MARCKS Regulation of Mucin Secretion by Airway Epithelium in Vitro

Abstract

We have reported previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. The results of those studies supported a mechanism whereby MARCKS, upon phosphorylation by protein kinase C (PKC), translocates from plasma membrane to cytoplasm, where its binding to membranes of intracellular mucin granules is a key component of the secretory pathway. It remains unknown how MARCKS is targeted to and/or preferentially attaches to mucin granule membranes. We hypothesized that the chaperone cysteine string protein (CSP) may play an important role in this process. CSP was shown to associate with membranes of intracellular mucin granules in well-differentiated normal human bronchial epithelial (NHBE) cells in vitro, as determined by ultrastructural immunohistochemistry and Western blotting of isolated granule membranes. CSP in these cells complexed with MARCKS, as shown by co-immunoprecipitation. Given reported associations between CSP and a second chaperone, heat shock protein 70 (HSP70), a role for HSP70 in the MARCKS-dependent secretory mechanism also was investigated. HSP70 appeared to form a trimeric complex with MARCKS and CSP associated with mucin granule membranes within airway epithelial cells. Transfection of the HBE1 human bronchial epithelial cell line with siRNAs targeting sequences of MARCKS, CSP, or HSP70 resulted, in each case, in significant knockdown of expression of these proteins and subsequent attenuation of mucin secretion. The results provide the first evidence that CSP and HSP70, and their interactions with MARCKS, are involved in mucin secretion.

MARCKS modulates radial progenitor placement, proliferation and organization in the developing cerebral cortex

“…F-actin crosslinking activity of MARCKS is also reduced following phosphorylation (Arbuzova et al, 2002). Phosphorylated MARCKS translocates either to the cytosol or nucleus, where it has been shown to co-localize with a diverse array of cellular complexes, including components of the secretory pathway as well as γ-tubulin (Park et al, 2008). Surprisingly, we found that phosphorylation by PKC is not crucial for the role of MARCKS in radial glial cell polarization.…”

“…When unphosphorylated, the PSD domain binds calmodulin with high affinity, crosslinks F-actin and binds to multivalent lipids, including PIP 2 and PS (Blackshear et al, 1992;Hartwig et al, 1992;Sundaram et al, 2004). Phosphorylation of the PSD domain decreases the electrostatic interaction of MARCKS with the plasma membrane, thus facilitating release of MARCKS from the membrane (Park et al, 2008;Swierczynski and Blackshear, 1995;. F-actin crosslinking activity of MARCKS is also reduced following phosphorylation (Arbuzova et al, 2002).…”

MARCKS as a Negative Regulator of Lipopolysaccharide Signaling

“…We propose that phosphorylation of MARCKS, triggered as an early event of the LPS stimulation, leads to its dissociation from the cell membrane and translocation to the endosomes, where it can bind to LPS and downregulate the excessive activation of TLR4. MARCKS translocation to endosomes may also involve additional chaperones, similarly as translocation to mucin granules mediated by cysteine string protein (63). Efficiency of the myristoylation-deficient A2/G2 mutant of MARCKS, which under the normal conditions resides in the cytoplasm and associates with endosomes upon LPS stimulation, further supports this mechanism.…”

Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages

“…MARCKS regulates the hypersecretion of mucus in respiratory diseases, such as asthma and cystic fibrosis (70,81). Phosphorylation of MARCKS allows movement to the cytoplasm, binding to chaperone HSP70, which engages the cysteine string protein located on the surface of the secretory vesicles containing mucin (69,82). Because apoE is structurally a mucin, we hypothesize a similar model for apoE secretion, and future research will delineate the exact chaperone molecules and mechanisms through which MARCKS regulates both basal and stimulated apoE secretion.…”