CRHSP-28 is a Ca2؉ -regulated heat-stable phosphoprotein, abundant in the apical cytoplasm of epithelial cells that are specialized in exocrine protein secretion. To define a functional role for the protein in pancreatic secretion, recombinant CRHSP-28 (rCRHSP-28) was introduced into streptolysin-O-permeabilized acinar cells, and amylase secretion in response to elevated Ca 2؉ was determined. Secretion was enhanced markedly by rCRHSP-28 over a time course that closely corresponded with the loss of the native protein from the intracellular compartment. No effects of rCRHSP-28 were detected until ϳ50% of the native protein was lost from the cytosol. Secretion was enhanced by rCRHSP-28 over a physiological range of Ca 2؉ concentrations with 2-3-fold increases in amylase release occurring in response to low micromolar levels of free Ca 2؉ . Further, rCRHSP-28 augmented secretion in a concentration-dependent manner with minimal and maximal effects occurring at 1 and 25 g/ml, respectively. Covalent cross-linking experiments demonstrated that native CRHSP-28 was present in a 60-kDa complex in cytosolic fractions and in a high molecular mass complex in particulate fractions, consistent with the slow leak rate of the protein from streptolysin-O-permeabilized cells. Probing acinar lysates with rCRHSP-28 in a gel-overlay assay identified two CRHSP-28-binding proteins of 35 (pp35) and 70 kDa (pp70). Interestingly, preparation of lysates in the presence of 1 mM Ca 2؉ resulted in a marked redistribution of both proteins from a cytosolic to a Triton X-100-insoluble fraction, suggesting a Ca 2؉ -sensitive interaction of these proteins with the acinar cell cytoskeleton. In agreement with our previous study immunohistochemically localizing CRHSP-28 around secretory granules in acinar cells, gel-overlay analysis revealed pp70 copurified with acinar cell secretory granule membranes. These findings demonstrate an important cell physiological function for CRHSP-28 in the Ca 2؉ -regulated secretory pathway of acinar cells.Exocrine cells specializing in protein secretion release a variety of factors necessary for normal function of the digestive, urogenital, respiratory, and ocular systems. Activation of these epithelia by neural and humoral agents stimulates the exocytosis of secretory granules at the apical plasma membrane in a process that is largely controlled by cellular Ca 2ϩ . In pancreatic acinar cells, Ca 2ϩ release is initiated in the apical pole and then propagates through the cell periphery to the basal cytoplasm. The cyclic reuptake and release of Ca 2ϩ from intracellular stores create an oscillatory mode of signaling with spatial and temporal characteristics that are unique to the specific type and concentration of physiologic stimulus (for review, see Refs. 1-3). Although the high concentrations of Ca 2ϩ generated in the apical cytoplasm are necessary for secretory granule trafficking and exocytosis to occur, a comprehensive understanding of the molecular events elicited by this ion is lacking.The secretory pathway in ac...

, indicating these molecules likely interact under physiological conditions. Immunofluorescence microscopy confirmed a dual localization of CRHSP-28 and annexin VI, which appeared in a punctate pattern in the supranuclear and apical cytoplasm of acini. Stimulation of cells for 5 min with the secretagogue cholecystokinin enhanced the colocalization of CRHSP-28 and annexin VI within regions of acini immediately below the apical plasma membrane. Tissue fractionation revealed that CRHSP-28 is a peripheral membrane protein that is highly enriched in smooth microsomal fractions of pancreas. Further, the content of CRHSP-28 in microsomes was significantly reduced in pancreatic tissue obtained from rats that had been infused with a secretory dose of cholecystokinin for 40 min, demonstrating that secretagogue stimulation transiently alters the association of CRHSP-28 with membranes in cells. Collectively, the Ca 2؉ -dependent binding of CRHSP-28 and annexin VI, together with their colocalization in the apical cytoplasm, is consistent with a role for these molecules in acinar cell membrane trafficking events that are essential for digestive enzyme secretion.

blewski. CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells. Am J Physiol Gastrointest Liver Physiol 285: G1300-G1309, 2003. First published July 31, 2003 10.1152 10. /ajpgi.00534.2002 -regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) is highly expressed in exocrine glands and was shown to regulate digestive enzyme secretion from pancreatic acinar cells. We found CRHSP-28 highly expressed in cultured mucosal secretory T84 cells, consistent with an important regulatory role in apical membrane trafficking. Stimulation of cells with carbachol (CCh) induced rapid, concentration-dependent phosphorylation of CRHSP-28 on at least two serine residues. Isoelectric focusing and immunoblotting were used to characterize cellular mechanisms governing CRHSP-28 phosphorylation. Phosphorylation depends on elevated cellular Ca 2ϩ , being maximally induced by ionomycin and thapsigargin and fully inhibited by BAPTA-AM. In vitro phosphorylation of recombinant CRHSP-28 was 10-fold greater by casein kinase II (CKII) than Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII). However, phosphopeptide mapping studies demonstrated that CaMKII induced an identical phosphopeptide profile to endogenous CRHSP-28 immunoprecipitated from T84 cells. Although calmodulin antagonists had no effect on CCh-stimulated phosphorylation, disruption of actin filaments by cytochalasin D inhibited phosphorylation by 50%. Confocal microscopy indicated that CRHSP-28 is expressed in perinuclear regions of cells and accumulates immediately below the apical membrane of polarized monolayers following CCh stimulation. CaMKII was also localized to the subapical cytoplasm and was clearly displaced following actin filament disruption. These data suggest that CRHSP-28 phosphorylation is regulated by a CaMKII-like enzyme and likely involves a translocation of the protein within the apical cytoplasm of epithelial cells. calcium signaling; membrane trafficking; protein kinase; casein kinase II; calcium/calmodulin-dependent protein kinase II

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