CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells.
Ca(2+)-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 Ca2+, being maximally induced by ionomycin and thapsigargin and fully inhibited by BAPTAAM. In vitro phosphorylation of recombinant CRHSP-28 was 10-fold greater by casein kinase II (CKII) than Ca2+/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.
Kaspar
Kala M
KM
Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Thomas
Diana D H
DD
Taft
William B
WB
Takeshita
Eriko
E
Weng
Ning
N
Groblewski
Guy E
GE
D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D045744:Cell Line, Tumor; D016764:Cell Polarity; D015638:Cytochalasin D / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D003599:Cytoskeleton / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007525:Isoelectric Focusing; D015739:Nocodazole / Q000494:pharmacology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011976:Receptors, Muscarinic / Q000502:physiology; D012694:Serine / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D014018:Tissue Distribution; D000096926:Benzenesulfonamides
D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin; D015638:Cytochalasin D; D004791:Enzyme Inhibitors; D015739:Nocodazole; D010750:Phosphoproteins; D011976:Receptors, Muscarinic; D012694:Serine; D013449:Sulfonamides; C102584:calcium-regulated heat-stable protein 28; C072105:KN 93; D000096926:Benzenesulfonamides