CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells

Abstract

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

Tumor protein D52 (TPD52): a novel B-cell/plasma-cell molecule with unique expression pattern and Ca2+-dependent association with annexin VI

“…Indeed, CRHSP28 has been recently shown to be serine phosphorylated upon increasing Ca 2ϩ levels in epithelial cells. 45 Finally, it has been reported that in rat pancreatic acinar cells, CRHSP28 can bind annexin VI in a calcium-dependent way and that this molecular complex plays an important role in acinar cell membrane trafficking events that are essential for digestive enzyme secretion. 14 Our finding-that in the Thiel human myeloma cell line, TPD52 associates to annexin VI in a Ca 2ϩ -dependent manner-raises the intriguing possibility that these molecules may act in concert to regulate secretory processes also in plasma cells.…”

Tumor protein D52 controls trafficking of an apical endolysosomal secretory pathway in pancreatic acinar cells

“…SDS-PAGE and immunoblotting were conducted as previously described (38). Anti-D52 (5 g/ml)-HA (1:1,000), -syntaxin 2-4 (1:1,000), -VAMP2 (1:2,000), and -VAMP8 (1:1,000), -syt1 (1:2,000), and -AP-3 (1:1,000) immunoreactivities were detected by using appropriate horseradish peroxidase-conjugated secondary antibodies.…”

“…D52 phosphorylation is Ca 2ϩ dependent and potentially mediated by the ␦6-isoform of CaM kinase II (15,38). In acinar cells, D52 phosphorylation is maximal within 2 min of secretagogue stimulation and then rapidly declines by 15 min (38 -39).…”

Positive Regulation of Apoptosis Signal-regulating Kinase 1 by hD53L1

“…It also has been reported that D52 tumor proteins are involved in Ca 2ϩ signaling to regulate proliferation and Ca 2ϩ -stimulated secretory activity (37,38). Recently, it has been reported that calcium/ calmodulin-dependent protein kinase II phosphorylates a Ca 2ϩ -regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) in cultured mucosal T84 cells (39). Thus, our finding that hD53L1, a member of the tumor protein D52 family, activates the ASK1 pathway suggests that it may play a role in the Ca 2ϩ -dependent ASK1-regulating pathway.…”

“…It is also possible that hD53L1 may require other cellular factor(s) or external stimuli to gain full activity. Calcium may also be involved in the regulation, since calcium/calmodulin-dependent protein kinase II acts on both ASK1 and D52 family (35,39). These possibilities suggest that the regulation mechanism of ASK1 by hD53L1 may be different from the mechanisms of ASK1 by Daxx and TRAF2.…”