Targeted phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibits localized Ca2+ release and shapes oscillatory Ca2+ signals
Targeted phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibits localized Ca2+ release and shapes oscillatory Ca2+ signals
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DOI：
10.1074/jbc.m004278200
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发表时间：
2000-10-27
影响因子：
4.8
通讯作者：
Yule, DI
中科院分区：
文献类型：
--
作者：
Giovannucci, DR;Groblewski, GE;Yule, DI
目前的研究提供了生化和功能证据，表明蛋白激酶 A (PBA) 靶向局部 Ca2+ 释放位点可对胰腺腺泡细胞中的 Ca2+ 信号进行快速、特异性磷酸化。通过监测笼中闪光光解引起的胰腺腺泡细胞中的 Ca2+ 动态，研究了肌醇 1,4,5-三磷酸 (InsP(3)) 受体的 PKA 依赖性磷酸化对 Ca2+ 释放的调节控制。 InsP(3) 在 PRA 激活之前和之后，通过数字成像和电生理学方法以高时间分辨率对 Ca2+ 动态进行成像。全细胞膜片钳技术用于引入笼状化合物并记录Ca2+激活的Cl-电流的活性，低浓度笼状InsP(3)的光解引起Cl-电流，该电流被二丁酰-cAMP或毛喉素处理所抑制。相比之下，PKA 激活剂对释放 Ca2+ 或光解释放较高浓度的 InsP(3) 引起的 Cl- 电流的激活没有显着的抑制作用。用 Rp-腺苷-3',5'-环单硫代磷酸酯（PKA 的选择性抑制剂）或 Ht31（一种已知可破坏 PKA 靶向的肽）治疗，可在很大程度上消除毛喉素诱导的 Ca2+ 释放抑制。使用免疫细胞化学方法揭示了 PKA 靶向 Ca2+ 动员位点的进一步证据，证明 PKA 的 R-II β 亚基。定位于腺泡细胞的顶端区域，并与 III 型 InsP(3) 受体共免疫沉淀，但不与 I 型或 II 型 InsP(3) 受体共免疫沉淀。最后，我们证明乙酰胆碱引起的信号传导模式可以通过提高 cAMP 水平转化为更“CCK 样”的模式。我们的数据提供了一种简单的机制，通过该机制可以形成不同的振荡 Ca2+ 模式。
Current study provides biochemical and functional evidence that the targeting of protein kinase A (PBA) to sites of localized Ca2+ release confers rapid, specific phosphoregulation of Ca2+ signaling in pancreatic acinar cells, Regulatory control of Ca2+ release by PKA-dependent phosphorylation of inositol 1,4,5-trisphosphate (InsP(3)) receptors was investigated by monitoring Ca2+ dynamics in pancreatic acinar cells evoked by the flash photolysis of caged InsP(3) prior to and following PRA activation, Ca2+ dynamics were imaged with high temporal resolution by digital imaging and electrophysiological methods. The whole cell patch clamp technique was used to introduce caged compounds and to record the activity of a Ca2+-activated Cl- current, Photolysis of low concentrations of caged InsP(3) evoked Cl- currents that were inhibited by treatment with dibutryl-cAMP or forskolin. In contrast, PKA activators had no significant inhibitory effect on the activation of Cl- current evoked by uncaging Ca2+ or by the photolytic release of higher concentrations of InsP(3). Treatment with Rp-adenosine-3',5'-cyclic monophoshorothioate, a selective inhibitor of PKA, or with Ht31, a peptide known to disrupt the targeting of PKA, largely abolished forskolin-induced inhibition of Ca2+ release. Further evidence for the targeting of PKA to the sites of Ca2+ mobilization was revealed using immunocytochemical methods demonstrating that the R-II beta subunit of PKA. was localized to the apical regions of acinar cells and co-immunoprecipitated with the type III but not the type I or type II InsP(3) receptors, Finally, we demonstrate that the pattern of signaling evoked by acetylcholine can be converted to one that is more "CCK-like" by raising cAMP levels. Our data provide a simple mechanism by which distinct oscillatory Ca2+ patterns can he shaped.