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

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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.