uncharacterized, 50-kDa protein kinase in cells (11, 12).
Phosphorylated I
B dissociates from its' cytoplasmic ligand NF
B
and is rapidly proteolyzed. NF
B then translocates to the nucleus and
stimulates gene transcription (16). However, the ability of PKC
(and
the 50-kDa effector kinase) to regulate activity of NF
B or the
cytokine mediator p85 I
B kinase (17) in a normal physiological
context remains to be established.
An 80-kDa protein named LIP binds PKC/
in vitro and in
serum-stimulated Cos cells that overexpress both LIP and PKC
/
(18). PKC
/
·LIP complex formation correlates with increased
kinase activity and NF
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·PKC
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
/
and
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.
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EXPERIMENTAL PROCEDURES |
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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 PKC
(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
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-
-D-galactopyranoside-inducible T7 RNA
polymerase. E. coli BL21 (DE3) transformed with recombinant
expression plasmid was grown and induced with
isopropyl-1-thio-
-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 8 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 atCell 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 [-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
[
-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
-galactosidase reporter enzyme that
accumulates in nuclei.
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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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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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 and
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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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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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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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 ,
/
, 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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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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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
-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
-galactosidase in the nucleus. Promoter activity is visualized by
histochemical staining for
-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.
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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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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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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 C (48). Formation of PICK1-PKC
complexes may selectively direct PKC
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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DISCUSSION |
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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 /
and PKC
. 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
/
and PKC
(Table I). Since neither
/
nor
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
/
and
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
/
, 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 Val; Ser
Thr; Arg
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 /
and
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 /
and
PKC isoforms are expressed in many
mammalian tissues (1-5). PKC
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
/
and
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 /
in the nucleus and PKC
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 PKC 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 PKC
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.
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ACKNOWLEDGEMENT |
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We thank Ann Marie Alba for expert secretarial assistance.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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