Kaspar Kala M, Thomas Diana D H, Taft William B, Takeshita Eriko, Weng Ning, Groblewski Guy E
Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Am J Physiol Gastrointest Liver Physiol. 2003 Dec;285(6):G1300-9. doi: 10.1152/ajpgi.00534.2002. Epub 2003 Jul 31.
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.
28 kDa的钙调节热稳定蛋白（CRHSP - 28；肿瘤蛋白D52家族成员）在外分泌腺中高表达，并已证明其可调节胰腺腺泡细胞的消化酶分泌。我们发现CRHSP - 28在培养的黏膜分泌性T84细胞中高表达，这与它在顶端膜转运中的重要调节作用一致。用卡巴胆碱（CCh）刺激细胞可诱导CRHSP - 28在至少两个丝氨酸残基上快速、浓度依赖性磷酸化。等电聚焦和免疫印迹用于表征调控CRHSP - 28磷酸化的细胞机制。磷酸化依赖于细胞内钙离子升高，离子霉素和毒胡萝卜素可最大程度诱导磷酸化，而BAPTA - AM可完全抑制。重组CRHSP - 28的体外磷酸化被酪蛋白激酶II（CKII）诱导的程度比钙离子/钙调蛋白依赖性蛋白激酶II（CaMKII）高10倍。然而，磷酸肽图谱研究表明，CaMKII诱导的磷酸肽图谱与从T84细胞免疫沉淀的内源性CRHSP - 28相同。尽管钙调蛋白拮抗剂对CCh刺激的磷酸化没有影响，但细胞松弛素D破坏肌动蛋白丝可使磷酸化抑制50%。共聚焦显微镜显示CRHSP - 28在细胞的核周区域表达，CCh刺激后在极化单层细胞的顶端膜下方立即积累。CaMKII也定位于顶端下方的细胞质中，肌动蛋白丝破坏后明显移位。这些数据表明CRHSP - 28磷酸化受一种类似CaMKII的酶调节，并且可能涉及该蛋白在上皮细胞顶端细胞质内的易位。
