Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.
- myristoylated alanine-rich C kinase substrate
- heat shock protein 70
myristoylated alanine-rich c kinase substrate (MARCKS) is a protein kinase C (PKC) substrate implicated in diverse biological processes including cell motility, phagocytosis, membrane trafficking, and secretion (3, 12, 24). Under normal conditions, intracellular MARCKS is attached to the cytoplasmic face of the plasma membrane. When phosphorylated by activated PKC, MARCKS translocates from the plasma membrane to the cytoplasm (2, 11). Previously, our laboratory demonstrated that MARCKS is a key regulatory protein in the mechanism of mucin secretion in airway epithelial cells. Upon phosphorylation by agents that stimulate secretion, phospho-MARCKS moves into the cytoplasm, where it binds to membranes of mucin granules that are stored in preparation for exocytotic release. Binding of MARCKS to these granule membranes appears to be a critical component of the mucin secretory pathway, as inhibition of binding correlates with decreased secretion (25).
The exact cellular mechanism(s) whereby MARCKS regulates mucin secretion, and especially other proteins that may interact with MARCKS in this process, have not been elucidated. Recently, association between MARCKS and the intracellular chaperone, heat shock protein 70 (HSP70) was demonstrated in airway epithelial cells (18, 21). This MARCKS-HSP70 complex appears to be involved in the MARCKS-regulated secretory mechanism because siRNA knockdown of HSP70 in a virally transformed human airway epithelial cell line attenuated mucin secretion (21).
In this report, mechanisms of interaction(s) between MARCKS and HSP70 were investigated utilizing well-differentiated primary cultures of normal human bronchial epithelial (NHBE) cells. A small-molecule HSP70 inhibitor, MAL3-101 (5, 8), and siRNA techniques in primary cells were utilized to examine effects of attenuating HSP70 function on mucin secretion and binding of HSP70 to MARCKS and to another chaperone also implicated in secretion, cysteine string protein (CSP). Previous studies have implicated the formation of a trimeric complex between MARCKS, HSP70, and CSP as important in the mechanism of mucin secretion, with CSP being a component of the mucin granule membrane and HSP70 binding to MARCKS and serving to target it to the granule membrane via interactions with CSP (12, 18, 21). In addition, intracellular trafficking of MARCKS and HSP70 in living NHBE cells was investigated via confocal microscopy utilizing fluorescently labeled HSP70 and MARCKS constructs. Related work published previously from this laboratory relied mostly on experiments done in virally transformed epithelial cell lines (18, 21). However, signaling pathways in these cell lines often are aberrant, and validation in primary cells is required. This is the first study to show MARCKS trafficking in living airway epithelial cells and the role of HSP70 in this process, as well as attenuation of mucin secretion by a pharmacological antagonist to HSP70. The results indicate that interactions between MARCKS and HSP70 (and CSP) appear to be integral components of the mucin secretory process in well-differentiated human airway epithelial cells.
MATERIALS AND METHODS
The small molecule pyrimidinone-peptoid HSP70 modulator MAL3-101 (8) used in this study was a gift from Dr. Peter Wipf (University of Pittsburgh, Pittsburgh, PA). It was solubilized in DMSO, first to a stock solution of 20 mg/ml and then further diluted to the indicated experimental concentrations. Antibodies against MARCKS (clone 2F12; mouse monoclonal) and CSP (rabbit polyclonal) were from Millipore (Billerica, MA). Mouse monoclonal anti-HSP70 antibody was from Affinity BioReagents (Golden, CO). Mouse monoclonal anti-green fluorescent protein (GFP) antibody was from Abcam (Cambridge, MA). Horseradish peroxidase (HRP)-conjugated goat anti-actin and HRP-conjugated goat anti-mouse IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated goat anti-rabbit antibody was from Cell Signaling (Danvers, MA). Chemically synthesized double-stranded siRNA duplexes with 2-nt 3′-dTdT overhangs were purchased from Dharmacon (Lafayette, CO) for the target gene HSP70 (HSP70 siRNA), target sequence (5′CTG GCC TTT CCA GGT GAT CAA3). An irrelevant RNA duplex used as a control siRNA (sequence 5′-AAUUCUCCGAA CGUGUCACGU-3′) with no significant homology to any mammalian gene sequence (CTL siRNA) was purchased from Ambion (Austin, TX). Silencing was quantified by immunoblotting.
Cell Culture and Transfection
Expansion, cryopreservation, and culture of NHBE cells in air-liquid interface were performed as described previously (15). Briefly, NHBE cells (Cambrex, San Diego, CA) were seeded in vented T75 flasks (500 cells/cm2) and cultured until cells reached 85–90% confluence. Cells then were dissociated by trypsin/EDTA and frozen as passage 2. Air-liquid interface culture was initiated by seeding passage 2 cells (2 × 104 cells/cm2) in Transwell clear culture inserts (Costar, Cambridge, MA) thinly coated with rat tail collagen, type I (Collaborative Biomedical, Bedford, MA). Cells were cultured submerged in medium at 37°C in an atmosphere of 5% CO2 for 5–7 days until nearly confluent. At that time, an air-liquid interface was created by removing the apical medium and feeding cells basolaterally. Medium was changed daily thereafter. Cells were cultured for an additional 14 days to allow full differentiation before being used for the indicated studies.
For studies involving transfections, NHBE passage 2 cells were directly seeded onto collagen-coated 35-mm plastic dishes with glass bottoms (MatTek, Ashland, MA) or plastic culture plates and cultured until cells reached 50–70% confluence. Cells were then transfected with the plasmids described below according to the manufacturer's instructions using FuGene6 reagent (Roche, Indianapolis, IN) or with double-stranded siRNAs targeting HSP70 or control siRNA by using the DharmaFECT DuoTransfection reagent (from Dharmacon) (1). After 48 h, cells were harvested and equivalent amounts of proteins separated by SDS/PAGE for immunoblot analysis. Other cells expressing fluorescently tagged proteins were directly processed for laser-scanning microscopy using a Zeiss LSM-510.
Measurement of Mucin Secretion by ELISA
Before collection of baseline and test mucin samples, accumulated mucin at the apical surface of the cells was removed by a wash with PBS, pH 7.2, containing 1 mM dithiothreitol. To collect the baseline secretion, cells were incubated with medium alone for 30 min, and secreted mucin in the apical medium was collected and reserved. Cells were rested for 24 h and then exposed to medium containing the selected stimulatory and/or inhibitory reagents (or appropriate controls) for a 15- or 30-min period, after which secreted mucin was collected and reserved as the test sample. Both baseline and test secretions were analyzed by double-sandwich ELISA using the pan-mucin antibody 17Q2 (1:1,000 dilution; Covance, Berkeley, CA) as the primary antibody (17). The ratio of test/baseline was used to quantify mucin secretion, allowing each culture well to serve as its own control and thus minimizing deviation caused by variability among culture wells. Levels of mucin secretion were reported as percentages of the medium or solvent control as reported previously (16).
Immunoprecipitation was performed using Dynal beads coated with protein A according to the manufacturer's instructions (Dynal, Great Neck, NY). Total protein was extracted from cells using an immunoprecipitation lysis buffer specifically designed to maintain protein-protein interactions (20 mM sodium phosphate, pH 7.5; 500 mM NaCl; 0.1% SDS; 1% NP-40; and protease inhibitors). Proteins were diluted to ∼1 mg/ml using PBS, and 5–10 μl of antibody was added to 1 ml cell lysate. The sample was incubated overnight at 4°C with gentle shaking, and an appropriate amount of Dynal beads coated with protein A was added to the antigen-antibody complex (∼50 μl of gel per 5 μg of antibody). The sample was incubated with gentle mixing for 2 h at room temperature, and the immobilized protein A-bound complexes were washed three times with 0.5 ml of the lysis buffer. Enhanced chemiluminescence reagents were used for antibody detection after blotting to nitrocellulose membranes. All immunoblots were quantified by densitometry with LabWorks (UVP, Upland, CA).
Design of DNA Constructs
Full-length MARCKS (4) and HSP70 were amplified by PCR to encode BamHI and EcoRI restriction sites at the 5′ and 3′ ends, respectively, with the terminator codon replaced with a sequence encoding a diglycine linker. The products were cut, purified, and ligated into a pEYFP vector (Clontech, Mountain View, CA) to generate a full-length MARCKS-yellow fluorescent protein (YFP) construct or into a pcDNA3.1-m cyan fluorescent protein (CFP) vector to generate a HSP70-mCFP construct. Both constructs were confirmed by restriction enzyme double digestion and DNA sequencing.
NHBE cells were seeded onto collagen-coated 35-mm plastic dishes with glass bottoms (MatTek) and cultured overnight. Cells were then transfected with MARCKS-YFP or and/or HSP70-CFP. After 48 h, cells expressing YFP- and/or CFP-tagged protein images were identified on either a Zeiss laser-scanning microscope (LSM-510) or an Olympus 1X81. Trafficking of MARCKS-YFP and HSP70-CFP, by living or fixed cells, was followed constitutively or after addition of stimulatory/inhibitory agents. All living cell imaging experiments were performed at 37°C.
Cytotoxicity of all reagents used was assessed using CytoTox 96 kits according to the manufacturer's instructions (Promega, Madison, WI). All experiments were performed with reagents at nontoxic concentrations.
Data were analyzed for significance using one-way ANOVA with Bonferroni posttest corrections. Differences between treatments were considered significant at P < 0.05.
The HSP70 Inhibitor, MAL3-101, Attenuates PMA-Stimulated Mucin Secretion
NHBE cells were preincubated with MAL3-101 (100 or 300 μM) for 15 min; phorbol 12-myristate 13-acetate (PMA) (100 nM) was then added, and cells incubated for an additional 30 min, at which time mucin secretion was measured by ELISA. As illustrated in Fig. 1, preincubation with MAL3-101 suppressed PMA-stimulated mucin secretion. The effects of MAL3-101 were not related to cytotoxicity based on results from Promega CytoTox 96 LDH release/retention assays (data not shown).
PMA-Enhanced Binding of HSP70, MARCKS, and CSP to Each Other in NHBE Cells is Inhibited by Preincubation with the HSP70 Inhibitor, MAL3-101
NHBE cells were preincubated with MAL3-101 (0–300 μM) for 15 min and then exposed to PMA (100 nM) for an additional 10 min. At this time, cell lysates were immunoprecipitated with the indicated antibody, resolved by SDS-PAGE, and immunoblotted with the second indicated antibody to detect coprecipitated proteins. As we have shown previously that both HSP70 and a second chaperone, CSP, are involved in the mucin secretion process (18, 21), we investigated whether PMA also enhanced binding of MARCKS and/or HSP70 to CSP. As illustrated in Fig. 2, PMA increased associations of each of the proteins with the others in the absence of MAL3-101; pretreatment with MAL3-101 attenuated, in a concentration-dependent manner, PMA-induced associations of MARCKS and HSP70 (Fig. 2A), MARCKS and CSP (Fig. 2B), and HSP70 and CSP (Fig. 2C).
MARCKS and HSP70 Trafficking in Living NHBE Cells
Protein expression and translocation in transfected NHBE cells.
To visualize localization and trafficking of MARCKS and HSP70 in living NHBE cells, we constructed a human full-length MARCKS construct tagged at the carboxyl terminus with YFP and a human full-length HSP70 construct tagged at the carboxyl terminus with CFP for transfection into NHBE cells for visualization by confocal microscopy. Fluorescence confirmed that each construct was transiently transfected into NHBE cells. Posttransfection, cells were lysed in RIPA buffer, and expression of each fusion protein was assayed by immunoblotting. The proteins ran at the predicted sizes (data not shown).
PMA promotes MARCKS translocation from plasma membrane to cytoplasm in living NHBE cells but does not affect HSP70 cytoplasmic localization.
MARCKS-YFP was expressed predominantly at the plasma membrane in NHBE cells after transfection (Fig. 3A). Treatment of cells with 100 nM PMA for 10 min led to internalization of much of the MARCKS-YFP into the cytoplasm (Fig. 3B). In contrast, HSP70-CFP was visualized throughout the cytoplasm, and treatment with 100 nM PMA did not appear to affect its cellular localization (Fig. 3, C and D).
Effects of HSP70 Inhibition on MARCKS Translocation in Living NHBE Cells
Treatment with MAL3-101.
Cells were preincubated with MAL3-101 (300 μM) for 15 min and then exposed to PMA (100 nM) for an additional 10 min, at which time the intracellular localization of MARCKS-YFP was observed. As indicated in Fig. 4, treatment with MAL3-101 appeared to inhibit translocation of MARCKS from plasma membrane to cytoplasm.
Treatment with HSP70 siRNA.
To modulate endogenous HSP70 levels, chemically synthesized double-stranded siRNA duplexes with 2-nt 3′-dTdT overhangs targeting HSP70 were used. NHBE cells were transfected with MARCKS-YFP and either HSP70 siRNA or a nontargeting control siRNA (CTL siRNA). Transfection of NHBE cells with HSP70 siRNA caused a concentration-dependent decrease in HSP70 levels that reached 70–80% at 200 nM; the control siRNA had no effect on HSP70 levels at these concentrations (Fig. 5, A and B). After 48 h, living NHBE cells were visualized for translocation of MARCKS-YFP via confocal microscopy. As illustrated in Fig. 5, C and D, transfection of cells with siRNA directed against HSP70 inhibited PMA-induced movement of MARCKS-YFP to the cytoplasm. In contrast, MARCKS-YFP expressed in cells transfected with control siRNA was localized to the plasma membrane and translocated to the cytoplasm after stimulation with PMA (Fig. 5, E and F).
Confocal Imaging of Cotransfected NHBE Cells Shows MARCKS-HSP70 Interactions
NHBE cells were cotransfected with MARCKS-YFP (green) and HSP70-CFP (red) and examined under confocal microscopy with and without stimulation with 100 nM PMA for 30 min before fixation. These studies were done with fixed cells, not live cells, as in the other experiments illustrated in Figs. 3⇑–5. Thus the exposure time to PMA was 30 min rather than 10 min as in living cells because studies done with living cells in our hands need to be done within 10–15 min, as cells tend to move out of the field of focus as well as lose some of their fluorescence. Thirty-minute exposure was chosen to allow for full interactions between the proteins. As indicated in Fig. 6, A and B, HSP70-CFP appears to be localized in the cytoplasm, and MARCKS-YFP is located primarily on the plasma membrane in resting cells, with little apparent interaction between these proteins (Fig. 6C). After exposure to PMA, HSP70 remains cytoplasmic although appearing somewhat more punctuate and perinuclear (Fig. 6D), whereas much of the MARCKS-YFP translocates to the cytoplasm (Fig. 6E). The merged image of these cells (Fig. 6F) shows many areas of orange (combination of green and red) reflecting colocalization of MARCKS-YFP and HSP70-CFP in the cytoplasm. As illustrated in Fig. 6G, this association of MARCKS-YFP and HSP70-CFP in the cytoplasm after PMA treatment was also demonstrated by coimmunoprecipitation:immunoprecipitation with an antibody to HSP70, and immunoblotting with an antibody to GFP (cross reacts with YFP) shows greatly enhanced binding of MARCKS-YFP with HSP70 (both exogenous and endogenous) after exposure to PMA.
Our laboratory was the first to demonstrate that MARCKS plays a major role in secretion of mucin from airway epithelial cells (16). The results of the studies presented here indicate that the intracellular chaperone protein, HSP70, is critical to MARCKS function in mucin secretion by these cells. With the use of well-differentiated NHBE cells, the results indicate that MARCKS associates with HSP70 when the cells are stimulated to secrete via activation of PKC by exposure to PMA. The importance of this interaction is demonstrated by studies using a small molecule pyrimidinone-peptoid hybrid HSP70 inhibitor, MAL3-101. Treatment of NHBE cells with MAL3-101 1) attenuates mucin secretion stimulated by exposure of the cells to PMA, as determined by ELISA, 2) attenuates PMA-enhanced interactions between HSP70, MARCKS, and CSP, as determined by coimmunoprecipitation, and 3) attenuates translocation of MARCKS from plasma membrane to cytoplasm and subsequent association with HSP70, as determined by confocal microscopy of NHBE cells transfected with fluorescently tagged MARCKS and HSP70 constructs. Additional studies utilizing siRNA against HSP70 showed that attenuating expression of HSP70 inhibited MARCKS translocation from membrane to cytoplasm in these cells, similar to the effects of MAL3-101. Thus we present strong evidence that HSP70 plays a major role in the mechanism of airway epithelial cell mucin secretion, and interactions between HSP70 and MARCKS appear key to this role.
In the mucin secretion studies reported in Fig. 1, looking at effects of MAL3-101 on stimulated mucin secretion by NHBE cells, we added 100 μM of 1,8-bromo-cGMP to enhance the stimulatory response. In previous studies, we found that PMA was a moderate enhancer of mucin secretion when added by itself, but that coaddition of 1,8-bromo-cGMP acted in a seemingly synergistic manner to optimally enhance the secretory response; thus maximal mucin secretion is generated with addition of both PMA and 8-bromo-cGMP (16). The reason for this is that 8-bromo-cGMP acts to increase production of the cGMP-NO pathway in these cells, which results in suppression of production of protein phosphatase type 2a, having the net effect of maintaining MARCKS phosphorylation and enhancing the stimulation of mucin secretion (16). Thus, in studies dealing with mucin secretion, we added the two kinase stimulators together to maximize the secretory response to better analyze the potential inhibitory effect of the HSP70 inhibitor, MAL3-101, as illustrated in Fig. 1.
In addition, these secretion studies were performed using an “artificial” PKC activator, PMA, to elicit a response. A question that could arise would be whether this mechanism also holds true when stimulation for secretion occurs with biologically relevant secretagogues. In previous studies, both in vitro, using uridine triphosphate (16) or human neutrophil elastase (HNE) (22) as a stimuli, and in vivo with HNE as a secretagogue (10), similar results implicating MARCKS protein in the response were obtained.
How do the MARCKS-HSP70 (and CSP) interactions regulate airway mucin secretion? HSPs, such as HSP70 (and its constitutive form, HSC70), belong to a family of molecular chaperones involved in a number of essential processes in cells, including protein folding, prevention of protein aggregation, direction of subcellular traffic, and rearrangement of multiple proteins, as well as exocytosis in different cell types (19). In previous studies, a role for HSP70 in airway mucin secretion was suggested, as siRNA-induced knockdown of HSP70 in the virally transformed human bronchial HBE1 cell line decreased mucin secretion. In those studies, it was observed that HSP70 appeared to form an intracellular trimeric protein complex with MARCKS and CSP after PMA stimulation (18, 21). Formation of this protein complex, however, did not appear to be a simple physiological event, whereas binding of MARCKS to HSP70 was shown to be a direct interaction between the two proteins. MARCKS would only bind to CSP after it first associated with HSP70, indicating that MARCKS-CSP binding is indirect and requires the presence of HSP70 (18).
The results reported here support the above concept. It is known that there is a specific and direct binding of CSP to HSP70 (and HSP70/HSC70 only, not any of the other HSPs) (26). The binding of CSP to HSP70 involves the “J” domain on the NH2 terminus of CSP, a 70-amino-acid region of homology shared with bacterial DnaJ, which specifically binds to HSP70-like proteins via a highly conserved histidine-proline-aspartic acid (HPD) motif and stimulates HSP70 ATPase activity (6, 27, 30). Thus DnaJ binding stimulates the switch of HSP70 from an ATP-bound state to an ADP-bound state, which stabilizes the binding of HSP70 to a “client” peptide (20). HSP70-like molecules contain three discrete domains that appear independent: there is a conserved 44-kDa ATPase domain at the NH2 terminus, an 18-kDa peptide-binding domain, and a 10-kDa helical lid domain at the COOH terminus. When ATP binds to the NH2-terminal domain, it stimulates a conformational change that exposes a peptide-binding channel, which then facilitates rapid binding and release of substrates by HSP70; when these substrates are released, there is a concomitant exchange of ADP for ATP (8, 9, 31).
DnaJ proteins are the largest class of HSP70 cochaperones, with more than 40 members in human cells (27). The compound MAL3-101, developed as a possible antitumor agent (since HSPs are highly expressed in proliferating tumor cells), is a small (∼930 kDa), membrane-permeable, pyrimidinone-peptoid hybrid molecule that modulates activity of HSP70 by specifically inhibiting J-domain stimulated HSP70 ATPase activity, but it does not affect other endogenous actions of HSP70 (5, 8, 23, 28, 29). The results reported here appear to reflect this action. Treatment of NHBE cells with MAL3-101 attenuated association of HSP70 with MARCKS and inhibited the ability of MARCKS to translocate to the cytoplasm. Because CSP appears to be a cytoplasmic protein associated with secretory granule membranes (21), the inhibitory effect of MAL3-101 on MARCKS-CSP interactions implicates indirect binding of MARCKS to CSP via HSP70 as a critical step in MARCKS regulation of mucin secretion in airway epithelium, as indicated above.
To look further at MARCKS and HSP70 intracellular trafficking, fluorescently tagged constructs of both proteins were transfected into living NHBE cells, and their movement and associations were monitored by confocal microscopy. MARCKS-YFP translocated from plasma membrane to cytoplasm within 10 min of PMA exposure. The HSP70-CFP was expressed in the cytoplasm, where it appeared associated with MARCKS after PMA stimulation (Fig. 6). Interestingly, MAL3-101 attenuated MARCKS translocation into the cytoplasm and resultant MARCKS-HSP70 associations, as did treatment of cells with HSP70 siRNA. MAL3-101 did not affect the cytoplasmic localization of HSP70.
Thus the key finding in this paper is that, in NHBE cells, MARCKS requires the presence of intact and functional HSP70 to translocate from membrane to cytoplasm and to regulate the process of mucin secretion. Without the ability to bind to HSP70, MARCKS either does not move from the membrane to the cytoplasm, or perhaps just cycles back to reattach to the plasma membrane. In previous studies, we have shown that MARCKS phosphorylation occurs within seconds after exposure of NHBE cells to PMA and that this phosphorylation is rapidly reversed so that by 10–15 min postexposure, MARCKS is no longer heavily phosphorylated and thus free to reattach to membranes, be they intracellular membranes such as those surrounding mucin granules or back to the plasma membrane (18, 22). One function of HSP70 in these cells could be to bind to MARCKS once it is dephosphorylated and becomes cytoplasmic and to keep it in the cytoplasm. Another possibility is that MARCKS simply requires the presence of active HSP70 to translocate to the cytoplasm and form the trimeric complex with CSP. The actual hypothetical mechanism by which HSP70, CSP, and MARCKS regulate mucin granule translocation and exocytotic secretion is illustrated in Fig. 1 of Ref. 12.
The results support this hypothesis in that the “purpose” of HSP70 in the MARCKS-regulated mucin secretory pathway appears to be, at least in part, to mediate and perhaps facilitate MARCKS translocation to the cytoplasm, where it is free to interact with membranes of intracellular mucin granules. As described above, HSP70 has a distinct and specific affinity for CSP, so an HSP70-MARCKS complex could be specifically targeted toward CSP due to this attraction. Because CSP is located in large part on membranes of intracellular mucin granules (21), these interactions could provide discrete binding sites for MARCKS on these granule membranes, allowing MARCKS to attach via its NH2-terminal region. Because MARCKS also binds actin and myosin at another site on the molecule (13, 16, 18), MARCKS could theoretically serve as a cross bridge between the cytoskeleton and mucin granules; thus contraction of the cytoskeleton would move the MARCKS-bound granule to the cell surface, where additional binding to docking and scaffolding proteins and ultimately exocytotic release of the granule contents into the airway lumen would occur.
Relatedly, it would seem that other molecules in addition to MARCKS, HSP70, CSP, actin, and myosin would be involved in exocytosis of mucin granules in airway epithelium and perhaps similar secretory cell types. For example, numerous soluble N-ethylmaleimide-sensitive factor attachment proteins, soluble N-ethylmaleimide-sensitive factor attachment protein receptors, vesicle-associated membrane proteins (especially VAMP 8), and mammalian uncoordinated (especially munc-18, the mammalian homolog of the unc-18 gene, also called nSec1 or rbSec1) (7, 14) have been implicated in the secretory process. Exactly how the above proteins function collectively to regulate the endocytotic process and whether or not their function in this process involves MARCKS or other chaperones require further elucidation.
This work was supported by NIH grant R37 HL36982 to K. Adler.
K. Adler holds 150,000 founders' shares of a start-up biotech, BioMarck, and serves as a scientific consultant and member of the scientific advisory board without monetary compensation. He receives more than $100,000 yearly in grants from NIH and the U.S. EPA. He serves as editor-in-chief of the American Journal of Respiratory Cell and Molecular Biology, for which he receives a stipend less than $100,000 yearly. None of the other authors have relevant financial disclosures, and all other authors have nothing to declare. North Carolina State University does not have a financial relationship with a commercial entity that has an interest in the subject matter or materials discussed in this study.
Author contributions: S.F., W.C., and X.-R.R. performed experiments; S.F. and A.L.C. analyzed data; S.F., A.L.C., and Q.Y. prepared figures; S.F. drafted manuscript; S.F., A.L.C., W.C., J.P., Q.Y., X.-R.R., and K.B.A. approved final version of manuscript; A.L.C., J.P., Q.Y., and K.B.A. edited and revised manuscript; J.P. and K.B.A. conception and design of research; Q.Y. and K.B.A. interpreted results of experiments.
The authors thank Dr. Laura J. Sommerville for assistance with the confocal microscopy. We also thank Dr. Peter Wipf of the University of Pittsburgh for the generous gift of the HSP70 inhibitor, MAL3-101.
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