1 Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114; and 2 UMR 8525, Lille II University, Institut de Biologie et Institut Pasteur de Lille, 59021 Lille, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We previously reported an increased
secretion of amyloid precursor-like protein 2 (APLP2) in the healing
corneal epithelium. The present study sought to investigate signal
transduction pathways involved in APLP2 shedding in vitro. APLP2 was
constitutively shed and released into culture medium in
SV40-immortalized human corneal epithelial cells as assessed by Western
blotting, flow cytometry, and indirect immunofluorescence. Activation
of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA)
caused significant increases in APLP2 shedding. This was inhibited by staurosporine and a PKC--specific, N-myristoylated peptide
inhibitor. Epidermal growth factor (EGF) also induced APLP2
accumulation in culture medium. Basal APLP2 shedding as well as that
induced by PMA and EGF was blocked by a mitogen-activated protein
kinase (MAPK) kinase inhibitor, U-0126. Our results suggest that MAPK activity accounts for basal as well as PKC- and EGF-induced APLP2 shedding. In addition, PKC-
may be involved in the induction of
APLP2 shedding in corneal epithelial cells.
amyloid precursor-like protein 2; ectodomain shedding; epidermal growth factor; protein kinase C; mitogen-activated protein kinase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE MAMMALIAN AMYLOID
PRECURSOR FAMILY is composed of three highly conserved
transmembrane glycoproteins, the amyloid precursor protein (APP) and
amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) (29,
73, 74). APP is the precursor of A peptides of
39-43 amino acids, which are the major component of
cerebrovascular and neuritic plaques or amyloid deposits found in the
brains of patients with Alzheimer's disease (18, 51).
Like APP, APLP2 is encoded by alternatively spliced mRNAs. One of the
spliced exons has structural/functional homology to the Kunitz-type
serine protease inhibitors (KPI) (66, 74), and one
controls the addition of chondroitin sulfate (CS) to the core protein
(54, 68, 69). Both APP and APLP2 are ubiquitously
expressed in mammalian tissues and cells, and a variety of
physiological and pathological roles have been suggested for these two
proteins (3, 10, 13, 25, 46, 51, 52).
The APP family proteins are members of a large, functionally and
structurally heterogeneous group of transmembrane proteins that undergo
proteolytic cleavage and release their ectodomains into the
extracellular milieu (termed ectodomain shedding or secretion) (34, 49, 55). Ectodomain shedding has attracted much
attention recently because the proteins concerned are involved in
pathophysiological processes such as neurodegeneration,
apoptosis, oncogenesis, and inflammation (26).
These shed proteins include receptors for tumor necrosis factor-
(TNF-
) and interleukin-6; growth factors/cytokines such as TNF-
,
transforming growth factor-
(TGF-
), epidermal growth factor
(EGF), and heparin-binding EGF (HB-EGF); and cell adhesion molecules
such as vascular cell adhesion molecule, E- and L-selectin, and
syndecan-1 and -4, for example (26, 65). Members of the
APP family may share significant overlap in function and expression.
Although their functions are unknown, members of the APP protein family
have features in common with cell adhesion molecules and CS
proteoglycans, important constituents of the cell surface and the
extracellular matrix (ECM) of mammalian cells (13, 49).
Thus the release of ectodomains of APP and APLP2 and their
incorporation into ECM proteins would change the properties of ECM and
microenvironments. Consistent with this, we observed APLP2 accumulation
at the wound bed in front of the leading edge of migratory epithelium
(22). Hence, shedding of APLP2 in corneal epithelial cells
may be an important regulatory step for APLP2 to function in modulation
of corneal epithelial migration and wound healing.
Shedding is believed to be under stringent regulation in vivo. One of
the best studied proteins for ectodomain shedding is APP. APP can be
processed by -secretase to generate a large secreted form and
nonamyloidogenic carboxy-terminal fragments or by a
-secretase to
generate A
(reviewed in Ref. 51). Processing of APP by
-secretase is known as ectodomain shedding and precludes A
formation (14, 56). This process is known to be regulated
by a growing list of neurotransmitters, growth factors, cytokines, and
hormones (reviewed in Ref. 38). In response to these
stimuli, several intracellular cascades have been implicated in the
regulation of APP processing. Phorbol esters that directly activate
most protein kinase C (PKC) isozymes are often used experimentally to
induce shedding; numerous studies have shown that activation of PKC by
phorbol 12-myristate 13-acetate (PMA) enhances APP cleavage in a
variety of cell lines as well as in primary neuronal cultures (33, 36, 37, 40, 57). Neurotransmitters and neuropeptides have been shown to stimulate G protein-coupled receptors and regulate APP processing by PKC-dependent signaling pathways (38, 42, 43).
PKC is composed of a family of closely related serine/threonine protein
kinases consisting of at least 12 isozymes (39). The role
of various PKC isozymes in regulating APP processing has not been
addressed extensively (38). PKC isozymes may directly activate a specific function of cells or trigger a cascade of protein
kinases that ultimately stimulates a specific cellular response
(39). Izumi et al. (27) showed that upon PMA
stimulation, PKC- phosphorylates ADAM-9, a member of a disintegrin
and metalloproteinase (ADAM) family, resulting in cleavage of the
membrane-anchored form and release of the soluble form of HB-EGF.
PKC-
and -
, but not PKC-
, have been implicated in regulation
of APP shedding in fibroblast cell lines (28, 60). The
underlying mechanisms used by these isozymes to activate this process
are not clear.
In addition to activation of PKC, several other intracellular signaling pathways have been implicated to act as intermediaries in PKC-independent neurotransmitter receptor regulation of APP processing, including the second messengers Ca2+, phospholipase A2, and cAMP-dependent protein kinase (7, 40-42, 45, 59). Furthermore, growth factors, including nerve growth factor (NGF) (12), EGF (58), and insulin (62), have been found to stimulate APP processing through tyrosine kinase receptors in a PKC-independent manner (reviewed in Ref. 38). Recent studies have suggested that the mitogen-activated protein kinase (MAPK) signaling cascade acts as an effector pathway for activating shedding metalloproteinase(s) and consequent ectodomain release (12, 15-17, 36). Thus it is interesting to determine whether PKC functions via activation of MAPK signaling pathways in mediating ectodomain shedding.
Unlike APP, the processing of APLP2 and its regulation have not been
extensively studied. APLP2 is suggested to be processed similarly to
APP, since such ectodomain shedding of APLP2 may influence the
amyloidogenic process of APP (35). However, the sequences
in the region in which the cleavage site for -secretase resides,
~100 amino acids amino-terminal to the putative membrane-spanning segment, are divergent between APP and APLP2 (48, 63, 72, 74). Recently, two widely different functions have been ascribed to APLP2 but not to other members of the APP family. They play a role
in chromosome replication and/or segregation (46, 72) and
in association with the major histocompatibility complex class I
molecule Kd (53). At least in the latter case,
the unique function of APLP2 is related to the processing of the
protein. Our observation that APLP2, but not APP, is upregulated in
healing corneal epithelial cells and that secreted APLP2 is accumulated
on the wound bed in front of the migratory epithelial sheet suggests
that ectodomain shedding of APLP2 may be involved in modulating corneal
epithelial wound healing (22). Thus differences in amino
acid sequences and in functions of these two closely related proteins
would imply subtle differences in regulation of APLP2 shedding from
that of APP. It is, therefore, of interest to understand regulation of APLP2 processing.
Recently, we identified multiple PKC isozymes expressed in human
corneal epithelial cells and found that EGF stimulation failed to
induce PKC isozyme translocation (Xu, Dartt, and Yu, unpublished observations). The aim of the current study was to begin to
understand the signaling mechanisms that result in the activation of
ectodomain shedding of APLP2. We have provided evidence that PKC- and
EGF-induced, as well as basal, APLP2 shedding is modulated by the MAPK
pathway. We have also shown that PKC- isozyme might be involved in
PMA-induced APLP2 shedding.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
PMA from Sigma (St. Louis, MO) was dissolved in dimethyl sulfoxide and
kept at 20°C. Stocks were diluted in culture medium before
experiments. Control media always contained equivalent concentrations
of the solvent. EGF was purchased from R&D Systems (Minneapolis, MN).
Staurosporine and U-0126 were from Calbiochem (San Diego, CA).
Fluorescein isothiocyanate (FITC)-streptavidin was from Jackson
ImmunoLab (West Grove, PA), rhodamine-phalloidin was from Molecular
Probes (Eugene, OR), and Vectashield mounting medium with
4',6-diamidino-2-phenylindole (DAPI) was from Vector Lab (Burlingame,
CA). All other chemicals were purchased from Sigma.
Cell culture and treatments. A stably SV40-transfected human corneal epithelial (THCE) cell line that continues to grow and exhibit a cobblestone-like appearance was generously provided by Dr. K. Araki-Sasaki (2). THCE cells were grown in a keratinocyte basic medium (KBM) supplemented with growth factors (KGM; BioWhittaker, Walkersville, MD) in a humidified 5% CO2 incubator at 37°C. Cells were cultured onto plastic dishes precoated with fibronectin collagen coating mix (Biological Research Faculty and Facility, Ijamsville, MD) and grown to 80% confluence in KGM. Before treatment, the KGM was replaced with KBM for 16 h (growth factor starvation overnight). Cells were then incubated in the presence or absence of test substances freshly dissolved in KBM.
Detection of APLP2 shedding by Western blotting. APLP2 shedding was quantitated after PMA (1 µM) or EGF (20 ng/ml) stimulation for 16 h. Staurosporine (40 nM), a broad-spectrum inhibitor of protein kinases, was applied 4 min before PMA treatment. After various time intervals, conditioned media were collected, cooled to 4°C, and centrifuged to discard debris. The supernatant fluids were concentrated with Nanosep microconcentrators (10-kDa molecular cutoff; Pall Filtron, Northborough, MA) and quantified. Cultured THCE cells were washed with phosphate-buffered saline [PBS; containing (in mM) 137 NaCl, 10.1 Na2HPO4, 1.8 KH2PO4, and 2.7 KCl at pH 7.2] and lysed in lysis buffer [containing (in mM) 50 Tris at pH 8.0, 150 NaCl, and 5 EDTA; 0.5% Triton X-100; the following protease inhibitors (in µg/ml): 50 pepstatin, 50 leupeptin, and 10 aprotinin; and 0.25 mM phenylmethylsulfonyl fluoride]. After centrifugation to remove detergent-insoluble material, the lysate was subjected to protein determination using a micro BCA (bicinchoninic acid) protein assay reagent kit (Pierce, Rockford, IL).
The detergent-soluble protein in cell lysates or the corresponding portion of conditioned medium was adjusted to equal amounts with Laemmli buffer and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 5-15% gradient minigels (Bio-Rad, Hercules, CA). After electrophoresis, proteins were transferred electrophoretically to a nitrocellulose membrane (Bio-Rad). Equal amounts of protein loading were confirmed by staining the membranes with Ponceau S. The nonspecific binding sites of membranes were blocked with 5% nonfat skim milk in Tris-buffered saline (20 mM Tris · HCl, pH 7.6, and 150 mM NaCl) containing 0.05% Tween 20 (TBS-T) at room temperature for 1 h and subsequently incubated with primary antibody against D2II (68) diluted in 5% nonfat milk in TBS-T at room temperature for 1 h. After being washed, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antiserum (Bio-Rad) at room temperature for 1 h. Immune complexes were visualized with enhanced chemiluminescence detection reagent (Pierce). The chemiluminescent images were captured using the Biological Detection System (Pittsburgh, PA) and quantified using NIH Image (version 1.61) software. The relative amount of APLP2 was the sum of all three bands in each lane. To ensure that experimental values were within the linear range of the assay, we analyzed several exposure times. Results were expressed as square pixels that varied between experiments, in part, because of differences in exposure times and treatment parameters. Thus at least three independent experiments were performed for each treatment, and a control was included in each experiment.Flow cytometric and immunofluorescence microscopic analyses of APLP2 shedding. Flow cytometry and indirect immunofluorescence were used to assess the loss of APLP2 from the plasma membrane. Growth factor-starved THCE cells were incubated with 1 µM PMA or 20 ng/ml EGF for 15 min. Cells were fixed with 4% paraformaldehyde freshly dissolved in PBS without permeabilization. Cells were incubated with D2II antibody and then with FITC-conjugated goat anti-rabbit IgG, both at room temperature, for 1 h. Surface fluorescence was analyzed with an Epics XL flow cytometer (Coulter, Miami, FL). Fluorophores were excited at 488 nm, and emission was collected at 530 nm for FITC. The stained cells were also viewed under a Nikon Eclipse E-800 microscope equipped with a Spot digital camera.
Effects of myristoylated pseudosubstrate-derived peptides and
MAPK inhibitor on APLP2 shedding.
Four N-myristoylated peptides derived from the pseudosubstrate
sequences of PKC- (myr-PKC-
; a 9-amino acid peptide
common to all three PKC isozymes), PKC-
(myr-PKC-
, a 14-amino
acid peptide unique to PKC-
), PKC-
(myr-PKC-
), and PKC-
(myr-PKC-
) were used to study the role of PKC isozymes in
PMA-induced APLP2 shedding. To allow intracellular accumulation of the
peptides, we preincubated THCE cells with 1 µM of each peptide for 60 min, and they continued in culture with PMA for 16 h. APLP2
shedding was measured by Western blotting as discussed.
Statistical analysis. Statistical parameters were ascertained with the StatView program (Abacus Concepts, Berkeley, CA), and results were expressed as means ± SE. Statistical differences between groups were determined using Student t-test or between multiple groups using ANOVA. Significance was set at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PMA- and EGF-induced APLP2 ectodomain shedding.
To study the extracellular cleavage of transmembrane APLP2, we
developed an assay that allowed a convenient and quantitative assessment of APLP2 shedding following stimulation (Fig.
1). The disappearance of cell-associated
APLP2 and appearance of secreted APLP2 were assessed by immunoblotting
using antibodies against the extracellular domain of APLP2 (Fig.
1A). In corneal epithelial cell lysate (cell associated),
APLP2 appeared as three bands with different mobilities, resulting from
differential glycosylation and processing of transmembrane APLP2
(22). A discrete band at ~105 kDa represented molecules
without processing, and a discrete ~120-kDa band represented
glycosylated but not CS-modified APLP2 (22, 68). The light
band of heterogeneous polypeptides with apparent molecular mass of
~130-200 kDa was the result of modification of APLP2 core
protein with CS glycosaminoglycan chains of various lengths. Similarly,
secreted APLP2 appeared in the medium as the three species but was
smaller in size because the secreted forms lack transmembrane and
cytoplasmic domains. The ~130- to 200-kDa CS glycosaminoglycan
chain-modified molecules in the medium were the predominant form of
secreted APLP2. The detection of secreted APLP2 allowed a quantitative
assessment of the transmembrane APLP2 cleavage, which correlated with
immunoreactivity of the soluble APLP2 as assessed by Western blotting.
|
|
PMA-induced APLP2 accumulation in media was due to ectodomain
shedding.
The APP family of proteins is known to be distributed in a variety of
cells at the cell surface and in intracellular spaces (13,
75). Staining of THCE cells with anti-D2II antibody directed against the ectodomain of APLP2 revealed two types of staining patterns: bright surface staining in certain cells and punctate staining in all cells. Treatment of THCE cells with PMA resulted in
reduction in cell surface immunoreactivity (bright surface staining),
probably due to shedding of cell surface APLP2 (Fig. 3A). However, punctate
staining in PMA-treated cells was still observed, but the intensity was
lower. Similarly, EGF also induced reduction of APLP2 cell surface
staining (data not shown).
|
Effect of PKC on APLP2 shedding.
Staurosporine, a nonselective protein kinase inhibitor for both
tyrosine and serine/threonine kinases (20, 47), was
applied to the culture medium before addition of PMA. Staurosporine
reduced PMA-induced shedding of APLP2 ectodomain in THCE cells (66%
inhibition, P < 0.05; Fig.
4), suggesting a possible pathway through
a PMA-responsive protein kinase such as PKC in PMA-induced APLP2
shedding.
|
Effects of N-myristoylated PKC pseudosubstrate peptides on
PMA-induced shedding of APLP2.
To characterize which PKC isozyme(s) mediates APLP2 shedding, we used
N-myristoylated peptide inhibitors derived from the pseudosubstrate
sequences of PKC-, -
, -
, and -
and examined their
effects on PMA-induced APLP2 shedding in THCE cells. We first
determined the permeability of THCE cells to pseudosubstrate-derived peptides. Biotin-conjugated peptides were synthesized and applied to
the cultured cells for 5 or 15 min, followed by detection of biotin in
cells. As shown in Fig. 5, within 5 min,
biotin staining was found in a region of THCE cells, suggesting that
myristoylated peptide inhibitors enter THCE cells within 5 min at a
certain region of the cell body; within 15 min, these peptides were
evenly distributed in cells (data not shown). A similar biotin staining pattern was observed for all four myr-PKC peptides (Fig. 5, myr-PKC-
and -
). These data suggest that the THCE cells are equally permeable to the different myristoylated peptide inhibitors.
|
|
Effects of MEK inhibition on PMA- or EGF-stimulated ERK
phosphorylation and APLP2 shedding.
Several signaling pathways are activated upon growth factor-induced
stimulation of tyrosine kinase receptors. The receptor-activated MAPK
cascade mediated by Ras, Raf, and MEK has been suggested to mediate
ectodomain shedding of several transmembrane proteins (11, 50,
70). We therefore tested whether PMA- or EGF-induced APLP2
shedding is also mediated through activation of MAPK. Upon MAPK
activation, ERK must be phosphorylated by MEK on both a tyrosine and a
threonine residue in the TEY motif (1, 44).
Phosphorylated ERK can be detected using antibodies against the
phosphorylated TEY consensus sequence. As shown in Fig.
7, there was basal phosphorylation of
MAPK in untreated growth factor-starved THCE cells. Addition of either
PMA or EGF to the cells resulted in an increase in ERK phosphorylation
(Fig. 7A). The presence of U-0126 (10 µM) significantly blocked PMA- or EGF-induced ERK phosphorylation as reflected by a
decreased ratio of phosphorylated ERK1/2 to ERK2 immunoreactivity (pERK/ERK ratio; P < 0.01 and P < 0.05 for PMA and EGF, respectively). In addition, U-0126 also
inhibited basal phosphorylation of ERK in THCE cells (P < 0.05).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have studied the physiological signaling mechanisms that lead
to ectodomain shedding of APLP2, a member of the APP family of proteins
that is upregulated in the healing corneal epithelium. Several
important observations have emerged from our studies. First, activation
of PKC with PMA and EGF stimulate ectodomain shedding of APLP2. Second,
PKC-, and probably PKC-
, may be responsible for PMA-induced APLP2
shedding. Third, PMA-induced cleavage of APLP2 occurs through MAPK
activation. Finally, activation of the MAPK signaling pathway accounts
for PKC- and EGF-induced, as well as the basal level of, APLP2 shedding.
Electrophoretic migration of APLP2 demonstrates that APLP2 recovered from the conditioned medium corresponds to the ectodomain, which lacks the cytoplasmic and transmembrane domains. In THCE cells, the APLP2 ectodomain is shed from cultured cells at a basal rate, but this shedding is markedly enhanced by activating PKC with PMA or by stimulation with EGF, a mitogenic growth factor that has been implicated in regulation of corneal wound healing (76). Thus we have concluded that APLP2 belongs to the class of transmembrane proteins that undergo proteolytic cleavage and release of their ectodomains into the extracellular milieu and that this ectodomain shedding is regulated by physiological effectors.
In a recent study, we showed PMA-induced translocation of four PKC
isozymes, PKC-, -
I, -
, and -
, in THCE cells (Xu, Dartt, and
Yu, unpublished observations). One or more of these isozymes is
likely to be involved in PMA-induced APLP2 shedding. Although there has
been considerable interest in the potential role of PKC isozymes in the
process of ectodomain shedding, isolating the role of the individual
isozymes has been proven to be complex because of overlapping
sensitivity to activators and inhibitors. In this study, we used
myristoylated peptides derived from PKC isozyme pseudosubstrate
sequences as isozyme-specific inhibitors (78).
Myristoylation of kinase pseudosubstrate peptides enhances their
ability to cross intact plasma membranes and thus inhibit intracellular
protein kinases. Cell permeability and isozyme selectivity of the
myristoylated peptides have been described in detail previously (61, 77, 78). In line with these previous studies, we
showed that these myristoylated pseudosubstrate peptides are readily transported into cultured corneal epithelial cells within 5 min of
incubation (Fig. 5) and that the myristoylated peptides do not
selectively inhibit translocation of their cognate PKC isoforms in THCE
cells (data not shown). This approach is becoming increasingly common
in identifying the cellular role(s) of particular protein kinases
(19, 32, 64, 67, 78). We showed that myr-PKC-
inhibitor
significantly blocked PMA-induced shedding of APLP2 in THCE cells,
whereas myr-PKC-
and -
had no apparent effects, suggesting
that PKC-
is utilized in PMA-induced shedding of APLP2. Izumi et al.
(27) showed that PKC-
is involved in PMA-induced ectodomain shedding of HB-EGF. We also observed in some experiments that PKC-
myristoylated pseudosubstrate peptide was as effective as PKC-
pseudosubstrate. However, sometimes PKC-
pseudosubstrate exhibited no effect on PMA-induced ectodomain shedding. The reason for
the inconsistent results of PKC-
pseudosubstrate is not clear but
may be related to the complicated mechanisms of action of these
pseudosubstrates, which currently are not fully understood (23,
78). Consistent with our results, PKC-
was
implicated in PMA-induced APP secretion in APP-transfected Chinese
hamster ovary (CHO) cells (28). The same study also
suggested a role for PKC-
in APP secretion in human Ntera 2 neurons
and in CHO cells. Several other studies also reported the involvement
of PKC-
in regulation of APP secretions in fibroblast cells
(4, 28, 30, 60). It is not clear whether the
difference in PKC-
involvement between previously reported
studies and our current studies is due to the different cell types used
or to the subtle differences between APLP2 and APP secretion
(28). A comparative study of PMA-induced secretion of
APLP2 with APP in epithelial and in neuronal cells is needed to clarify
this issue.
Consistent with the previous studies of APP secretion mediated by
-secretase (12, 36), our results assessing ERK
phosphorylation and using the highly specific MEK inhibitor U-0126
suggest a key role for MAPK signaling cascade in regulating APLP2
shedding. Inhibition of this pathway completely blocked EGF-induced
ectodomain shedding and partially decreased the basal level of
ectodomain cleavage in cultured THCE cells. Inhibition of basal
shedding of APLP2 by U-0126 is different from that of TGF-
, which is
mediated through p38 MAPK signaling pathway (15),
suggesting that certain differences exist for the regulation of
transmembrane protein shedding between different cell types or
different shed proteins. We also observed that ERK is phosphorylated at
a basal level without growth factor stimulation, and this basal
phosphorylation may be responsible for the basal level of APLP2
shedding. It is not clear whether the basal level of APLP2 shedding is
a result of cell culture conditions and SV40 transfection or a
reflection of a constitutive level of shedding under normal
physiological conditions in vivo.
In THCE cells, the induction of MAPK signaling accounted for the activation of ectodomain shedding by PMA, a potent activator of PKC. Inhibition of PMA-induced shedding by U-0126 suggests that APLP2 shedding is not a direct consequence of PKC activity but, rather, results from PKC-induced activation of MAPK. The fact that PKC activation induces MAPK signaling suggests that in corneal epithelial cells, PKC may play a role in the activation of MAPK signaling by growth factor receptors. However, our observations that EGF stimulation does not induce translocation of PKC isozymes (Xu, Dartt, and Yu, unpublished observations) and that the specific PKC inhibitors calphostin C and chelerythrine chloride fail to block EGF-induced APLP2 secretion (data not shown) suggest that EGF stimulation is PKC independent. To date, the natural stimuli that induce PKC activation that in turn mediates APLP2 secretion in corneal epithelial cells is not known.
Because MAPK signaling is induced by growth factors, various tyrosine kinases, and oncogenes (9, 50, 70), a large variety of stimuli and changes in the cellular environment may lead to ectodomain shedding of APLP2 and other transmembrane proteins through the same signaling pathways (15). Our data demonstrate that EGF is one of such growth factors that activate MAPK and promote APLP2 secretion. NGF is another factor known to stimulate APP secretion (12, 36, 38), while insulin induces APP secretion by MAPK-independent pathways. The best-characterized means of stimulating the MAPK pathway is by activation of receptor tyrosine kinases (8). Our data indicated that the "direct route" of ERK activation by receptor tyrosine kinases is necessary and sufficient for growth factor, such as EGF, stimulation of APLP2 secretion in epithelial cells.
The mechanism by which the MAPK pathway regulates APLP2 shedding is
unknown. It is possible that upregulation of APLP2 by EGF and/or PMA
treatment contributes to the increased accumulation of APLP2 in culture
medium. We observed depletion of cell surface APLP2 within 15 min (Fig.
3) of PMA stimulation. We also reported that complete PMA-induced
translocation of PKC isozymes occurs within 10 min (Xu, Dartt, and Yu,
unpublished observations). The time course of APLP2 shedding in
response to PMA stimulation suggests that activation of MAPK induces
APLP2 shedding, at least in part, by increasing secretase activity.
This increased cleavage activity may also shed newly inserted APLP2
from cell surface, thus reducing or preventing possible internalization
of the molecules in activated cells. These suggestions are consistent
with a recent observation that the majority of secreted APP originates
from the intracellular compartment (28). As expected, the
enzyme response for APLP2 shedding in corneal epithelial cells is
sensitive to metalloproteinase inhibitor GM-6001 (Xu, Dartt, and Yu,
unpublished observations). Two ADAM family members,
TNF--converting enzyme (TACE) (6) and ADAM-10
(31), have been shown to contribute to the cleavage of
APP. Recently, using an APP mutant construct specifically targeted to
the trans-Golgi network (TGN), Skovronsky et al. (57)
demonstrated that regulated
-secretase (TACE/ADAM-10) APP cleavage
occurs in the TGN. Although our study cannot distinguish
intracellular or cell surface cleavage, the facts that cell surface
staining of APLP2 is diminished upon both PMA and EGF stimulation
within 15 min and that the time required for one-half of the total
APLP2 to be secreted under normal conditions is 2 h
(34) imply that at least some activation of APLP2 cleavage
enzyme occurs at the cell surface.
The facts that EGF activates ectodomain shedding of APLP2 in vitro and that secreted APP and APLP2 are accumulated in the traumatic brain and wounded corneas, respectively, suggest physiological relevance for the release of APP family from cells. Recently, the EGF receptor was found to be activated in wounded corneal epithelia, a step that appears to be necessary for epithelial cell migration and wound closure (76). This EGF receptor activation is consistent with increased expression and secretion of APLP2 in migratory epithelial cells in vivo (22). Our previous studies revealed that the majority of APLP2 molecules in the corneal epithelium contain a KPI domain and are posttranslationally modified by the addition of CS (22). The KPI domain is known to inhibit certain serine proteases including plasmin, which is involved in corneal wound healing (24, 71). Thus release of APLP2 derivatives and their incorporation into ECM may inhibit matrix-associated serine proteinase activity in wounded corneas. Hence, wound-induced shedding may provide a mechanism to balance proteinase activity during ECM remodeling. CS proteoglycans in the ECM of the central nervous system, including neurocan and phosphocan, are believed to modulate cell adhesion, axonal growth, and guidance during neural development (5, 21). Our observation that CS-modified APLP2 promotes cell adhesion and migration in vitro and is concentrated in front of the migratory epithelial sheet in vivo suggests a similar role for the protein in epithelial wound healing of the cornea. As such, regulation of APLP2 ectodomain shedding as demonstrated here may be of physiological significance for the protein to function in epithelial cell migration and wound healing. Further studies are needed to clarify the functional role of the ectodomain shedding process and of shed molecules of the APP-like proteins in regulating corneal epithelial wound healing or other biological processes.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Eye Institute Grant EY-10869 (to F.-S. X. Yu).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: F.-S. X. Yu, Schepens Eye Research Institute, 20 Staniford St., Boston, MA 02114 (E-mail: fushinyu{at}vision.eri.harvard.edu).
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.
Received 13 October 2000; accepted in final form 5 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, N,
Maller J,
Tonks N,
and
Sturgill T.
Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase.
Nature
343:
651-653,
1990[ISI][Medline].
2.
Araki-Sasaki, K,
Ohashi Y,
Sasabe T,
Hayashi K,
Watanabe H,
Tano Y,
and
Handa H.
An SV40-immortalized human corneal epithelial cell line and its characterization.
Invest Ophthalmol Vis Sci
36:
614-621,
1995[Abstract].
3.
Bayer, TA,
Cappai R,
Masters CL,
Beyreuther K,
and
Multhaup G.
It all sticks together-the APP-related family of proteins and Alzheimer's disease.
Mol Psychiatry
4:
524-528,
1999[ISI][Medline].
4.
Benussi, L,
Govoni S,
Gasparini L,
Binetti G,
Trabucchi M,
Bianchetti A,
and
Racchi M.
Specific role for protein kinase C alpha in the constitutive and regulated secretion of amyloid precursor protein in human skin fibroblasts.
Neurosci Lett
240:
97-101,
1998[ISI][Medline].
5.
Bovolenta, P,
and
Fernaud-Espinosa I.
Nervous system proteoglycans as modulators of neurite outgrowth.
Prog Neurobiol
61:
113-132,
2000[ISI][Medline].
6.
Buxbaum, JD,
Liu KN,
Luo Y,
Slack JL,
Stocking KL,
Peschon JJ,
Johnson RS,
Castner BJ,
Cerretti DP,
and
Black RA.
Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated -secretase cleavage of the Alzheimer amyloid protein precursor.
J Biol Chem
273:
27765-27767,
1998
7.
Buxbaum, JD,
Ruefli AA,
Parker CA,
Cypess AM,
and
Greengard P.
Calcium regulates processing of the Alzheimer amyloid protein precursor in a protein kinase C-independent manner.
Proc Natl Acad Sci USA
91:
4489-4493,
1994[Abstract].
8.
Cobb, MH,
and
Goldsmith EJ.
How MAP kinases are regulated.
J Biol Chem
270:
14843-14846,
1995
9.
Cohen, P.
The search for physiological substrates of MAP and SAP kinases in mammalian cells.
Trends Cell Biol
7:
353-361,
1997[ISI].
10.
Coulson, EJ,
Paliga K,
Beyreuther K,
and
Masters CL.
What the evolution of the amyloid protein precursor supergene family tells us about its function.
Neurochem Int
36:
175-184,
2000[ISI][Medline].
11.
Denhardt, DT.
Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.
Biochem J
318:
729-747,
1996[ISI][Medline].
12.
Desdouits-Magnen, J,
Desdouits F,
Takeda S,
Syu LJ,
Saltiel AR,
Buxbaum JD,
Czernik AJ,
Nairn AC,
and
Greengard P.
Regulation of secretion of Alzheimer amyloid precursor protein by the mitogen-activated protein kinase cascade.
J Neurochem
70:
524-530,
1998[ISI][Medline].
13.
Dodart, JC,
Mathis C,
and
Ungerer A.
The -amyloid precursor protein and its derivatives: from biology to learning and memory processes.
Rev Neurosci
11:
75-93,
2000[ISI][Medline].
14.
Esch, FS,
Keim PS,
Beattie EC,
Blacher RW,
Culwell AR,
Oltersdorf T,
McClure D,
and
Ward PJ.
Cleavage of amyloid beta peptide during constitutive processing of its precursor.
Science
248:
1122-1124,
1990[ISI][Medline].
15.
Fan, H,
and
Derynck R.
Ectodomain shedding of TGF- and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades.
EMBO J
18:
6962-6972,
1999
16.
Fitzgerald, ML,
Wang Z,
Park PW,
Murphy G,
and
Bernfield M.
Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase.
J Cell Biol
148:
811-824,
2000
17.
Gechtman, Z,
Alonso JL,
Raab G,
Ingber DE,
and
Klagsbrun M.
The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading.
J Biol Chem
274:
28828-28835,
1999
18.
Goedert, M,
Sisodia SS,
and
Price DL.
Neurofibrillary tangles and -amyloid deposits in Alzheimer's disease.
Curr Opin Neurobiol
1:
441-447,
1991[Medline].
19.
Graham, MA,
Rawe I,
Dartt DA,
and
Joyce NC.
Protein kinase C regulation of corneal endothelial cell proliferation and cell cycle.
Invest Ophthalmol Vis Sci
41:
4124-4132,
2000
20.
Groundwater, P,
Solomons K,
Drewe J,
and
Munawar M.
Protein tyrosine kinase inhibitors.
Prog Med Chem
33:
233-329,
1996[Medline].
21.
Grumet, M,
Friedlander DR,
and
Sakurai T.
Functions of brain chondroitin sulfate proteoglycans during developments: interactions with adhesion molecules.
Perspect Dev Neurobiol
3:
319-330,
1996[ISI][Medline].
22.
Guo, J,
Thinakaran G,
Guo Y,
Sisodia SS,
and
Yu FX.
A role for amyloid precursor-like protein 2 in corneal epithelial wound healing.
Invest Ophthalmol Vis Sci
39:
292-300,
1998[Abstract].
23.
Harris, TE,
Persaud SJ,
and
Jones PM.
Pseudosubstrate peptide inhibitors of -cell protein kinases: altered selectivity after myristoylation.
Mol Cell Endocrinol
155:
61-68,
1999[ISI][Medline].
24.
Hayashi, K,
Berman M,
Smith D,
Ghatit A,
Pease S,
and
Kenyon K.
Pathogenesis of corneal epithelial defects: role of plasminogen activator.
Curr Eye Res
10:
381-398,
1991[ISI][Medline].
25.
Homayouni, R,
Rice DS,
Sheldon M,
and
Curran T.
Disabled-1 binds to the cytoplasmic domain of amyloid precursor-like protein 1.
J Neurosci
19:
7507-7515,
1999
26.
Hooper, N,
Karran E,
and
Turner A.
Membrane protein secretase.
Biochem J
321:
265-279,
1997[ISI][Medline].
27.
Izumi, Y,
Hirata M,
Hasuwa H,
Iwamoto R,
Umata T,
Miyado K,
Tamai Y,
Kurisaki T,
Sehara-Fujisawa A,
Ohno S,
and
Mekada E.
A metalloprotease-disintegrin, MDC9/meltrin-/ADAM9 and PKC
are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor.
EMBO J
17:
7260-7272,
1998
28.
Jolly-Tornetta, C,
and
Wolf BA.
Regulation of amyloid precursor protein (APP) secretion by protein kinase C alpha in human ntera 2 neurons (NT2N).
Biochemistry
39:
7428-7435,
2000[ISI][Medline].
29.
Kang, J,
Lemaire H,
Masters C,
and
Grzeschik K.
The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor.
Nature
325:
733-736,
1987[ISI][Medline].
30.
Kinouchi, T,
Sorimachi H,
Maruyama K,
Mizuno K,
Ohno S,
Ishiura S,
and
Suzuki K.
Conventional protein kinase C (PKC)- and novel PKC
, but not -
, increase the secretion of an N-terminal fragment of Alzheimer's disease amyloid precursor protein from PKC cDNA transfected 3Y1 fibroblasts.
FEBS Lett
364:
203-206,
1995[ISI][Medline].
31.
Lammich, S,
Kojro E,
Postina R,
Gilbert S,
Pfeiffer R,
Jasionowski M,
Haass C,
and
Fahrenholz F.
Constitutive and regulated -secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease.
Proc Natl Acad Sci USA
96:
3922-3927,
1999
32.
Laudanna, C,
Mochly-Rosen D,
Liron T,
Constantin G,
and
Butcher EC.
Evidence of zeta protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis.
J Biol Chem
273:
30306-30315,
1998
33.
Lee, RK,
Wurtman RJ,
Cox AJ,
and
Nitsch RM.
Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors.
Proc Natl Acad Sci USA
92:
8083-8087,
1995[Abstract].
34.
Lo, A,
Thinakaran G,
Slunt H,
and
Sisodia S.
Metabolism of the amyloid precursor-like protein 2 in MDCK cells. Polarized trafficking occurs independent of the chondroitin sulfate glycosaminoglycan chain.
J Biol Chem
270:
12641-12645,
1995
35.
Lyckman, AW,
Confaloni AM,
Thinakaran G,
Sisodia SS,
and
Moya KL.
Post-translational processing and turnover kinetics of presynaptically targeted amyloid precursor superfamily proteins in the central nervous system.
J Biol Chem
273:
11100-11106,
1998
36.
Mills, J,
Laurent Charest D,
Lam F,
Beyreuther K,
Ida N,
Pelech SL,
and
Reiner PB.
Regulation of amyloid precursor protein catabolism involves the mitogen-activated protein kinase signal transduction pathway.
J Neurosci
17:
9415-9422,
1997
37.
Mills, J,
and
Reiner PB.
Phorbol esters but not the cholinergic agonists oxotremorine-M and carbachol increase release of the amyloid precursor protein in cultured rat cortical neurons.
J Neurochem
67:
1511-1518,
1996[ISI][Medline].
38.
Mills, J,
and
Reiner PB.
Regulation of amyloid precursor protein cleavage.
J Neurochem
72:
443-460,
1999[ISI][Medline].
39.
Nishizuka, Y.
Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J
9:
484-496,
1995
40.
Nitsch, RM,
Deng M,
Growdon JH,
and
Wurtman RJ.
Serotonin 5-HT2a and 5-HT2c receptors stimulate amyloid precursor protein ectodomain secretion.
J Biol Chem
271:
4188-4194,
1996
41.
Nitsch, RM,
Deng A,
Wurtman RJ,
and
Growdon JH.
Metabotropic glutamate receptor subtype mGluR1 stimulates the secretion of the amyloid
-protein precursor ectodomain.
J Neurochem
69:
704-712,
1997[ISI][Medline].
42.
Nitsch, RM,
Kim C,
and
Growdon JH.
Vasopressin and bradykinin regulate secretory processing of the amyloid protein precursor of Alzheimer's disease.
Neurochem Res
23:
807-814,
1998[ISI][Medline].
43.
Nitsch, RM,
Slack BE,
Wurtman RJ,
and
Growdon JH.
Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors.
Am J Hum Genet
51:
998-1014,
1992[ISI][Medline].
44.
Pague, D,
Rossomondo A,
Martino P,
Erickson A,
Her J,
Shabanowitz J,
Hunt D,
Weber M,
and
Sturgill T.
Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).
EMBO J
10:
885-892,
1991[Abstract].
45.
Racchi, M,
Baetta R,
Salvietti N,
Ianna P,
Franceschini G,
Paoletti R,
Fumagalli R,
Govoni S,
Trabucchi M,
and
Soma M.
Secretory processing of amyloid precursor protein is inhibited by increase in cellular cholesterol content.
Biochem J
322:
893-898,
1997[ISI][Medline].
46.
Rassoulzadegan, M,
Yang Y,
and
Cuzin F.
APLP2, a member of the Alzheimer precursor protein family, is required for correct genomic segregation in dividing mouse cells.
EMBO J
17:
4647-4656,
1998
47.
Ruegg, U,
and
Burgess G.
Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases.
Trends Pharmacol Sci
10:
218-220,
1989[ISI][Medline].
48.
Sandbrink, R,
Masters CL,
and
Beyreuther K.
Similar alternative splicing of a non-homologous domain in beta A4-amyloid protein precursor-like proteins.
J Biol Chem
269:
14227-14234,
1994
49.
Schubert, D,
Jin LW,
Saitoh T,
and
Cole G.
The regulation of amyloid beta protein precursor secretion and its modulatory role in cell adhesion.
Neuron
3:
689-694,
1989[ISI][Medline].
50.
Seger, R,
and
Krebs E.
The MAPK signaling cascade.
FASEB J
9:
726-735,
1995
51.
Selkoe, D.
Cell biology of the amyloid -protein precursor and the mechanism of Alzheimer's disease.
Annu Rev Cell Biol
10:
373-403,
1994[ISI].
52.
Selkoe, DJ.
The cell biology of -amyloid precursor protein and presenilin in Alzheimer's disease.
Trends Cell Biol
8:
447-453,
1998[ISI][Medline].
53.
Sester, M,
Feuerbach D,
Frank R,
Preckel T,
Gutermann A,
and
Burgert HG.
The amyloid precursor-like protein 2 associates with the major histocompatibility complex class I molecule Kd.
J Biol Chem
275:
3645-3654,
2000
54.
Shioi, J,
Pangalos MN,
Ripellino JA,
Vassilacopoulou D,
Mytilineou C,
Margolis RU,
and
Robakis NK.
The Alzheimer amyloid precursor proteoglycan (appican) is present in brain and is produced by astrocytes but not by neurons in primary neural cultures.
J Biol Chem
270:
11839-11844,
1995
55.
Sisodia, SS.
Beta-amyloid precursor protein cleavage by a membrane-bound protease.
J Neurosci Res
31:
428-442,
1992[ISI][Medline].
56.
Sisodia, SS,
Koo EH,
Beyreuther K,
Unterbeck A,
and
Price DL.
Evidence that -amyloid protein in Alzheimer's disease is not derived by normal processing.
Science
248:
492-495,
1990[ISI][Medline].
57.
Skovronsky, DM,
Moore DB,
Milla ME,
Doms RW,
and
Lee VM.
Protein kinase C-dependent -secretase competes with
-secretase for cleavage of amyloid-
precursor protein in the trans-Golgi network.
J Biol Chem
275:
2568-2575,
2000
58.
Slack, BE,
Breu J,
Muchnicki L,
and
Wurtman RJ.
Rapid stimulation of amyloid precursor protein release by epidermal growth factor: role of protein kinase C.
Biochem J
327:
245-249,
1997[ISI][Medline].
59.
Slack, BE,
Breu J,
Petryniak MA,
Srivastava K,
and
Wurtman RJ.
Tyrosine phosphorylation-dependent stimulation of amyloid precursor protein secretion by the m3 muscarinic acetylcholine receptor.
J Biol Chem
270:
8337-8344,
1995
60.
Slack, BE,
Nitsch RM,
Livneh E,
Kunz GM, Jr,
Breu J,
Eldar H,
and
Wurtman RJ.
Regulation by phorbol esters of amyloid precursor protein release from Swiss 3T3 fibroblasts overexpressing protein kinase C alpha.
J Biol Chem
268:
21097-21101,
1993
61.
Sohn, UD,
Zoukhri D,
Dartt D,
Sergheraert C,
Harnett KM,
Behar J,
and
Biancani P.
Different protein kinase C isozymes mediate lower esophageal sphincter tone and phasic contraction of esophageal circular smooth muscle.
Mol Pharmacol
51:
462-470,
1997
62.
Solano, DC,
Sironi M,
Bonfini C,
Solerte SB,
Govoni S,
and
Racchi M.
Insulin regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase-dependent pathway.
FASEB J
14:
1015-1022,
2000
63.
Sprecher, CA,
Grant FJ,
Grimm G,
OHara PJ,
Norris F,
Norris K,
and
Foster DC.
Molecular cloning of the cDNA for a human amyloid precursor protein homolog: evidence for a multigene family.
Biochemistry
32:
4481-4486,
1993[ISI][Medline].
64.
Standaert, ML,
Bandyopadhyay G,
Perez L,
Price D,
Galloway L,
Poklepovic A,
Sajan MP,
Cenni V,
Sirri A,
Moscat J,
Toker A,
and
Farese RV.
Insulin activates protein kinases C- and C-
by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes.
J Biol Chem
274:
25308-25316,
1999
65.
Subramanian, S,
Fitzgerald M,
and
Bernfield M.
Regulated shedding of syndecan-1 and -4 ectodomains by thrombin and growth factor receptor activation.
J Biol Chem
272:
14713-14720,
1997
66.
Tanzi, R,
McClatchey A,
Gusella J,
and
Neve R.
Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease.
Nature
331:
528-530,
1988[ISI][Medline].
67.
Thiam, K,
Loing E,
Zoukhri D,
Rommens C,
Hodges R,
Dartt D,
Verwaerde C,
Auriault C,
Gras-Masse H,
and
Sergheraert C.
Direct evidence of cytoplasmic delivery of PKC-, -
and -
pseudosubstrate lipopeptides: study of their implication in the induction of apoptosis.
FEBS Lett
459:
285-290,
1999[ISI][Medline].
68.
Thinakaran, G,
and
Sisodia SS.
Amyloid precursor-like protein 2 (APLP2) is modified by the addition of chondroitin sulfate glycosaminoglycan at a single site.
J Biol Chem
269:
22099-22104,
1994
69.
Thinakaran, G,
Slunt HH,
and
Sisodia SS.
Novel regulation of chondroitin sulfate glycosaminoglycan modification of amyloid precursor protein and its homologue, APLP2.
J Biol Chem
270:
16522-16525,
1995
70.
Van der Geer, P,
Hunter T,
and
Lindberg R.
Receptor protein-tyrosine kinases and their signal transduction pathways.
Annu Rev Cell Biol
10:
251-337,
1994[ISI].
71.
Van Nostrand, WE,
Schmaier AH,
Siegel RS,
Wagner SL,
and
Raschke WC.
Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid -protein precursor.
J Biol Chem
270:
22827-22830,
1995
72.
Von der Kammer, H,
Loffler C,
Hanes J,
Klaudiny J,
Scheit KH,
and
Hansmann I.
The gene for the amyloid precursor-like protein APLP2 is assigned to human chromosome 11q23-q25.
Genomics
20:
308-311,
1994[ISI][Medline].
73.
Wasco, W,
Bupp K,
Magendantz M,
Gusella JF,
Tanzi RE,
and
Solomon F.
Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid beta protein precursor.
Proc Natl Acad Sci USA
89:
10857-10861,
1992[Abstract].
74.
Wasco, W,
Gurubhagavatula S,
Paradis MD,
Romano DM,
Sisodia SS,
Hyman BT,
Neve RL,
and
Tanzi RE.
Isolation and characterization of APLP2 encoding a homologue of the Alzheimer's associated amyloid beta protein precursor.
Nat Genet
5:
95-100,
1993[ISI][Medline].
75.
Yamazaki, T,
Koo EH,
and
Selkoe DJ.
Cell surface amyloid -protein precursor colocalizes with
1 integrins at substrate contact sites in neural cells.
J Neurosci
17:
1004-1010,
1997
76.
Zieske, JD,
Takahashi H,
Hutcheon AE,
and
Dalbone AC.
Activation of epidermal growth factor receptor during corneal epithelial migration.
Invest Ophthalmol Vis Sci
41:
1346-1355,
2000
77.
Zoukhri, D,
Hodges RR,
Dicker DM,
and
Dartt DA.
Role of protein kinase C in cholinergic stimulation of lacrimal gland protein secretion.
FEBS Lett
351:
67-72,
1994[ISI][Medline].
78.
Zoukhri, D,
Hodges R,
Sergheraert C,
Toker A,
and
Dartt D.
Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion.
Am J Physiol Cell Physiol
271:
C263-C269,
1996.