MARCKS and related chaperones bind to unconventional myosin V isoforms in airway epithelial cells.
We have shown previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule in the process of mucin secretion by airway epithelial cells, and that part of the secretory mechanism involves intracellular associations of MARCKS with specific chaperones: heat shock protein 70 (Hsp70) and cysteine string protein (CSP). Here, we report that MARCKS also interacts with unconventional myosin isoforms within these cells, and further molecular interactions between MARCKS and these chaperones/cytoskeletal proteins are elucidated. Primary human bronchial epithelial cells and the HBE1 cell line both expressed myosin V and VI proteins, and both MARCKS and CSP were shown to bind to myosin V, specifically Va and Vc. This binding was enhanced by exposing the cells to phorbol-12-myristate-13-acetate, an activator of protein kinase C and stimulator of mucin secretion. Binding of MARCKS, Hsp70, and CSP was further investigated by His-tagged pull down assays of purified recombinant proteins and multiple transfections of HBE1 cells with fusion proteins (MARCKS-HA; Flag-Hsp70; c-Myc-CSP) and immunoprecipitation. The results showed that MARCKS binds directly to Hsp70, and that Hsp70 binds directly to CSP, but that MARCKS binding to CSP appears to require the presence of Hsp70. Interrelated binding(s) of MARCKS, chaperones, and unconventional myosin isoforms may be integral to the mucin secretion process.
Lin
Ko-Wei
KW
Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, 27606, USA.
Fang
Shijing
S
Park
Joungjoa
J
Crews
Anne L
AL
Adler
Kenneth B
KB
D001980:Bronchi / Q000473:pathology*; D004847:Epithelial Cells / Q000166:cytology*; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D018832:Molecular Chaperones; D009077:Mucins / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000737:chemistry*; D024701:Myosin Type V / Q000737:chemistry*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011485:Protein Binding; D020033:Protein Isoforms; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D018832:Molecular Chaperones; D009077:Mucins; D020033:Protein Isoforms; C089785:myosin VI; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D013755:Tetradecanoylphorbol Acetate