IPLA2B FORMS A SIGNALING COMPLEX W/ THE CAMKIIB EXP IN PANCREATIC ISLET B-CELLS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。分泌胰岛素的胰岛B细胞表达含有钙调蛋白结合位点和蛋白质相互作用结构域的VIA组Ca 2+非依赖性磷脂酶A2（iPLA 2B）。以iPLA 2B cDNA为诱饵，通过酵母双杂交筛选cDNA文库，鉴定出Ca 2 +/钙调素非依赖性蛋白激酶IIB（CaMKIIB）是一个与iPLA 2B相互作用的蛋白。从大鼠胰岛文库中克隆CaMKIIB cDNA揭示了一种显性CaMKIIB同种型mRNA由成人胰岛表达，并且在脑或新生胰岛中未观察到，并且大鼠和人B细胞表达的同种型具有高度保守性。二元双杂交试验使用编码这种亚型的DNA作为诱饵和iPLA 2B DNA作为猎物，证实了酶的相互作用，如用CaMKIIB作为猎物和iPLA 2B诱饵的试验一样。His标记的CaMKIIB固定在金属亲和基质上结合iPLA 2B，并且这不需要外源性钙调蛋白，并且不被钙调蛋白拮抗剂或Ca 2+螯合剂EGTA阻止。这两种酶的活性增加后，他们的协会，和iPLA 2B反应产物降低CaMKIIB活性。iPLA 2B抑制剂溴烯醇内酯和CaMKIIB抑制剂KN 93都减少了INS-1胰岛素瘤细胞的花生四烯酸释放，并且都抑制胰岛素分泌。CaMKIIB和iPLA 2B可以从INS-1细胞中共免疫沉淀，而毛喉素可以增强葡萄糖诱导的胰岛素分泌，增加免疫沉淀复合物的丰度。这些发现表明iPLA 2B和CaMKIIB在B细胞中形成信号传导复合物，这与两种酶参与胰岛素分泌的报道一致，并且它们的表达在胰腺祖细胞分化为内分泌祖细胞时被共诱导。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Insulin-secreting pancreatic islet B-cells express a Group VIA Ca2+-independent phospholipase A2 (iPLA2B) that contains a calmodulin binding site and protein interaction domains. We identified Ca2+/calmodulindependent protein kinase IIB (CaMKIIB) as a potential iPLA2B-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA2B cDNA as bait. Cloning CaMKIIB cDNA from a rat islet library revealed that one dominant CaMKIIB isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human B-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA2B DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIB as prey and iPLA2B bait. His-tagged CaMKIIB immobilized on metal affinity matrices bound iPLA2B, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca2+ chelator EGTA. Activities of both enzymes increased upon their association, and iPLA2B reaction products reduced CaMKIIB activity. Both the iPLA2B inhibitor bromoenol lactone and the CaMKIIB inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIB and iPLA2B can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA2B and CaMKIIB form a signaling complex in B-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.