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J. Biol. Chem., Vol. 280, Issue 8, 6840-6849, February 25, 2005

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Group VIA Phospholipase A₂ Forms a Signaling Complex with the Calcium/Calmodulin-dependent Protein Kinase II Expressed in Pancreatic Islet -Cells^*

Zhepeng Wang¶, Sasanka Ramanadham¶, Zhongmin Alex Ma||, Shunzhong Bao¶, David J. Mancuso¶**, Richard W. Gross¶**, and John Turk¶¶¶

From the Mass Spectrometry Resource, Divisions of Endocrinology, Diabetes, and Metabolism and of ^**Bioorganic Chemistry and Molecular Pharmacology, Departments of ^¶Medicine, Chemistry, and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^||Division of Experimental Diabetes and Aging, Mount Sinai School of Medicine, New York, New York 10029

Insulin-secreting pancreatic islet -cells express a Group VIA Ca²⁺-independent phospholipase A₂ (iPLA₂) that contains a calmodulin binding site and protein interaction domains. We identified Ca²⁺/calmodulindependent protein kinase II (CaMKII) as a potential iPLA₂-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA₂ cDNA as bait. Cloning CaMKII cDNA from a rat islet library revealed that one dominant CaMKII 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 -cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA₂ DNA as prey confirmed interaction of the enzymes, as did assays with CaMKII as prey and iPLA₂ bait. His-tagged CaMKII immobilized on metal affinity matrices bound iPLA₂, 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 iPLA₂ reaction products reduced CaMKII activity. Both the iPLA₂ inhibitor bromoenol lactone and the CaMKII inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKII and iPLA₂ 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 iPLA₂ and CaMKII form a signaling complex in -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.

Received for publication, May 12, 2004 , and in revised form, November 22, 2004.

^* This work was supported by United States Public Health Service Grants R37-DK34388 (to J. T.), P01-HL57278 (to R. W. G.), P41-RR00954, P60-DK20579, and P30-DK56341, by an award (to S. R.) from the American Diabetes Association, and by an award (to Z. A. M.) from the Juvenile Diabetes Research Foundation (No. 1-2002-646). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶¶ To whom correspondence should be addressed: Box 8127, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8190; Fax: 314-362-8188; E-mail: jturk{at}wustl.edu.

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