MARCKS protein has been shown to be a central molecule involved in airway mucin secretion in vitro
and in vivo
). Here, we show that the cytoskeletal protein, myosin V, is expressed in human airway epithelial cells and associates with MARCKS. Exposure of these cells to PMA enhances MARCKS interactions with myosin V, specifically the Va and Vc isoforms. Previously, associations of MARCKS with intracellular chaperones, including Hsp70 and CSP, were shown to be integral to the secretory process (5
). In this report, studies using bacterial and mammalian expression systems revealed that, within human airway epithelial cells, MARCKS appears to bind directly to Hsp70, that Hsp70 binds directly to CSP, but MARCKS does not bind directly to CSP, suggesting that MARCKS–CSP associations are indirect and may require the presence of Hsp70. Interestingly, CSP also binds to myosin V, and exposure of cells to the protein kinase C activator, PMA, enhances interactions of both CSP and MARCKS with myosin V. The results of these studies provide additional evidence of interactions between MARCKS and the chaperones, Hsp70 and CSP, and provide the first evidence that MARCKS interacts specifically with the cytoskeletal protein myosin V.
Previous work from our laboratory, using a radiolabeled immunoprecipitation assay and matrix-assisted laser desorption ionization/time of flight mass spectrometry/internal sequence analysis (MALDI-TOF), showed that MARCKS associates with myosin in the cytoplasm of NHBE cells (2
). Mass spectrometric proteomic analysis of tryptic peptides from isolated mucin granule membranes from airway epithelial cells also implicated myosin as one of the proteins that associates with these granules (10
). Since MARCKS is an actin-binding and crosslinking protein, actin/myosin-dependent contraction in concert with MARCKS may mediate granule movement to the cell periphery as an initial step in the exocytotic release of mucin.
The myosin investigated in airway epithelium here, unconventional myosin V, is a nonmuscle type, F-actin–based motor protein. There are three distinct subclasses of myosin V in vertebrates: myosins Va, Vb, and Vc (8
). Myosin V is a dimeric protein (12
) and myosin Va is known to have a high affinity for F-actin (13
). Myosin Va has been implicated in regulating traffic of synaptic vesicles in neurons, secretory granules in neuroendocrine cells, insulin granules in β-cells, and melanosome trafficking in melanocytes (14
). Myosin Vc is a newly discovered subtype abundantly expressed in epithelial and secretory tissues (8
). It is present on zymogen granules of pancreatic acinar cells, and could play a role in exocrine secretion from this organ (20
). Myosin VI is another unconventional myosin implicated in organelle trafficking (21
). In our studies, myosin Va, myosin Vc, and myosin VI all were expressed in both NHBE and HBE1 cells (). Both MARCKS and CSP were shown to bind to myosin Va and Vc, and this binding was enhanced by exposure to PMA (). As myosin Vc has been shown to be physically associated with mucin granule membranes in airway epithelial cells via mass spectrometric analysis (10
), it may represent an important MARCKS-binding protein that could be involved in airway mucin secretion. Binding of MARCKS to myosin isoforms might involve the tail domain of the myosin, as this region of the molecule has been shown previously to be involved in exocytosis of large dense core vesicles in neurons (24
). Studies involving silencing of the different myosin isoforms or transfection of dominant negative mutants of these proteins are presently underway in our laboratory to determine the role of MARCKS-myosin interactions in the process of mucin secretion.
The results of this study also support our previous findings that MARCKS, Hsp70, and CSP associate in airway epithelial cells. The binding solutions used here include nonionic NP-40 and modified RIPA buffer. Since RIPA buffer is a strong solution for immunoprecipitation, the fact that MARCKS, Hsp70, and CSP still associate after treatment with RIPA suggests strong interactions between these proteins. To determine whether these protein–protein interactions are truly specific interactions, and to understand how these proteins actually bind, in vitro binding assays of purified recombinant proteins and co-immunoprecipitation of overexpressed tagged proteins in mammalian cells were performed. Apparent direct binding between MARCKS and Hsp70 in vitro was observed by generating purified recombinant proteins and analyzing their binding properties using His-tagged pull-down assays (). Purified GST protein did not bind to purified Hsp70, which not only provided a negative “technique” control for the binding assay, but also supports the idea that the binding between MARCKS and Hsp70 could be direct and specific.
We further confirmed binding of these proteins in a mammalian overexpression system. Stepwise binding events were examined by multiple transfections of HBE1 cells with specifically designed fusion proteins (MARCKS–HA; Flag–Hsp70; c-Myc–CSP) and immunoprecipitations. Since the amount of overexpressed fusion proteins is much higher than other endogenous proteins, the positive results from these immunoprecipitation experiments strongly indicate direct binding of the proteins. As mentioned above, the results indicate that MARCKS-Hsp70 binding is direct, as is Hsp70-CSP binding, but MARCKS binding to CSP appears to be indirect (). It should be pointed out that reflects results in HBE1 cell lysates in which tagged MARCKS and CSP are highly overexpressed, with probably hundredfolds increase over endogenous levels of the proteins and of the Hsp70 that is present in these cells. The data thus support a mechanism whereby interaction of MARCKS and CSP requires the presence of Hsp70; cytosolic MARCKS may bind to Hsp70, and this MARCKS/Hsp70 complex then may be targeted to cytoplasmic mucin granules through interactions with granule-associated CSP.
HBE1 cells exposed to the PKC activator and mucin secretagogue, PMA, show phosphorylation of MARCKS in a concentration-dependent manner (). The formation of MARCKS–CSP and MARCKS–Hsp70 complexes was increased by exposure to PMA, and CSP was shown to interact with phosphorylated MARCKS (). The data shown in reflect results of experiments performed in whole cell lysates with only endogenous levels of proteins expressed. Therefore, it is probable that endogenously present Hsp70 enables CSP to interact with MARCKS, although not directly as indicated in the purified protein experiments shown above. In addition, shows that as PMA increases the phosphorylation of MARCKS (panel A), the levels of Hsp70 are also increased (panel D). Consequently, the levels of CSP that interact with MARCKS are also increased, perhaps due to the increased availability of Hsp70 acting as the common binding target for both MARCKS and CSP ().
These findings suggest a functional role for interactions between MARCKS, Hsp70, and CSP in several cellular events, and support a mechanism of mucin secretion involving MARCKS phosphorylation and important roles for these chaperones in the secretory process (1
). Additional proteins and intracellular signaling steps involved in the process remain to be elucidated.