A role for MAP kinase in regulating ectodomain shedding of APLP2 in corneal epithelial cells

Ke-Ping Xu1, Driss Zoukhri1, James D. Zieske1, Darlene A. Dartt1, Christian Sergheraert2, Estelle Loing2, and Fu-Shin X. Yu1

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
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ABSTRACT
INTRODUCTION
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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-epsilon -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-epsilon 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 Abeta 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-alpha (TNF-alpha ) and interleukin-6; growth factors/cytokines such as TNF-alpha , transforming growth factor-alpha (TGF-alpha ), 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 alpha -secretase to generate a large secreted form and nonamyloidogenic carboxy-terminal fragments or by a beta -secretase to generate Abeta (reviewed in Ref. 51). Processing of APP by alpha -secretase is known as ectodomain shedding and precludes Abeta 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-delta 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-alpha and -epsilon , but not PKC-delta , 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 alpha -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-epsilon isozyme might be involved in PMA-induced APLP2 shedding.


    MATERIALS AND METHODS
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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.

The D2II anti-extracellular domain of APLP2 was a gift from Drs. Gopal Thinakaran and Sangram S. Sisodia (Dept. of Pharmacological and Physiological Sciences, University of Chicago) (68). Antibodies against extracellular-signal related kinase (ERK) 2 (p42 MAPK) and phosphorylated ERK1/2 (p42/p44 MAPK) were from Santa Cruz Biotechnology (Santa Cruz, CA).

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-alpha beta gamma (myr-PKC-alpha beta gamma ; a 9-amino acid peptide common to all three PKC isozymes), PKC-alpha (myr-PKC-alpha , a 14-amino acid peptide unique to PKC-alpha ), PKC-delta (myr-PKC-delta ), and PKC-epsilon (myr-PKC-epsilon ) 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.

To determine whether THCE cells are permeable to the different inhibitor peptides, we biotinylated the myristoylated peptides (67). THCE cells cultured in eight-chamber wells were incubated for 5 or 15 min at 37°C with 5 µM of different biotinylated PKC pseudosubstrate peptides. Cells were washed twice with PBS and then fixed with PBS containing 2.5% paraformaldehyde and 0.01% glutaraldehyde for 15 min at room temperature. Fixed cells were permeabilized with 0.3% Triton X-100 in PBS for 10 min and blocked for 10 min at room temperature with PBS containing 1.5% bovine serum albumin (BSA). Cells were then incubated for 1 h at room temperature with FITC-streptavidin and rhodamine-phalloidin in PBS containing 1.5% BSA for 1 h. The slides were mounted with Vectashield mounting medium with DAPI and examined under a Nikon Eclipse E-800 fluorescence microscope equipped with a Spot digital camera.

To assess the role of MAPK, we pretreated THCE cells with 10 µM U-0126, a potent and specific inhibitor of the upstream MAPK regulator, MAPK kinase (MEK) 1/2, for 15 min and then treated with PMA or EGF for 10 min. The stimulation was stopped by the addition of 5 vols of ice-cold KBM. The cells were then washed with PBS, collected in lysis buffer, and subjected to Western blotting using a mouse monoclonal antibody against phosphorylated ERK1/2 and a mouse monoclonal antibody against ERK2.

To determine the function of ERK phosphorylation, we carried out PMA or EGF stimulation for 16 h in the presence of U-0126. At the end of incubation, culture media were collected and APLP2 secretion was determined by Western blotting.

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.


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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.


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Fig. 1.   Phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF) accelerate amyloid precursor-like protein 2 (APLP2) shedding. A: representative Western blot of cell-associated APLP2 and secreted APLP2. SV40-transfected human corneal epithelial (THCE) cells were starved overnight in keratinocyte basic medium (KBM) and then treated with PMA (1 µM) or EGF (20 ng/ml) in KBM for 16 h. KBM alone was used as control (Cont). The harvested conditioned media were concentrated, separated by SDS-PAGE, and probed with DII2 antiserum. B: image analysis of the effect of PMA or EGF on APLP2 shedding (appeared in medium). There were significant increases of secreted APLP2 (the sum of 3 APLP2 immunoreactive bands) in cultured media after PMA or EGF treatment (**P < 0.01) compared with control. Data are means ± SE of 3 experiments.

Using this assay, we quantified PMA- and EGF-stimulated cleavage of transmembrane APLP2 and ectodomain release (Fig. 1B) by measuring the relative amount of APLP2 in culture medium (the sum of 3 shifted bands). Shedding of APLP2 in PMA- and EGF-treated THCE cells was significantly increased as assessed by Western blotting and image analysis. Treatment with PMA or EGF resulted in a large increase (5- or 3-fold, respectively, P < 0.01) in secreted APLP2. Concomitant with this increase, a decrease of the cellular transmembrane APLP2 in PMA- or EGF-treated THCE cells was evident compared with control (Fig. 1A).

A time-course analysis showed that PMA increased soluble APLP2 immunoreactivity in the culture medium starting within 15 min (Fig. 2A), resulting in a significant increase of APLP2 secretion over basal levels in a time-dependent manner (P < 0.05). The highest level of APLP2 accumulation in the culture medium was observed at 12 h. The effect of PMA was also concentration dependent, with 1 µM PMA treatment resulting in a 4.8-fold increase in shedding over the control (Fig. 2B).


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Fig. 2.   Time and concentration dependence of PMA-induced ectodomain shedding of APLP2. A: THCE cells were stimulated with PMA (1 µM) for the indicated times. Secreted APLP2 was assessed as described in Fig. 1. Basal secretion of APLP2 in culture medium without PMA stimulation was also evaluated. There were significant increases of APLP2 shedding over basal levels upon stimulation with PMA (*P < 0.05). Data are means ± SE of 6 experiments. B: THCE cells were stimulated for 16 h with indicated concentrations of PMA.

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).


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Fig. 3.   PMA-induced shedding of APLP2 correlates with the loss of cell surface staining of APLP2. A: THCE cells were incubated with or without (control) PMA for 15 min and then stained with DII2 antibody without permeabilization. The bound antibodies were visualized after incubation with FITC-conjugated goat anti-rabbit IgG. Bar, 10 µm. B: THCE cells were treated with no addition (control), PMA, or EGF for 15 min, sequentially probed with DII2 and FITC- conjugated antibody, and finally analyzed by flow cytometry. The mean fluorescence intensity for each sample is shown beside the key. PMA and EGF treatment decreased mean fluorescence intensity of APLP2.

Loss of cell surface APLP2 immunoreactivity was also assessed by flow cytometry (Fig. 3B). After PMA or EGF treatment, the relative cell number at high-intensity fluorescence peak (102 to 103) was decreased, concomitant with an increase in cell number at low-intensity fluorescence peaks, indicating APLP2 cleavage and consequent release upon PMA or EGF stimulation.

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.


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Fig. 4.   A nonselective protein kinase inhibitor, staurosporine, blocked PMA-activated shedding of APLP2. Growth factor-starved THCE cells were pretreated with staurosporine (40 nM, 4 min) and then washed with basic culture medium. PMA (1 µM) was added for 16 h. The conditioned medium was subjected to Western blotting analysis with DII2 antibody. A: representative Western blotting of APLP2 fragments released alone (control) or in the presence of staurosporine pretreatment with (P+ST) or without PMA (ST). B: image analysis of the effect of staurosporine on basal and PMA-induced APLP2 release. The increase of APLP2 shedding in the presence of PMA was significantly inhibited by staurosporine (*P < 0.05). Data are means ± SE of 3 experiments.

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-alpha beta gamma , -alpha , -delta , and -epsilon 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-alpha and -epsilon ). These data suggest that the THCE cells are equally permeable to the different myristoylated peptide inhibitors.


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Fig. 5.   THCE cells were permeable to myristoylated peptides derived from the pseudosubstrate domains of specific protein kinase C (PKC) isoforms. THCE cells were treated with biotinylated myristoylated pseudosubstrate peptides of PKC-alpha (middle) or PKC-epsilon (right), with no peptides added as control (left), for 5 min and then fixed and permeabilized as described in MATERIALS AND METHODS. Cellular localization of biotin-conjugated peptides was revealed with FITC-streptavidin, F-actin with rhodamine-phalloidin, and nuclei with 4',6-diamidino-2-phenylindole (DAPI). Triple exposure shows the overlapping fluorescence of the myristoylated pseudosubstrate peptides (green), microfilaments (red), and nuclear DAPI staining (blue).

PMA-induced shedding was significantly inhibited by the myristoylated peptide corresponding to the pseudosubstrate sequence of PKC-epsilon (myr-PKC-epsilon , 37% decrease compared with PMA, P < 0.05) but was not inhibited by myr-PKC-alpha beta gamma and -alpha (Fig. 6). Together, these data suggest that PKC-epsilon is involved in stimulating APLP2 shedding, whereas PKC-alpha , -beta , and -gamma are not. In a different set of experiments, myr-PKC-delta either inhibited APLP2 shedding as effectively as myr-PKC-epsilon or did not inhibit it; the reason for this variability is not clear.


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Fig. 6.   Myristoylated pseudosubstrate peptide of PKC-epsilon (myr-PKC-epsilon ) inhibits PMA-induced APLP2 shedding in THCE cells. A: representative Western blot of PMA-induced APLP2 accumulation in conditioned media of THCE cells treated with myr-PKC-alpha beta gamma , -alpha , -delta , and -epsilon . Growth factor-starved THCE cells were pretreated with myr-PKC peptides (1 µM) for 60 min, and then PMA was added along with myr-PKC peptides for 16 h. Conditioned media were collected, concentrated, and probed with D2II antibody. B: image analysis of the effect of myr-PKC on PMA-induced APLP2 release. The increase of APLP2 shedding in the presence of PMA was significantly inhibited only by myr-PKC-epsilon (*P < 0.05). Data are means ± SE of 3-6 experiments.

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).


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Fig. 7.   U-0126 inhibits PMA- or EGF-induced extracellular signal-regulated kinase (ERK) activation. A: representative Western blot of ERK phosphorylation by PMA or EGF. Serum-starved THCE cells were pretreated with (+) or without (-) U-0126 (10 µM, 15 min) and then stimulated with (+) or without (-) PMA (1 µM) or EGF (20 ng/ml) for 10 min. Cell lysates were subjected to Western blotting analysis with ERK2 or phosphorylated ERK (pERK, p44 and p42) antibodies. B: image analysis of mitogen-activated protein kinase (MAPK) phosphorylation induced by PMA or EGF. Results are expressed as the ratio of phosphorylated ERK1/2 to ERK2 immunoreactivity (pERK/ERK). PMA- or EGF-induced increase in the phosphorylation state of ERK was significantly antagonized by U-0126, a pharmacological inhibition of MAPK kinase (MEK) (**P < 0.01 and *P < 0.05 for PMA and EGF, respectively). Data are means ± SE of 3 experiments.

Addition of U-0126 (10 µM) also reduced PMA- or EGF-induced soluble APLP2 to a level similar to or slightly below the basal level (Fig. 8, P < 0.05), suggesting that both PMA- and EGF-induced cleavage of transmembrane APLP2 are due to activation of the MAPK pathway. A similar effect was also observed with PD-98059, another specific inhibitor of MEK (data not shown). Inhibition of the MAPK pathway also significantly affected the basal level of APLP2 cleavage in the absence of exogenous stimulation. Together, these results show that MAPK phosphorylation is related to APLP2 accumulation in the cultured medium of control, PMA- or EGF-treated THCE cells.


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Fig. 8.   U-0126 inhibits PMA- or EGF-induced APLP2 shedding. A: representative Western blot of the effect of U-0126 on PMA- or EGF-stimulated APLP2 accumulation in conditioned media for 16 h. THCE cells were treated with (+) or without (-) U-0126 (10 µM) for 15 min before the addition of PMA (1 µM) or EGF (20 ng/ml). APLP2 secretion was detected by D2II antibody. B: image analysis of APLP2 shedding. PMA- and EGF-induced, as well as basal (control), secretion of APLP2 in THCE cells was significantly blocked by the MEK inhibitor U-0126 (*P < 0.05). Data are means ± SE of 3 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-epsilon , and probably PKC-delta , 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-alpha , -beta I, -delta , and -epsilon , 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-epsilon inhibitor significantly blocked PMA-induced shedding of APLP2 in THCE cells, whereas myr-PKC-alpha beta gamma and -alpha had no apparent effects, suggesting that PKC-epsilon is utilized in PMA-induced shedding of APLP2. Izumi et al. (27) showed that PKC-delta is involved in PMA-induced ectodomain shedding of HB-EGF. We also observed in some experiments that PKC-delta myristoylated pseudosubstrate peptide was as effective as PKC-epsilon pseudosubstrate. However, sometimes PKC-delta pseudosubstrate exhibited no effect on PMA-induced ectodomain shedding. The reason for the inconsistent results of PKC-delta 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-epsilon 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-alpha in APP secretion in human Ntera 2 neurons and in CHO cells. Several other studies also reported the involvement of PKC-alpha in regulation of APP secretions in fibroblast cells (4, 28, 30, 60). It is not clear whether the difference in PKC-alpha 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 alpha -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-alpha , 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-alpha -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 alpha -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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 alpha -secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem 273: 27765-27767, 1998[Abstract/Free Full Text].

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[Free Full Text].

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 beta -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-alpha and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades. EMBO J 18: 6962-6972, 1999[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

18.   Goedert, M, Sisodia SS, and Price DL. Neurofibrillary tangles and beta -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[Abstract/Free Full Text].

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 beta -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[Abstract/Free Full Text].

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-gamma /ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J 17: 7260-7272, 1998[Abstract/Free Full Text].

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)-alpha and novel PKCepsilon , but not -delta , 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 alpha -secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. Proc Natl Acad Sci USA 96: 3922-3927, 1999[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

41.   Nitsch, RM, Deng A, Wurtman RJ, and Growdon JH. Metabotropic glutamate receptor subtype mGluR1alpha stimulates the secretion of the amyloid beta -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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

51.   Selkoe, D. Cell biology of the amyloid beta -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 beta -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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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 beta -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 alpha -secretase competes with beta -secretase for cleavage of amyloid-beta precursor protein in the trans-Golgi network. J Biol Chem 275: 2568-2575, 2000[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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-zeta and C-lambda 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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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-alpha , -epsilon and -zeta 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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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 beta -protein precursor. J Biol Chem 270: 22827-22830, 1995[Abstract/Free Full Text].

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 beta -protein precursor colocalizes with beta 1 integrins at substrate contact sites in neural cells. J Neurosci 17: 1004-1010, 1997[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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.


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