Functional role of J domain of cysteine string protein in Ca2+-dependent secretion from acinar cells

• Published in: American journal of physiology: Gastrointestinal and liver physiology 2009-05, Vol.296 (5), p.G1030-G1039
• Main Authors: Weng, Ning, Baumler, Megan D, Thomas, Diana D H, Falkowski, Michelle A, Swayne, Leigh Anne, Braun, Janice E A, Groblewski, Guy E
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Enzymes - metabolism; Exocytosis; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Hormones and Signaling; HSC70 Heat-Shock Proteins - metabolism; HSP40 Heat-Shock Proteins - chemistry; HSP40 Heat-Shock Proteins - genetics; HSP40 Heat-Shock Proteins - metabolism; In Vitro Techniques; Male; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutation; Pancreas, Exocrine - enzymology; Pancreas, Exocrine - metabolism; Peptide Fragments - metabolism; Protein Structure, Tertiary; R-SNARE Proteins - metabolism; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins - metabolism; Secretory Pathway; Secretory Vesicles - enzymology; Secretory Vesicles - metabolism; SNARE Proteins - metabolism
• ISSN: 0193-1857; 1522-1547
• DOI: 10.1152/ajpgi.90592.2008

The heat shock protein 70 family members Hsc70 and Hsp70 are known to play a protective role against the onset of experimental pancreatitis, yet their molecular function in acini is unclear. Cysteine string protein (CSP-alpha) is a zymogen granule (ZG) membrane protein characterized by an NH(2)-terminal "J domain" and a central palmitoylated string of cysteine residues. The J domain functions as a cochaperone by modulating the activity of Hsc70/Hsp70 family members. A role for CSP-alpha in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP-alpha truncations into isolated acini following their permeabilization with Perfringolysin O. Incubation of acini with CSP-alpha(1-82), containing the J domain, significantly augmented Ca(2+)-stimulated amylase secretion. Effects of CSP-alpha(1-82) were concentration dependent, with a maximum 80% increase occurring at 200 microg/ml of protein. Although CSP-alpha(1-82) had no effects on basal secretion measured in the presence of < or =10 nM free Ca(2+), it did significantly augment GTP-gammaS-induced secretion under basal Ca(2+) conditions by approximately 25%. Mutation of the J domain to abolish its cochaperone activity failed to augment Ca(2+)-stimulated secretion, implicating the CSP-alpha/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP-alpha physically associates with vesicle-associated membrane protein 8 (VAMP 8) on ZGs, and the CSP-alpha-VAMP 8 interaction was dependent on amino acids 83-112 of CSP-alpha. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP-alpha colocalization with VAMP 8. These data establish a role for CSP-alpha in regulating digestive enzyme secretion and suggest that CSP-alpha and Hsc70 modulate specific soluble N-ethylmaleimide-sensitive attachment receptor interactions necessary for exocytosis.