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HORMONES AND SIGNALING

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

¹Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin; ²Hotchkiss Brain Institute, Department of Physiology and Biophysics, University of Calgary, Alberta, Canada

Submitted 13 October 2008 ; accepted in final form 19 February 2009

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-) is a zymogen granule (ZG) membrane protein characterized by an NH₂-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- in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP- truncations into isolated acini following their permeabilization with Perfringolysin O. Incubation of acini with CSP-_1-82, containing the J domain, significantly augmented Ca²⁺-stimulated amylase secretion. Effects of CSP-_1-82 were concentration dependent, with a maximum 80% increase occurring at 200 µg/ml of protein. Although CSP-_1-82 had no effects on basal secretion measured in the presence of 10 nM free Ca²⁺, it did significantly augment GTP-S-induced secretion under basal Ca²⁺ conditions by 25%. Mutation of the J domain to abolish its cochaperone activity failed to augment Ca²⁺-stimulated secretion, implicating the CSP-/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP- physically associates with vesicle-associated membrane protein 8 (VAMP 8) on ZGs, and the CSP--VAMP 8 interaction was dependent on amino acids 83-112 of CSP-. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP- colocalization with VAMP 8. These data establish a role for CSP- in regulating digestive enzyme secretion and suggest that CSP- and Hsc70 modulate specific soluble N-ethylmaleimide-sensitive attachment receptor interactions necessary for exocytosis.

heat shock protein 70; zymogen granules