title: MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro.
abstract: 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.
authors: Department of Molecular Biomedical Sciences, North Carolina State University, College of Veterinary Medicine, Raleigh, NC 27607, USA.
authors: Shijing
authors: S
authors: Fang
authors: Anne L
authors: AL
authors: Crews
authors: Wei
authors: W
authors: Chen
authors: Joungjoa
authors: J
authors: Park
authors: Qi
authors: Q
authors: Yin
authors: Xiu-Rong
authors: XR
authors: Ren
authors: Kenneth B
authors: KB
authors: Adler
mesh_terms: D001483:Base Sequence; D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D050956:HSP40 Heat-Shock Proteins / Q000502:physiology; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D018613:Microscopy, Confocal; D009077:Mucins / Q000378:metabolism*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D021381:Protein Transport; D011744:Pyrimidinones / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D009077:Mucins; D011744:Pyrimidinones; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C088460:cysteine string protein; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D013755:Tetradecanoylphorbol Acetate
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