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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。胰岛素分泌的胰岛B细胞通过钙非依赖性磷脂酶A2(IPLA2B)表达A组，iPLA2B含有钙调蛋白结合位点和蛋白相互作用结构域。我们以iPLA2B为诱饵，通过酵母双杂交筛选文库，确定了CaMKIIB是一个潜在的与iPLA2B相互作用的蛋白。从大鼠胰岛文库中克隆CaMKIIB基因，发现一个主要的CaMKIIB亚型在成年胰岛中表达，在脑和新生胰岛中不表达，并且在大鼠和人B细胞中表达的亚型具有高度的保守性。以编码该异构体的DNA为诱饵，以iPLA2B DNA为猎物的双杂交分析证实了酶的相互作用，以CaMKIIB为猎物和iPLA2B为诱饵的分析也证实了酶的相互作用。固定在金属亲和基质上的His标记的CaMKIIB与iPLA2B结合，这不需要外源钙调蛋白，也不能被钙调素拮抗剂或钙离子螯合剂EGTA阻止。两种酶结合后活性增加，iPLA2B反应产物降低CaMKIIB活性。IPLA2B抑制剂溴烯醇内酯和CaMKIIB抑制剂KN93均可减少INS-1胰岛素瘤细胞花生四烯酸的释放，并抑制胰岛素的分泌。CaMKIIB和iPLA2B可以从INS-1细胞中免疫共沉淀，Forsklin可以放大葡萄糖诱导的胰岛素分泌，增加免疫可沉淀复合体的丰度。这些发现表明，iPLA2B和CaMKIIB在B细胞中形成了一个信号复合体，这与报道的两种酶参与胰岛素分泌以及它们的表达是在胰腺前体细胞分化为内分泌前体细胞时共同诱导的报道一致。

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.