From the Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912-3175
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ABSTRACT |
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In order to understand the regulatory role of
protein kinase C (PKC) in secretory epithelia, it is necessary to
identify and characterize specific downstream targets. We previously
identified one such protein in studies of gastric parietal cells. This
protein was referred to as pp66 because it migrated with an apparent
molecular mass of 66 kDa on SDS-polyacrylamide gels. The
phosphorylation of pp66 is increased by the cholinergic agonist,
carbachol, and by the PKC activator, phorbol-12-myristate-13-acetate,
in a calcium-independent manner. In this study, we have purified pp66
to homogeneity and cloned the complete open reading frame.
GenBankTM searches revealed a 45% homology with the
Dictyostelium actin-binding protein, coronin, and ~67%
homology with the previously cloned human and bovine coronin-like
homologue, p57. pp66 appears to be most highly expressed in the
gastrointestinal mucosa and in kidney and lung. Confocal microscopic
studies of an enhanced green fluorescent protein fusion construct of
pp66 in cultured parietal cells and in Madin-Darby canine kidney cells
indicate that pp66 preferentially localizes in F-actin-rich regions. On
the basis of our findings, we propose that pp66 may play an important,
PKC-dependent role in regulating membrane/cytoskeletal
rearrangements in epithelial cells. We have tentatively named this
protein coroninse, because it appears to be highly
expressed in secretory epithelia.
Actin-binding proteins play important roles in regulating diverse
activities in nonmuscle cells including, for example, cytoskeletal remodeling, signal transduction, cell adhesion, migration, and motility
(see Refs. 1-3 for reviews). Coronin, which is expressed by the
unicellular eukaryote, Dictyostelium discoideum (4), is one
such protein. There is compelling evidence that coronin is involved in
the regulation of variety of actin-associated activities in
Dictyostelium, including phagocytosis, cytokenesis, and
cellular locomotion (5, 6). Coronin translocates into the phagocytic cup region during phagocytosis and is recruited to the leading edge of
newly formed cell processes in migrating cells (4, 5). Moreover, in
coronin null mutants, the rate of phagocytosis is reduced by ~70%
(6), and these mutants also exhibit reduced chemotaxis and motility
(7). Since Dictyostelium takes up all nutrients by
endocytosis and since coronin has been found to be present in the
actin-coated, endosomal phagocytic compartment that also contains
V-type H+-ATPase subunits and Rab proteins, it has been
suggested that coronin also plays a role in regulating endocytotic
processes (8).
Recently, a mammalian homologue of coronin, p57, was isolated from calf
thymocytes as a 57-kDa contaminant of phospholipase C-containing
cellular fractions (9). The deduced amino acid sequence of cDNA
from both the human peripheral blood leukocyte HL60 cell line and
bovine spleen indicated ~40% homology between p57 and coronin. p57,
which is also referred to as mammalian coronin, appears to be highly
conserved among mammalian species, with 95% identity between the human
and bovine homologues (9). Although the function of p57 has not been
defined, the homology between coronin and p57 and the observation that
p57 co-precipitates with F-actin in vitro has prompted
Suzuki and colleagues (9) to propose that this protein may also play a
role in regulating cell migration and motility. Western blot analyses
have shown that p57 is highly expressed in immune tissues but weakly
expressed in lung and intestine. There is also little to no detectable
expression of p57 in stomach, kidney, liver, and skeletal muscle (9). More recently, a 57-kDa protein in neutrophils was found to bind to the
COOH-terminal region of the oxidase-specific protein, p40phox,
and to accumulate around phagocytic cups. On the basis of partial peptide sequence analysis, this protein was identified as the human
coronin homologue, p57 (10). Thus, the mammalian p57 protein may, like
coronin in Dictyostelium, play an important role in regulating phagocytosis in the mammalian immune system.
In the present study, we report the isolation, cloning, and
characterization of a new mammalian coronin family member, which we
have named coroninse because, in contrast to p57, it is
highly expressed in a variety of secretory-type epithelial tissues
including stomach, intestine, kidney, and lung.
Coroninse contains numerous PKC phosphorylation
consensus sites as well as a putative membrane-spanning region,
characteristic of type 1b membrane proteins, near the amino terminus.
Interestingly, although this latter region was not previously
identified in either coronin or p57, it is highly conserved in all
three protein sequences.
Coroninse was initially identified as pp66, a
cholinergically regulated, PKC-dependent phosphoprotein in
the HCl-secreting gastric parietal cell (11, 12). In this study, we
present evidence that pp66/coroninse is primarily located
in an intracellular canalicular region of parietal cells, which is the
site of active HCl secretion. In the parietal cell, the onset of HCl
secretion is correlated with dramatic morphological transformations in
which internal tubulovesicles containing the proton-transporting
H+,K+-ATPase appear to fuse, by an
exocytotic-like process, with the canalicular membrane to form
elongated microvilli that extend into the canalicular lumen (13). The
cessation of secretion is correlated with the endocytotic-like
retrieval and return of the H+,K+-ATPase to
subcanalicular tubulovesicles (13). Because coroninse appears to localize in an intracellular region associated with active
membrane fusion/retrieval events and because the phosphorylation of
pp66/coroninse is increased by the secretory agonist
carbachol, we hypothesize that this novel phosphoprotein may play a
role in directing membrane/cytoskeletal rearrangements in the gastric parietal cell. Coroninse might also play a similar role in
other ion-transporting secretory epithelial cells such as kidney and lung in which secretion is regulated by exocytotic/endocytotic cycles
that direct ion transporters and channels to and from the active sites
(14-16). In addition, other data presented in this study indicate that
coroninse translocates to the leading edge of actively
developing cellular processes. Thus, this novel
PKC1-dependent
phosphoprotein may also play a role in regulating epithelial cell adhesion.
Cell Isolation and Culture--
Parietal cells were isolated
from the gastric mucosae of pathogen-free, male New Zealand White
rabbits (2-4 kg) using sequential Pronase/collagenase digestion (17).
These cells were enriched to 70-85% purity by density gradient
separation (Accudenz®, Accurate Chemical, Westbury, NY) or
to 95-98% purity using combined density gradient separation followed
by centrifugal elutriation (17). Freshly isolated cells were used for
metabolic labeling experiments and for pp66 isolation and purification.
For transfection experiments, cells were placed in primary culture by
plating on Matrigel®-coated glass coverslips or
glass-bottomed 35-mm culture dishes immediately after isolation
(18).
Metabolic 32P Labeling and Two-dimensional Gel
Analyses--
Endogenous ATP pools (95-98% enriched) were labeled
with 32P as described previously (11, 12, 17, 19). In
brief, cells were equilibrated in a low bovine serum albumin,
phosphate-free incubation medium containing 1.5-2 mCi/ml
[32P]orthophosphate for 90 min, 37 °C. After exposure
to various agents as indicated under "Results," cells were rapidly
pelleted, washed once in ice cold phosphate-buffered saline, and then
lysed in hot SDS/ Phosphoamino Acid Analysis of pp66 Isolated from
32P-Labeled Parietal Cells--
For phosphoamino acid
analysis, pp66 was phosphorylated in 32P-labeled parietal
cells by maximal stimulation with carbachol (100 µM, 10 min). Cells were rapidly pelleted, washed with ice-cold phosphate-buffered saline, extracted, and subjected to two-dimensional gel electrophoresis as described above. 32P-Labeled pp66
was detected by autoradiography, excised from dried gels, subjected to
acid hydrolysis under N2, and resolved by high voltage,
two-dimensional electrophoresis as described previously (20).
Ninhydrin-stained phosphoamino acid standards were used to locate
radiolabeled amino acids, which were detected by autoradiography (20).
pp66 Purification, Tryptic Digestion, and Peptide
Microsequencing--
pp66 was purified to apparent homogeneity using a
combination of detergent extraction and preparative two-dimensional gel electrophoresis. 32P-Labeled parietal cells (~100 × 106) were diluted to ~2 × 106 cells/ml,
temperature-equilibrated (30 min, 37 °C), and then stimulated for 10 min with 1 µM phorbol-12-myristate-13-acetate to induce
maximal pp66 phosphorylation. Cells were rapidly pelleted, rinsed one
time with ice-cold extraction buffer (as above), and then extracted in
the same buffer containing 0.5% Triton X-100, 1% Empigen BB
(Calbiochem) (v/v) for 15 min on ice. Detergent-insoluble pellets were
lysed in reducing solubilizing buffer and subjected to preparative
two-dimensional gel electrophoresis (19, 20). Gels were stained with a
modified Coomassie Blue stain and endogenously 32P-labeled
pp66 located by autoradiography. Co-migrating Coomassie Blue-stained
spots were excised, pooled, and concentrated to a single band on an 8%
SDS-polyacrylamide gel electrophoresis minigel. This band was then
excised and subjected to "in gel" tryptic digestion (19, 20).
Peptides from tryptic digests were resolved on a reversed phase high
pressure liquid chromatography column, and selected peaks were
sequenced at the Emory University Microsequencing Facility (Atlanta, GA).
Molecular Cloning of pp66 from Parietal Cell
mRNA--
Initially, a reverse transcription-PCR strategy,
employing peptide sequence information obtained from tryptic digests of
the purified pp66 protein, was used to generate a specific cDNA
product. Messenger RNA was prepared from >95% pure gastric parietal
cells, using biotinylated oligo(dT) and streptavadin
Magnesphere® particles from the Poly(A)Ttract System 1000 (Promega, Madison, WI). This mRNA was then used in a 3'-rapid
amplification of cDNA ends system (Life Technologies, Inc.) to
synthesize a tagged cDNA as described previously (19, 20). To
prepare a specific probe for pp66, two degenerate primers were
synthesized using peptide sequences obtained from the tryptic digest as
a template and using OligoPrimer Analysis software (version 5.0;
National Biosciences, Inc., Plymouth, MN) to assist in primer design.
The sense primer (24-fold degeneracy) was based on the amino acid
sequence IVTWHPT (ATHGTGACTTGGCAYCCNAC, SP-406). The antisense primer
(64-fold degeneracy) was based on the amino acid sequence CEIARFYK
(TTRTARAAICKGGCNATCTCRCA, ASP-997). Approximately 40 ng of cDNA
were amplified (95 °C for 20 s, 48 °C for 20 s, and
72 °C for 45 s; 35 cycles) in a reaction mixture containing
these primers (800 nM), 10 mM KCl, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO4,
10 mM (NH4)2SO4, 0.1%
Triton X-100, and 0.5 units of Pfu DNA polymerase
(Stratagene, La Jolla, CA). After amplification of the cDNA, a
620-base pair PCR product was generated, subcloned into pCR-Script SK+
(Stratagene). Sequencing was performed by the Molecular Biology Core
Facility at the Medical College of Georgia using an ABI Prism 377 automated DNA sequencer and ABI Prism Cycle Sequencing Dye Terminator
Ready Reaction kits.
Parietal Cell cDNA Library Screening--
To obtain the
complete open reading frame of pp66, the 620-base pair fragment
obtained in the initial PCR amplification of cDNA with SP-406 and
ASP-997 was PCR-labeled with digoxigenin dNTPs (Life Technologies) and
used as a probe to screen 500,000 plaques from a Uni-ZAP cDNA
library (Stratagene; derived from rabbit mRNA isolated from 98%
pure parietal cells) as described previously (19, 20). Four positive
plaques were identified by chemiluminescence (CSPD®
ready-to-use reagent; Boehringer Mannheim). The two longest inserts were selected for sequencing.
Molecular Cloning of Calf Spleen p57--
Total RNA was isolated
from calf spleen, and tagged cDNA was synthesized by 3'-rapid
amplification of cDNA ends as described above for pp66 cloning.
Sense (amino acid positions 1-6; ATGAGCCGGCAGGTGGGTC) and antisense
(amino acid positions 457-462; CTTGGCCTGGACTGTCTCC) primers were
designed based on the calf spleen sequence (GenBankTM
accession number D44496) and synthesized in the Medical College of
Georgia Molecular Biology Core Facility. p57 double-stranded DNA was
generated using PCR amplification with Pfu DNA polymerase (Stratagene). The PCR-generated band of the expected size was subcloned
into pCR-Script. Positive plaques were detected by blue-white screening
(21), and cDNA from plasmids was sequenced as described above.
Molecular Cloning of Rabbit Spleen p57--
Tagged cDNA was
synthesized from total RNA isolated from rabbit spleen by the same
methods used to prepare bovine spleen cDNA. Highly conserved
regions of p57 cDNA were identified by comparing the bovine and
human sequences in GenBank (accession numbers D44496 and D44497,
respectively) to the rabbit pp66 sequence. The 19-mer sense primer
(TTCTTTGACCCTGACACCA) was based on amino acid positions 277-282. The
17-mer antisense primer (amino acid positions 433-439) was designed to
anneal to p57 and not pp66. A 495-base pair product was generated by
PCR amplification as described for bovine spleen p57. The resulting
product was subcloned and sequenced as described above.
Northern Blot Analyses of pp66 and p57 mRNA
Distribution--
A 32P-labeled probe for pp66 mRNA
was prepared by radiolabeling the cDNA encoding the entire open
reading frame of pp66 by random priming with
[ In Vitro Phosphorylation of Native pp66 with Recombinant
PKC Transfection and Expression of EGFP-pp66 Fusion Protein in
Cultured Parietal Cells--
Parietal cells plated on
Matrigel®-coated 35-mm glass-bottomed dishes were cultured
for ~24 h as described previously (18) and then transiently
transfected with either pEGFP-N2 NH2-terminal protein
fusion vector (CLONTECH) or the same vector into
which the complete open reading frame for pp66 containing the
initiating ATG codon had been cloned, in frame, into the multiple
cloning site. Vectors were purified using an EndofreeTM
plasmid kit from Qiagen (Valencia, CA). Transfections were performed with either the cationic lipid, EffecteneTM, and a
manufacturer-supplied enhancer (Qiagen) at a ratio of 1:8 (DNA to
enhancer) and 1:25 (DNA to Effectene) or DMRIE-C (Life Technologies)
using 0.8 µg of DNA per 5 µg DMRIE-C. Transfection efficiencies
with these reagents ranged from 5 to 20% in different cell preparations.
Localization of EGFP-pp66 Fusion Protein in Living and Fixed
Cells: Comparison with Localization of Selected Cytoskeletal and
Organelle Markers--
EGFP-pp66 fusion protein was localized by
confocal microscopy in living and paraformaldehyde-fixed cells. In the
latter case, cells were rinsed in phosphate-buffered saline and fixed
for 15 min at room temperature in freshly prepared 4%
paraformaldehyde. For F-actin localization, fixed cells were
permeabilized with Triton X-100 (0.15%, 4 min) and then stained with
AlexaTM 568 phalloidin (Molecular Probes, Inc., Eugene, OR)
as per the manufacturer's instructions. Cells containing the
fluorescent EGFP-pp66 fusion protein were optically sectioned using a
krypton/argon laser at 488-nm excitation wavelength, and fluorescence
was detected with a photomultiplier at a 530-nm (emission filter, long
pass) emission wavelength. Cells counterstained for F-actin were
scanned at 488-568-nm excitation using a 565-nm beam splitter and
530 ± 30-nm (short pass) and 590-nm (long pass) wavelength
emission filters to detect the EGFP and Alexa 568 phalloidin signals,
respectively. For each dual label experiment, laser power, gains, and
photomultiplier tube settings were adjusted so that there was no
cross-over between the signals. Three-dimensional image reconstructions
(0.2-0.4-µm sections) were performed using Molecular Dynamics
ImageSpaceTM software running on a Silicon Graphics O2
computer platform. For Golgi localization, living cells were loaded
with the fluorescent Golgi marker, Bodipy FL C5 ceramide (1 µM), for 30 min at 37 °C and then scanned at 488-nm
excitation and 530-nm emission wavelengths. Mitochondria were localized
in living cells using the potential-sensitive fluorescent dye,
Mitotracker (Molecular Probes; 500 nM, 30 min, 37 °C;
568-nm excitation and 600-nm emission wavelengths).
Characterization of pp66 Phosphorylation Response in Vivo and in
Vitro--
We previously demonstrated that carbachol increases pp66
phosphorylation in parietal cells and that this response is 1) mimicked by the PKC activator, phorbol-12-myristate-13-acetate; 2)
calcium-independent; and 3) inhibited by the bisindolylmaleimide PKC
inhibitor, Ro 318220 (11, 12). Since these studies focused solely on
the 5-min time point, we expanded the analysis in this study to
determine more precisely the temporal pattern of
carbachol-dependent pp66 phosphorylation. The data in Fig.
1A show that pp66
phosphorylation is maximal within 5 min of stimulation. In addition,
although the level of phosphorylation declined after approximately 15 min of stimulation, it remained significantly elevated above control for at least 30 min. Fig. 1B shows the autoradiographic
localization of pp66 in analytical two-dimensional gels before and
after carbachol stimulation of 32P-labeled parietal cells,
and data in Fig. 1C show that carbachol stimulation leads to
a strong phosphorylation of pp66 on serine residues with a barely
detectable level of phosphorylation on threonine residues and no
detectable phosphorylation on tyrosine residues.
Purification, Sequencing, and Cloning of pp66: Sequence Comparisons
with Human and Bovine p57 Coronin-like Protein and
Coronin--
Several experimental approaches were undertaken in the
attempt to identify pp66. In situ 32P-labeling
studies indicated that the phosphorylated form of pp66 was enriched in
a Triton X-100-insoluble cell fraction; however, because pp66 was not
only NH2-terminally blocked but also highly resistant to
extraction from polyvinylidine fluoride membranes, it was not possible
to isolate a sufficient amount of pp66 from this fraction to obtain
reliable sequence information (not shown). Subsequently, a combined
detergent extraction (0.5% Triton X-100 plus 1% Empigen BB) was found
to yield a detergent-insoluble fraction that was highly enriched in
pp66. pp66 was identified as a row of two major and two minor Coomassie
Blue-stained spots with the major spots migrating immediately basic to
the minor spots that co-migrated with endogenously
32P-labeled pp66 (Fig. 2).
Amino acid analyses of the major spots confirmed that they were
isoforms of the same protein (not shown). At least two additional minor
32P-labeled spots that were more acidic than the two major
phosphorylated spots could be detected in preparative gels (Fig. 2,
arrows) as well as in analytical gels with longer exposure
times (not shown).
Of the seven peptides sequenced from these tryptic digests, a
GenBankTM search indicated that these peptides possessed
varying degrees of homology with the predicted amino acid sequences of
previously cloned human and bovine p57 actin-binding proteins
(GenBankTM accession numbers X89109, U34690, and D44497;
Figs. 3 and
4). The p57 protein from human and bovine
immune tissues (9) bears ~64% similarity and 42% sequence identity
with coronin, an actin-binding protein in D. discoideum
(GenBankTM accession number X61480). Interestingly, coronin
has also been detected in Triton X-100-insoluble cell fractions
(4).
Since one of the sequenced pp66 peptides bore no apparent homology to
either p57 or coronin sequences and since there was strong identity
(95%) between the human and bovine p57 sequences, the rabbit cDNA
was cloned to determine whether or not the pp66 protein was indeed the
rabbit homologue of the mammalian p57 coronin-like protein. As shown in
Fig. 3, the predicted amino acid sequence of rabbit pp66 shares 67%
identity to the predicted human sequence. In contrast, pp66 shares a
somewhat higher identity with coronin than does p57 (Fig. 4) (45 versus 40%, respectively). Interestingly, in comparing the
pp66 and p57 sequences, it is apparent that the COOH-terminal region of
pp66 is significantly more degenerate than the NH2-terminal
region. Moreover, the predicted amino acid sequence for pp66 is 22 amino acids longer than p57. A search of the nonredundant data base of
the GenBankTM expressed sequence tag division further
revealed that the highest scoring expressed sequence tags for both
human and bovine p57 were completely different from those for rabbit
(Table I). Finally, a Blast sequence
alignment (BLASTP, version 2.0.4) indicated that the rabbit pp66
protein shares greater homology with the putative human IR10 protein
(GenBank accession number Z31590) than does the human p57 protein (49 versus 45% homology). The function of the IR10 protein is
unknown. However, the fact that the IR10 sequence has been mapped to a
candidate genomic region of the nevoid basal cell carcinoma syndrome
(9q22.3 chromosomal region (23)) may prove to be of future importance
in unraveling the function(s) of pp66.
Potential Phosphorylation Sites and Other Motifs Present in
pp66--
Although a standard Prosite Pattern search detects only two
WD (or G
Based on further analyses with Prosite Motif, there are 12 potential
PKC phosphorylation sites within the predicted pp66 amino acid
sequence, seven of which are also present in the human p57 sequence
(Fig. 3). Seven of the 11 PKC consensus sequences in pp66 contain
serine residues, and, of these, three are conserved as compared with
the human sequence (amino acid residues 243-245, 291-293, and
391-393). There is also a single putative threonine-containing cyclic
AMP-dependent protein kinase phosphorylation consensus sequence (residues 206-209) that is conserved between pp66 and the
human and bovine sequences. Since this motif may also serve as a PKC
phosphorylation site (26) and since there is no evidence for
cAMP-dependent pp66 phosphorylation in rabbit parietal
cells, it is unlikely that this site is phosphorylated by cyclic
AMP-dependent protein kinase upon activation of the
cholinergic signaling pathway.
Within the deduced pp66 protein sequence, there are a number of other
motifs of unknown significance including six potential casein kinase II
phosphorylation sites (amino acid residues 100-103, 164-167,
311-314, 330-333, 381-384, and 391-394), six
N-myristoylation sites (residues 96-101, 101-106,
188-193, 208-213, 326-331, and 443-448), and two glycosylation
sites (residues 181-184 and 187-190). If, in addition to
phosphorylation, pp66 undergoes additional post-translational
modifications such as myristoylation, this may explain why this
protein, which has a predicted Mr of 53,609, migrates on SDS-polyacrylamide gel electrophoresis gels with and apparent molecular mass of 66 kDa.
Based on further analysis with PSORT, version 6.4 (27), pp66 contains a
putative transmembrane region (residues 47-63) typical of a type 1b
membrane protein with a long cytoplasmic tail (residues 64-486). The
presence of such a transmembrane region might explain the detergent
insolubility of pp66, since interconnected cytoskeletal proteins and
tightly associated integral membrane proteins are known to co-pellet as
Triton X-100-insoluble structures. Secondary structure analyses using
both the Paircoil algorithm of Berger et al. (28) and the
coiled-coil algorithm of Lupas (29) and both weighted and unweighted
MTK and MTIDK matrices with a window size of 21 amino acids predicted
the presence (p > 0.9) of a highly coiled region at
the end of the cytoplasmic tail (residues 444-484). Interestingly, all
of these structural features are conserved in the human p57 protein
(Fig. 3). There are also two conserved dileucine residues within the
putative cytoplasmic tails of pp66 and p57 (Fig. 3). There are several
lines of evidence that dileucine motifs can serve as endocytotic
targeting signals (30).
Northern Blot Analyses of pp66 and p57 mRNA Expression--
In
high stringency Northern blot analyses of total RNA from a variety of
tissues, the pp66 message was found to be widely distributed and of a
similar size (~1.8 kilobases) (Fig. 5,
A and C). The level of pp66 expression was
relatively high in the fundic mucosa of rabbit stomach, intestinal
mucosa, kidney, and lung as well as in spleen and adrenal. In contrast,
pp66 mRNA expression was low in heart, smooth muscle, and brain and
almost undetectable in liver, pancreas, and skeletal muscle. Within the gastric epithelium, more detailed Northern blot analyses of mRNA from parietal cells, chief cells, and gastric glands (which contain ~50% parietal and 50% chief cells plus a few endocrine-like cells) indicated that pp66 mRNA expression was higher in parietal cells as
compared with chief cells (Fig. 5D). Since our chief cell
preparations contained approximately 20% parietal cells, it is
unclear whether or not the apparent expression of pp66 message in this
cell type is the result of parietal cell contamination. In contrast,
parietal cells used for mRNA isolation were 95-98% enriched.
Thus, it is highly unlikely that the apparent high level of expression
in parietal cells is the result of contamination by some other cell type.
In parallel Northern blot analyses using a full-length p57 probe
generated as described under "Materials and Methods," a similarly sized message of approximately 1.6 kilobases was readily detected in
bovine and rabbit spleen but not in rabbit parietal cells even after
extended exposure times (Fig. 5B). Thus, pp66 but not p57 appears to be expressed in parietal cells, whereas both messages are
present in spleen.
Partial Molecular Cloning of a Rabbit p57 Homologue--
To
provide further confirmation that pp66 and p57 are different coronin
isoforms, primers specific for bovine p57 were used to partially clone
the p57 isoform from rabbit spleen RNA as described under "Materials
and Methods." DNA sequencing of the resultant PCR product (accession
number AF100414) indicated 89% identity between the predicted bovine
p57 amino acid sequence and the putative rabbit p57 sequence as
compared with 67% identity between the putative rabbit p57 sequence
and the rabbit pp66 sequence (not shown).
Expression and Localization of pp66-EGFP Fusion Protein in Parietal
Cells--
In order to define the subcellular localization of pp66,
parietal cells were transfected with plasmids containing cDNA
encoding for pp66-EGFP fusion protein. Within 24 h after
transfection, a vesicular pattern of pp66-EGFP expression was readily
detected in living parietal cells (Fig.
6A). In contrast, in control
experiments in which plasmids containing EGFP alone were transfected,
the resulting EGFP expression pattern was diffusely cytosolic, as expected (Fig. 6B). Incubation of transfected cells with
Triton X-100 had no effect on the pp66-EGFP signal (Fig.
6C) but led to the rapid disappearance (within 2 min) of the
EGFP signal in controls (Fig. 6D). Thus, the pp66-EGFP
fusion protein appears to behave like the native pp66 protein in that
both the expressed and native proteins are highly resistant to Triton
X-100 solubilization. Since the pattern of pp66-EGFP distribution was
different from both Golgi and mitochondrial staining (not shown), it is
unlikely that there is significant pp66-EGFP expression within these
cellular organelles.
In other experiments with paraformaldehyde-fixed parietal cells,
pp66-EGFP was found to be highly expressed in close proximity to the
F-actin-rich intracellular canaliculus both before (Fig. 7A) and after carbachol (100 µM) stimulation (Fig. 7B). Although less
prominent, pp66-EGFP expression was also detected immediately below the
F-actin-containing plasma membrane in these cells (Fig. 7). In
contrast, in polarized Madin-Darby canine kidney cells in which F-actin
is predominately localized at the plasma membrane, pp66-EGFP expression
appeared to be localized exclusively in this region (not shown). As
shown in Fig. 7B, a moderate level of pp66-EGFP expression
over a period of 24 h does not suppress the rapid morphological changes known to be induced by carbachol, including the
expansion of the intracellular canaliculus (18).2 During
the course of these latter experiments, we serendipitously discovered
that carbachol also induces a time-dependent formation of
actin-rich filopodia and membrane ruffles in parietal cells (Fig.
8). The confocal microscopic images
depicted in Fig. 8A were obtained by scanning
paraformaldehyde-fixed cells stained for F-actin at the level of
extracellular matrix attachment. F-actin staining is strong at the cell
membranes and within intracellular canaliculi in unstimulated cells in
which no filopodia were detected (Fig. 8, left
panel). In contrast, 1 h after carbachol addition, a
time when the acid secretory response to this agonist has been shown to
have declined to near basal levels (22), a number of newly formed
filopodia are apparent (Fig. 8, right panel). In parietal cells transfected with pp66-EGFP, there was also a
movement of this fluorescent fusion protein into the actin-rich
filopodia and an apparent enrichment at the leading edges of these cell processes (Fig. 8B). Carbachol induced the
formation of cell processes within 15 min; however, most of the
processes formed within this time frame were relatively short. After a
1-h exposure to carbachol, the majority of the newly formed processes
were longer, and some cells also formed curtain-like lamelopodia and
exhibited membrane ruffling (see legend to Fig. 8). Similar results
were obtained with the PKC activator, phorbol-12-myristate-13-acetate.
However, with phorbol-12-myristate-13-acetate the formation of
filopodia was more dramatic, and a high percentage of cells formed
curtain-like lamelopodia and exhibited membrane ruffling (not
shown).
In this study, we report the isolation, partial sequencing, and
cloning of a novel cholinergically regulated, coronin-like 66-kDa
signaling phosphoprotein, which we have tentatively named coroninse. The high degree of predicted secondary structure
conservation in coroninse as compared with p57 and coronin
strongly suggests that these proteins might serve similar functions
within different cell types. For example, the recruitment of coronin
into the actin network of the leading edge of migrating cells (5)
offers intriguing parallels with our finding that
EGFP-coroninse translocates to F-actin-rich, nascent cell
processes formed in response to carbachol stimulation. Thus, one role
for coroninse may be an involvement in the cytoskeletal
reorganization of epithelial cells, which is similar to the role played
by coronin in Dictyostelium. The uniqueness of our findings
with coroninse lies in the fact that this protein is
regulated in vivo by PKC-dependent changes in the state of phosphorylation, an observation that has not been reported
for either coronin or p57. Although not yet proven, we hypothesize that
coroninse serves as a direct substrate for PKC in
vivo because of the large number of PKC phosphorylation consensus sequences present in this protein and our observations that
coroninse is phosphorylated by a cholinergically activated
serine/threonine kinase in intact parietal cells. Because the
phosphorylation of coroninse in intact cells is
calcium-independent and because coroninse can serve as an
in vitro substrate for PKC The presence of proton pumps in the contractile vacuole of
Dictyostelium (31) as well as the presence of
V-H+ ATPase subunits and Rab proteins in the endocytic
vesicle compartment of this organism offer intriguing parallels with
the gastric parietal cell in which Rab proteins are present within
internal tubulovesicular membranes containing the
H+,K+-ATPase (32, 33) and in which activation
of this proton pump appears to depend upon reversible fusion of
tubulovesicles with the apically oriented canalicular membrane
(13).
The proposal that vesicular acidification regulates the association of
coronin with endosomal vesicles (8) has a precedent in that the WD
repeat motif-containing peptide component of intra-Golgi transport
(COPI) vesicles, In contrast to coronin, no definite function has yet been assigned to
the human or bovine p57 proteins. However, a protein that may be p57
has recently been found to accumulate around phagocytic cups and to be
associated with a p67phox and p40phox
protein-containing complex in human neutrophils (10). However, unlike
coronin, the p57/coronin-like protein in neutrophils does not appear to
disperse from around the phagocytic vacuole. Grogan and colleagues (10)
have speculated that the sustained localization of p57/coronin near
this vacuole may be important for the translocation of the
p40phox and p67phox proteins to their site of action.
Since we have also not detected any movement of
coroninse-EGFP around F-actin-rich intracellular canaliculi
in actively secreting parietal cells, coroninse, like the
neutrophil protein, may serve as a stable but regulated anchor for
other translocating proteins involved in ion transport in this cell
type. It should be emphasized, however, that this apparent lack of
translocation of coroninse within the canalicular region is
in direct contrast to the movement of coroninse into
nascent filopodia formed in response to carbachol stimulation. Thus, it remains to be determined whether there is a more subtle relocation of
coroninse that is associated with secretion that was not
detected by our methodology.
The conserved, highly coiled tail region at the far carboxyl terminus
of coronin has been identified as an actin-binding region (4). Given
the conservation of this predicted secondary structure in
coroninse as well as in p57, this region may well serve a
similar function in these mammalian proteins. Moreover, since all three proteins have a putative transmembrane region near the amino terminus, they are all likely to be inserted into membrane vesicles with the long
tails projecting into the cytoplasm. This structural arrangement would
clearly allow for interactions with F-actin as well as with other
proteins. The presence of WD repeat motifs, which have been shown to be
involved in a wide range of protein-protein interactions (24), further
supports this assumption. In contrast, the degeneracy in the carboxyl
terminus of p57 and coroninse could allow for different
protein-protein interactions. Given such a scenario, the changes in the
state of phosphorylation of coroninse could serve, for
example, to regulate protein associations within vesicles.
The localization of two dileucine sequence motifs within the putative
cytosolic tail of coroninse provides additional support for
the assumption that coroninse is targeted to specific
internal membrane compartments, since there is evidence that the
dileucine motif can serve as the internal functional equivalent to
tyrosine-containing targeting signals within coated pits of the plasma
membrane (30). The presence of these and other potential endocytosis
motifs in coroninse is particularly intriguing given recent
findings that alteration of a tyrosine-based endocytosis motif within
the
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
-mercaptoethanol and subjected to analytical or
preparative two-dimensional gel electrophoresis (isoelectric focusing;
pH 5-8; 9% SDS-polyacrylamide gel electrophoresis) as described
previously (11, 19). The 66-kDa phosphoprotein was identified by
autoradiographic comparisons with unstimulated controls. Changes in
phosphorylation were quantitated using Bio Image two-dimensional gel
analyzer software (19, 20) or a Molecular Dynamics PhosphorImager.
-32P]dCTP (specific activity, 6000 Ci/mmol; Amersham
Pharmacia Biotech) using a Ready-To-Go kit (Amersham Pharmacia
Biotech). Total RNA was isolated from various tissues using an RNA
STAT-60 kit (Tel-Test, Friendswood, TX), separated on 1.25%
formaldehyde-agarose gels containing 1 µg/ml ethidium bromide, and
transferred to Magnagraph (Micron, Inc., Westboro MA) nylon membranes
and probed as described previously (19, 20). Membranes were washed
under high stringency conditions (0.1× SSPE, 1% SDS, 65 °C) and
then subjected to autoradiography (Hyperfilm-MP; Amersham Pharmacia
Biotech) at
70 °C with intensifying screens.
--
Parietal cells were extracted with Triton X-100 and
Empigen BB as described above. The detergent-insoluble portion of these extracts (50 µg/assay tube) was resuspended in phosphorylation buffer
(final concentration: 25 mM HEPES, pH 7.4, 10 mM Mg(C2H3O2), 0.3 mM dithiothreitol, 10 µg of phosphatidylserine, 2 µg of
1,2-dioleoyl-sn-glycerol, and 0.1 mM
[
-32P]ATP) and incubated with or without (control)
recombinant PKC
(50 units; PanVera, Madison, WI) for 5 min at
30 °C. Reactions were stopped by the addition of hot
SDS/
-mercaptoethanol and subjected to analytical two-dimensional gel
electrophoresis and autoradiography as described above. pp66 was
identified based on co-migration of in vitro radiolabeled
spots with authentic pp66 labeled in vivo by stimulation of
intact 32P-labeled parietal cells with carbachol.
RESULTS
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Fig. 1.
Characterization of carbachol-stimulated pp66
phosphorylation in intact cells. A, time course of
carbachol-stimulated pp66 phosphorylation. 32P-Labeled
parietal cells were incubated with or without 10 µM
carbachol, fixed, and subjected to analytical two-dimensional gel
electrophoresis and autoradiography. Phosphorylation of pp66 was
quantified as described under "Materials and Methods." Values are
means ± S.E. for n = 4-6 cell preparations (*,
p < 0.01; **, p < 0.001).
B, typical migration pattern of pp66 isoforms on analytical
two-dimensional gels. Depicted are relevant portions of an
autoradiograph from experiments shown in A. Left panel,
control; right panel, 10 µM carbachol, 15 min.
The arrowhead indicates the position of the more basic of
the two pp66 isoforms that were quantitated. C, pp66 is
phosphorylated on serine and threonine residues following cholinergic
stimulation with carbachol (10 µM, 5 min).
Left, autoradiograph of two-dimensional thin layer
chromatograph. Dotted lines indicate location of
ninhydrin-stained phosphoamino acids. Right, ninhydrin
stain. Data are representative of two independent experiments.
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Fig. 2.
Purification of pp66 isoforms on preparative
two-dimensional gels. Individual spots were identified as pp66
isoforms by amino acid analysis. Boxes surround pp66
isoforms. Representative of a total of 15 such gels used to obtain
amino acid sequence from tryptic digests of excised pp66 spots.
A, Coomassie Blue stain. B, autoradiograph.
C, overlay of autoradiograph and gel.
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Fig. 3.
Amino acid sequence alignment of rabbit pp66
with human p57 showing similarities and differences in selected
motifs. Underlined regions represent amino
acid sequences obtained from tryptic digests of pp66. Shaded
boxes, PKC phosphorylation site motifs; open arrows,
region of pp66 that was initially PCR-cloned using primers based on
amino acid sequence information. This region was then used to screen a
parietal cell library from which the completed sequence was obtained.
Thick underline, transmembrane regions; open box,
WD repeat regions; underlined italic type, dileucine signal
motifs; double underlines, human p57 leucine zipper motif;
thick arrows, coiled-coil regions.
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Fig. 4.
Multiple sequence alignment comparing the
predicted amino acid sequences of bovine p57, rabbit pp66, and coronin
with human p57. Shaded areas indicate
regions of identity. Note the high degree of identity between the human
and bovine p57 sequences (95%) as compared with pp66 (67%). Note also
that the degree of homology between pp66 and p57 is substantially lower
near the COOH-terminal end as compared with the NH2
terminus. GenBankTM accession numbers for the human p57,
bovine p57, and coronin sequences used in this analysis were X89109,
D44496, and X61480, respectively.
Comparison of expressed sequence tags (ESTs) with best fits from the
NH2 terminus to the COOH terminus for pp66 and human and bovine
p57
) repeats in the human p57 sequence and only a single such
repeat in the rabbit pp66 and coronin sequences, further analysis with
the more powerful Prosite Profilescan (Swiss Institute for Experimental
Cancer Research server) detects three WD repeats within a single WD
repeat region in all three proteins (Fig. 3). The WD repeat region,
which was first identified in the
-subunit of heterotrimeric
GTP-binding proteins, has since been detected in a number of proteins
involved in the regulation of a variety of functions including signal
transduction, vesicular trafficking, cytoskeletal function, gene
regulation, and RNA processing (24). In contrast to p57 and coronin,
pp66 does not possess a leucine zipper motif, which is present both in
the human and bovine p57 sequences (Fig. 3, amino acid residues
433-454). There are also a number of potentially important motifs in
pp66 that do not appear in p57 based on a Blocks analysis using Prosite
Motifinder, version 15 (25). These include 1) a WNN motif (70%
identity; residues 182-191) that is present in the ephrin protein
family, members of which have been shown to associate with receptor
tyrosine kinases; 2) a DGR motif that is highly conserved in all actin
isoforms found in vertebrates and invertebrates (67% identity;
residues 209-263); 3) A YGR motif found in clathrin adapter complex
proteins, which have been shown to be involved in linking clathrin to
receptors present in coated vesicles (58% identity; residues
275-293); 4) an LRL-like motif that is present in the dynamin family,
members of which have been associated with endocytotic processes (70% identity; residues 392-417); and 5) a motif found in the AAA family of
ATPases, one member of which is the vesicular fusion protein, NSF (72%
identity; residues 366-402). Finally, a Prosite Prints analysis
detected a potential fibronectin type III3 fingerprint (residues
162-180). The fibronectin superfamily consists of 45 different
families of proteins, many of which are involved in cell adhesion or
are associated with receptor protein-tyrosine kinases or cytokine receptors.
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Fig. 5.
Expression of pp66 and p57 transcripts in
various tissues and cells. A, autoradiographs of
Northern blot analyses detected a single message for pp66 in rabbit
tissues with the highest level of expression in gastric fundic mucosa,
intestinal mucosa, kidney, and lung. Expression levels were also
relatively high in spleen and adrenal as compared with smooth and
skeletal muscle, for example. B, Northern blot analyses with
a full-length probe for bovine p57 detected a smaller sized message in
calf and rabbit spleen but not in rabbit parietal cells. C,
Northern blot analyses of calf spleen with a full-length probe for pp66
detected the same sized message as in rabbit parietal cells.
D, mRNA expression levels in rabbit parietal cells,
chief cells, and isolated gastric glands. A-C, positions of
28 and 18 S ribosomal RNAs in Northern blots of total RNA (20 µg/sample) are indicated, and ethidium bromide staining of 18 S
ribosomal RNA on each blot is included for comparison. D, 2 µg of mRNA were loaded per sample. Data are representative of two
or three independent experiments.
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Fig. 6.
Expression patterns of pp66-EGFP and EGFP
fusion protein constructs in live parietal cells before and after
detergent extraction. Parietal cells were cultured for 24 h
and then transfected with the cationic lipid, Effectene. Fluorescent
image sections (0.4 µm) were acquired ~24 h later by confocal
microscopy, and image sections were reconstructed as three-dimensional
images as described under "Materials and Methods." Reference images
acquired in a single in-focus image are to the right of the
corresponding fluorescent images. A, cell transfected with
pp66-pEGFP-N2 vector. B, control cell transfected with
pEGFP-N2 vector alone. C, cell transfected with
pp66-pEGFP-N2 vector after a 10-min incubation with 0.15% Triton
X-100. D, control cell transfected with pEGFP-N2 vector
alone after a 5-min incubation with 0.15% Triton X-100. Similar
expression patterns were obtained in eight independent experiments.
Bars, 5 µm.
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Fig. 7.
Intracellular localization of pp66-EGFP and
F-actin in parietal cells. Cells were transfected with the
pp66-pEGFP-N2 vector as in Fig. 9. Approximately 24 h after
transfection, unstimulated cells and cells treated with 10 µM carbachol for 15 min, were fixed and then stained for
F-actin with Alexa 568 phalloidin and scanned by confocal microscopy as
described under "Materials and Methods." Green,
pp66-EGFP fluorescence; red, F-actin. A, images
from an unstimulated cell. Left, three-dimensional
reconstruction of 0.4-µm image sections; center,
z-section through cell at the point in the cell shown in the
left panel is indicated by an arrow;
right, in-focus reference image. B, images from a
carbachol-stimulated cell. Left, section through the center
of the cell. Note the enlarged internal F-actin-enriched canaliculus as
compared with the collapsed F-actin-rich canaliculus in the
unstimulated cell. A portion of a nontransfected cell is shown on the
left. This cell serves as an internal control showing
absence of fluorescence "bleed-through" of the pp66-EGFP signal.
Right, reference image acquired at the same image plane as
the corresponding fluorescent image section. Similar results were
obtained in five independent experiments. Bars, 5 µm.
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Fig. 8.
Carbachol induces the formation of filopodia
in cultured parietal cells and pp66-EGFP migrates to the leading edge
of these cell processes. Cells transfected with pp66-pEGFP-N2
vector and nontransfected cells were fixed and stained for F-actin as
described in the legend to Fig. 7. Confocal image sections were
acquired at the level of attachment of cells to extracellular matrix.
A, nontransfected cells stained for F-actin.
Left, a group of four unstimulated cells with no detectable
filopodia. Intracellular canaliculi are prominent in three of the four
cells. Right, lower magnification of three cells that were
fixed 90 min after stimulation with 10 µM carbachol.
Filopodia are evident, and the cell in the lower
left quadrant exhibits membrane ruffling.
Bars, 10 µm. B, section of a transfected cell
imaged at higher magnification showing pp66 signal at the leading edge
of nascent cell processes and around F-actin-enriched internal
canaliculus. First panel, overlay of F-actin
(red) and pp66 signals (green); second
panel, pp66 signal; third panel, F-actin signal;
fourth panel, reference image. Similar results were obtained
in four independent experiments. Bars, 10 µm.
DISCUSSION
, it is possible that coroninse is a direct in vivo substrate for this
specific calcium-independent PKC isoform. The unequivocal demonstration
of these latter points will require further detailed studies to
identify and characterize the specific in vivo
phosphorylation sites.
'-COP, undergoes reversible,
pH-dependent associations with Golgi membranes (34). Since
'-COP has recently been shown to be a PKC
-selective receptor for
activated protein kinase C (35), this interaction could have potential
relevance to our findings that coroninse, which is also
a WD repeat-containing protein, is regulated within the PKC signaling
pathway. Another potentially relevant finding is that there is a fairly
well conserved clathrin adaptor motif in coroninse. Since
clathrin and clathrin adaptors have recently been localized to
tubulovesicular membranes in parietal cells (36), it is possible that
there is a regulated interaction between coroninse and
clathrin or a related protein that serves to direct, for example, the
endocytic retrieval of the H+,K+-ATPase from
the apical membrane to subapical tubulovesicular membranes.
-subunit of the H+,K+-ATPase leads to
constitutive acid secretion in transgenic mice (37). Finally, since PKC
activation has been associated mainly with the inhibition of HCl
secretion (22, 38), increases in coroninse phosphorylation
might also serve to increase the endocytotic retrieval of the
ion-transporting H+,K+-ATPase or some other
secretion-related ion channel or transporter from the apical membrane.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jan Pohl at the Emory University Microsequencing Facility for outstanding assistance in the amino acid analysis and tryptic digestion of native pp66 and the sequencing of peptides derived from this protein. We thank Dr. Terry Stoming and Carolyn Leithner at the Medical College of Georgia Molecular Biology Core Facility for DNA sequencing, Dr. James Goldenring for supplying the Madin-Darby canine kidney cells, and Beth Bowling at the Medical College of Georgia for help in the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R37 DK31900 (to C. S. C.) and F32 DK 09447 (to J. A. P.).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) AF056312 and AF100414.
To whom correspondence should be addressed: Inst. of Molecular
Medicine and Genetics, Sanders R & E Bldg., Rm. CB 2803, Medical College of Georgia, Augusta, GA 30912-3175. Tel.: 706-721-0681; Fax:
706-721-7915; E-mail: cchew{at}mailer.mcg.edu.
The abbreviations used are: PKC, protein kinase C; EGFP, enhanced green fluorescent protein; PCR, polymerase chain reaction.
2 J. A. Parente, Jr., X. Chen, C. Zhou, A. C. Petropoulos, and C. S. Chew, unpublished observations.
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REFERENCES |
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