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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

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phospholipases A₂ (PLA₂)¹ are a diverse group of enzymes that catalyze hydrolysis of sn-2 fatty acid substituents from glycerophospholipid substrates to yield a free fatty acid and a 2-lysophospholipid (1). The Group VIA PLA₂ designated iPLA₂ has a molecular mass of 84–88 kDa and does not require Ca²⁺ for catalysis (2). Various splice variants of iPLA₂ are expressed at high levels in testis (3), brain (4), and pancreatic islet -cells (5), among other tissues.

Certain nutrients, hormones, neurotransmitters, and pharmacologic agents stimulate insulin secretion from -cells, and the dominant physiologic insulin secretagogue is D-glucose. A series of signals that result from glucose-induced ATP production and alterations of intracellular redox potentials trigger insulin secretion via a rise in cytosolic [Ca²⁺], and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an important -cell [Ca²⁺] sensor and mediator of Ca²⁺-dependent events in insulin secretion (6–11). Much evidence (12–22) suggests that iPLA₂ also participates in insulin secretion, including the facts that the mechanism-based bromoenol lactone (BEL) inhibitor of iPLA₂ suppresses glucose-induced hydrolysis of arachidonate from islet membrane phospholipids, the rise in -cell cytosolic [Ca²⁺], and insulin secretion (19–22).

Depleting intracellular Ca²⁺ stores activates iPLA₂ in -cells and vascular smooth muscle cells (23, 24), and iPLA₂ participates in store-operated entry of Ca²⁺ from the extracellular space (25), which is thought to be involved in glucose-induced insulin secretion (26–31). Regulating store-operated calcium (SOC) entry requires that intracellular Ca²⁺ stores communicate with plasma membrane ion channels, and calmodulin participates in cross-talk between Ca²⁺ stores and SOC channels (25, 32). Lipid signaling molecules (e.g. lysophospholipids) and Ca²⁺-sensitive kinases and phosphatases (e.g. CaMKII and calcineurin) are also proposed to affect these interactions (9, 10, 25, 32). Mechanisms whereby iPLA₂ participates in glucose-induced rises in -cell cytosolic [Ca²⁺] and insulin secretion are likely to involve Ca²⁺-sensitive regulation of modulatory and effector proteins by phosphorylation-dephosphorylation events (9, 10), and iPLA₂ activity is also affected by local [Ca²⁺] increments that relieve its tonic inhibition by Ca²⁺/calmodulin (2, 8, 25).

The amino acid sequence of iPLA₂ contains an ankyrin repeat domain with eight strings of a repetitive motif of about 33 amino acid residues each (34). Ankyrin repeats link integral membrane proteins to the cytoskeleton and mediate protein-protein interactions in signaling (34–38). Ankyrin binds to inositol trisphosphate receptors (37), for example, which are located on Ca²⁺-containing vesicles that release intracellular Ca²⁺ when -cells are stimulated with glucose (26–31). Ankyrin G also associates with skeletal muscle postsynaptic membranes and sarcoplasmic reticulum (38), and CaMKII participates in regulating local [Ca²⁺] gradients in subcellular zones involved in Ca²⁺ signaling. CaMKII is an important Ca²⁺ signaling effector and serves as a gauge that temporally integrates [Ca²⁺] signal intensities (39), and calmodulin participates in several Ca²⁺-dependent processes in insulin secretion by -cells (40, 41). Calmodulin and iPLA₂ interact functionally (2, 8, 23, 24, 33), and the iPLA₂ domain from residues 650–722 contains a calmodulin binding site (2).

During cell signaling, iPLA₂ translocates to membranes (22, 25, 32) where it interacts with regulatory proteins to effect cellular activation. To identify proteins that interact with iPLA₂ to understand better its role in signaling, we performed yeast two-hybrid screening and have found that iPLA₂ interacts with the specific CaMKII isoform expressed in pancreatic islet -cells. This interaction is demonstrated by multiple independent techniques, and the interaction affects both iPLA₂ and CaMKII activities, thereby defining a signaling complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—The materials [-³²P]ATP, 55 mCi/mmol (16:0/[¹⁴C]18:2)glycerophosphocholine [1-palmitoyl-2-[¹⁴C]linoleoyl-sn-glycero-3-phosphocholine, rainbow molecular mass standards, enhanced chemiluminescence (ECL) reagent, and [³H]arachidonic acid were obtained from Amersham Biosciences. SDS-PAGE supplies were purchased from Bio-Rad. Coomassie reagent was obtained from Pierce. Alkaline phosphatase and peroxidase-conjugated goat anti-rabbit IgG antibodies were obtained from Roche Applied Science. Protease inhibitor mixture, kanamycin, ampicillin, ATP, calmodulin, autocamtide-3, arachidonic acid, lysophosphatidylcholine, calmodulin kinase II inhibitor KN93, common reagents, and salts were obtained from Sigma. Tetracycline was obtained from Invitrogen. Gentamicin and cell culture media were obtained from the Tissue Culture Support Center (Washington University, St. Louis, MO). TALON metal affinity resin, a rat brain cDNA library, AH109 yeast cells, and media for yeast two-hybrid screening were obtained from Clontech (Palo Alto, CA). Polyclonal antibodies to iPLA₂ and CaMKII were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The calmodulin inhibitor W13 was obtained from Calbiochem. The iPLA₂ suicide substrate BEL was obtained from Cayman Chemical Company (Ann Arbor, MI). Avian myeloblastosis virus reverse transcriptase was obtained from Roche Applied Science.

Screening of a Rat Brain cDNA Library in the Yeast Two-hybrid System—A rat brain cDNA library cloned into pACT2 vector (containing the LEU2 gene for selection) to produce fusion between protein-encoding DNA sequences and the DNA activation domain of GAL4 was used as prey. To produce the bait construct, full-length iPLA₂ cDNA cloned from rat pancreatic islets was ligated into the SalI-EcoRI sites of the two-hybrid BD pAS2-1 vector, which contained a TRP1 gene for selection, resulting in in-frame fusion of iPLA₂ with the DNA binding domain of the yeast GAL4 protein. The fidelity of constructs was confirmed by automated sequencing. The yeast strain AH109 was used for screening assays, and this strain contains HIS3 and lacZ reporter genes. Expression of each of these genes is regulated by a distinct GAL4-responsive promoter under control of a GAL4-responsive upstream activation site. Lack of autonomous activation by the iPLA₂/DNA binding domain fusion product was demonstrated by plating cells transformed with bait alone on media lacking histidine. In these assays, both bait and prey plasmids were transformed simultaneously into AH109 yeast cells, which were plated on medium lacking leucine, tryptophan, and histidine and allowed to grow at 30 °C for 4 days. Putative positive colonies were lifted onto filter paper and incubated with the chromogenic substrate X-gal. Interactions were confirmed when the blue -galactosidase reaction product was evident after 4 h of incubation at room temperature. Plasmids were then recovered from yeast and transformed into DH5 bacterial cells using ampicillin for selection. Isolated plasmids were sequenced, and BLAST searches were performed against GenBank (NIH genetic sequence data base) to identify putative iPLA₂-interacting proteins.

Molecular Cloning of CaMKII cDNA from a Rat Islet Library—Total RNA was isolated from adult rat islets as described previously (5). First strand cDNA was transcribed with avian myeloblastosis virus reverse transcriptase. PCR was performed using a pair of gene-specific primers designed from regions of cDNA sequence that are conserved in the mouse and rat brain CaMKII cDNA sequences (sense, 5'-ATCGCCACCGCCATGGCCACC-3'; antisense, 5'-CAGGCGCAGCTCTCACTGCAG-3'). A PCR band of 1,650 bp was gel purified, ligated into pGEM-T vector, and transformed into DH5 cells for amplification. DNA was purified and sequenced using T3 and T7 primers and gene-specific primers.

Binary Yeast Two-hybrid Assays—The iPLA₂ cDNA was cloned from an adult rat islet library (5). Full-length iPLA₂ cDNA was ligated into BD vector pAS2-1 or AD vector pACT2 and used as bait or prey. Full-length CaMKII cDNA was cloned into the AD vector pACT2 or BD vector pAS2-1 and used as prey or bait. Both bait and prey plasmids were transformed simultaneously into AH109 yeast cells, which were plated on restriction medium. After incubation (30 °C, 4 days), colonies were lifted onto filter paper and screened as described above. Colonies that produced the blue -galactosidase reaction product were considered positive for the interaction between iPLA₂ and CaMKII.

Cloning and Expression of His-tagged CaMKII, His-tagged iPLA₂, and FLAG-tagged Proteins in Sf9 Cells—Recombinant proteins were expressed in Spodoptera frugiperda (Sf9) cells using the Bac-to-Bac baculovirus expression system (Invitrogen) following the manufacturer's instructions, as described in detail elsewhere (2, 23, 33, 42). cDNA containing the entire coding sequence of His-tagged CaMKII, His-tagged iPLA₂, or FLAG-tagged iPLA₂ was cloned into the SalI-EcoRI site of the pFastBac-1 vector. The sequence of the insert was verified, and the plasmid was then transformed into DH10Bac cells. Recombinant bacmid DNA was isolated using an alkaline lysis protocol modified for high molecular weight plasmid purification. PCR analysis was performed with purified bacmid DNA and pUC/M13 forward and reverse primers to characterize the inserts in the recombinant bacmid DNA. The recombinant baculovirus was produced by transfecting the recombinant bacmid DNA into Sf9 cells. The baculovirus was amplified and used to infect Sf9 cell cultures to express the recombinant proteins (2, 23, 33, 42).

Immunoblotting Analyses—Proteins were analyzed by SDS-PAGE and transferred to a nylon membrane that was subsequently blocked with 5% nonfat dry milk for 1 h. The membrane was washed and incubated for 1 h with polyclonal antibody (1:200) to iPLA₂ or CaMKII. The membrane was then incubated with secondary antibody (1:30,000) coupled to horseradish peroxidase, and the antibody complex was visualized by ECL.

Interaction of CaMKII with iPLA₂ and Protein Pull-down Assays—In some experiments, both iPLA₂ and His-tagged CaMKII proteins were coexpressed in Sf9 cells. The Sf9 cell cytosol containing iPLA₂ and His-tagged CaMKII proteins was mixed with TALON metal affinity resin in the presence or absence of added Ca²⁺/calmodulin, the calmodulin antagonist W13, or the Ca²⁺ chelator EGTA and incubated (room temperature, with shaking, for 1 h). The mixture was washed with 10 bed volumes of wash buffer (50 mM Na₂HPO₄, 500 mM NaCl, pH 7.8) twice and transferred onto a gravity-flow column. The His-tagged CaMKII was eluted with elution buffer (50 mM Na₂HPO₄, 300 mM NaCl, and 200 mM imidazole, pH 7.8) and collected in 0.5-ml fractions. Desorbed proteins were visualized by immunoblotting analyses with antibodies to iPLA₂ or CaMKII.

In other experiments, iPLA₂ protein was first expressed in Sf9 cells and purified as described previously (23, 33). Cytosol was prepared from Sf9 cells infected with baculovirus containing cDNA that encoded His-tagged CaMKII and mixed with 1 ml of TALON metal affinity resin, as described above. The resin was washed and mixed with purified iPLA₂ protein. The mixture was then incubated (30 min at room temperature with shaking), washed three times, and loaded onto a 5-ml gravity-flow column. Bound proteins were desorbed with elution buffer, collected in 0.5-ml fractions, and analyzed by immunoblotting with antibodies specific for iPLA₂ or CaMKII.

Immunoprecipitation of FLAG-tagged iPLA₂ and CaMKII Expressed in Sf9 Cells—Sf9 cells expressing FLAG-tagged iPLA₂, CaMKII, or both were harvested by centrifugation, washed with phosphate-buffered saline, resuspended in cell lysis buffer supplemented with protease inhibitors, and homogenized by sonication. Cytosol was prepared by centrifugation (15,000 x g, 20 min) and incubated with 100 µl of anti-FLAG M2 affinity resin (2 h, 4 °C, gentle rotation) in the presence or absence of 10 mM Ca²⁺ chelator EGTA. Immunoprecipitated material was recovered by centrifugation and washed four times with wash buffer. Samples immunoprecipitated with anti-FLAG affinity resin were eluted with elution buffer (0.1 M glycine, pH 3.5). Aliquots (30 µl) were analyzed by 10% SDS-PAGE, transferred onto a nylon membrane, and blotted with iPLA₂ or CaMKII antibodies (1:200) followed by horseradish peroxidase-conjugated secondary antibodies (1:30,000).

Enzyme Activity Assays—For CaMKII activity assays, sample buffer (50 mM Pipes, 10 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM ATP, 0.75 mM CaCl₂, 20 µg/ml calmodulin, 20 µM autocamtide-3, 2 µCi of [-³²P]ATP, pH 7.4) was mixed with CaMKII (final volume 50 µl) and incubated (30 °C, 3 min). Assays were initiated by adding His-tagged CaMKII and terminated by adding 100 mM EDTA. Aliquots (30 µl) of the mixture were placed on Whatman P-81 phosphocellulose paper, which was washed with 75 mM H₃PO₄ and air-dried. Phosphorylated autocamtide-3 (a CaMKII model substrate) was quantified by liquid scintillation counting of ³²P. Control assays were performed without added Ca²⁺/calmodulin and with 10 mM EGTA.

The iPLA₂ activity assays were performed as described previously (5, 22). Briefly, 100 µl of sample was added to assay buffer containing 10 mM EGTA. Reactions were initiated by injecting 5 µl of 1-palmitoyl-2-[1-¹⁴C]palmitoyl-sn-glycerol-3-phosphorylcholine (specific activity 50 mCi/mmol, final concentration 5 µM) in ethanol. The assay mixture was incubated (37 °C, 5 min, with shaking), and the reaction was terminated by adding 100 µl of butanol. A 25-µl aliquot of the butanol layer was analyzed by Silica Gel G TLC as described previously (19). The amount of ¹⁴C-labeled free fatty acid was determined by liquid scintillation spectrometry.

[³H]Arachidonic Acid Release Measurements—INS-1 insulinoma cells (5 x 10⁵ cells/well) were prelabeled for 20 h with 0.5 µCi/ml [³H]arachidonic acid and placed in serum-free medium for 1 h. The cells were washed three times with glucose-free RPMI 1640 medium to remove unincorporated radiolabel. Cells were treated with 20 µM BEL or 8 µM KN93 for 30 min before adding RPMI 1640 medium containing 0.5% bovine serum albumin and incubating for 1 h. The medium was then removed and replaced with fresh medium of identical composition, and the cells were incubated for 40 min. Supernatants and cells were separated by centrifugation (500 x g, 5 min) and assayed for ³H content by liquid scintillation spectrometry.

Coimmunoprecipitation of iPLA₂ and CaMKII from INS-1 Cells— Immunoprecipitation was performed with a protein A-agarose slurry that had been washed twice with phosphate-buffered saline, mixed with a 10-µl solution of antibody to CaMKII or to iPLA₂, and incubated (room temperature, 40 min). The mixture was centrifuged, and the supernatant was discarded. The agarose-antibody complex in the precipitate was washed three times with phosphate-buffered saline, mixed with INS-1 cell cytosol, and incubated (overnight, 4 °C, with shaking). Immunoprecipitates were collected by centrifugation, washed, boiled for 5 min in SDS-PAGE sample loading buffer, and analyzed by SDS-PAGE. Proteins were transferred to nylon membranes, and immunoblotting was performed with primary antibody to iPLA₂ or to CaMKII and secondary antibody coupled to horseradish peroxidase, as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Yeast Two-hybrid Screening Indicates That CaMKII Is an iPLA₂-interacting Protein—To identify proteins that interact with iPLA₂, a yeast two-hybrid screen of a rat brain cDNA library was performed using iPLA₂ cDNA cloned from a rat islet cDNA library as bait. The commercially available rat brain cDNA library was used for screening because there are many biochemical similarities between brain and islets, including high iPLA₂ expression (19–22, 34). Colonies were identified that activated transcription of both the HIS3 gene (permitting autotrophic selection) and the lacZ reporter gene (permitting X-gal analysis) in the presence of bait. Such colonies were purified by culture after serial dilution, and the sequences of their cDNA inserts were determined. One colony contained cDNA that encoded 241 residues of rat brain CaMKII N-terminal amino acid sequence (residues 34–274). Several other colonies also contained inserts with the CaMKII sequence. This interaction was examined further because of its likely functional importance, which is suggested by the facts that calmodulin is an important -cell Ca²⁺-binding protein (43) and that -cells express high levels of CaMKII (44, 48), which regulates voltage-operated Ca²⁺ channels involved in insulin secretion (7, 45–47). Insulinoma cell secretion is also potentiated by overexpressing CaMKII (49) or iPLA₂ (22), and iPLA₂ binds calmodulin (2, 34).

Cloning CaMKII from a Rat Islet cDNA Library Reveals Tissue Specificity and a Developmental Profile of CaMKII Isoform Expression—Pancreatic islets express distinct CaMKII isoforms, and adult rat islets express predominantly the CaMKII isoform (48, 49). To determine whether CaMKII isoform(s) expressed in rat islets, like those in rat brain, also interact with iPLA₂, we cloned CaMKII cDNA from adult rat islets. Reverse transcription-PCR was performed using RNA isolated from rat islets as template and a pair of primers designed from regions of cDNA sequence which are conserved in rat and mouse CaMKII. The PCR product was cloned. Sequencing the insert revealed a putative initiation codon (ATG) at the 5'-end, a stop codon (TGA) at the 3'-end, and the entire coding sequence in an intervening single open reading frame. Fig. 1 illustrates the nucleotide and deduced amino acid sequences of the CaMKII isoform cloned from adult rat islets (ACaMKII). Fig. 2A illustrates sequence alignments for CaMKII from rat brain (BCaMKII), adult rat islets (ACaMKII), human -cells (HCaMKII), and neonatal rat islets (NCaMKII).

View larger version (83K): [in this window] [in a new window]   FIG. 1. Nucleotide and deduced amino acid sequences of the CaMKII isoform cloned from an adult rat pancreatic islet cDNA library. The autophosphorylation sites are displayed in bold type. The minimal CaM binding sequence is shaded.

View larger version (54K): [in this window] [in a new window]   FIG. 2. Alignment of deduced amino acid sequences of CaMKII isoforms cloned from rat brain (B), adult rat islets (A), adult human -cells (H), and neonatal rat islets (N). A compares aligned sequences of CaMKII isoforms from various sources. The variable region is shaded. B is a graphical alignment of the sequences. The region of difference in amino acid sequence between ACaMKII and HCaMKII is denoted by an asterisk (*).

To search for other subtypes of CaMKII in adult rat islets, we performed a series of reverse transcription-PCR experiments using RNA from adult rat islets as template and primers designed from various regions of the ACaMKII sequence, but we observed no other CaMKII subtype in adult rat islets (not shown). Adult rat pancreatic islets and adult human -cells thus express mRNA that encodes a CaMKII isoform that differs from those in adult brain or in neonatal islets, and the latter two isoforms also differ from each other. There is thus both tissue specificity and a developmental profile of CaMKII isoform expression, but there is little rat-to-human species heterogeneity in the CaMKII isoform expressed in adult pancreatic islet -cells.

Binary Yeast Two-hybrid Assays Confirm the Interaction between iPLA₂ and CaMKII—To confirm the interaction between iPLA₂ and ACaMKII observed in yeast two-hybrid screening experiments, binary yeast two-hybrid assays were performed. We first used ACaMKII as bait and iPLA₂ as prey. When bait or prey alone was transformed into yeast cells, no colonies grew in medium lacking leucine, tryptophan, and histidine, but when both bait and prey were transformed simultaneously into yeast cells, colonies formed and produced blue reaction products when treated with the chromogenic substrate X-gal (Fig. 3B, left column) that reflect interaction between ACaMKII and iPLA₂. When the bait and prey DNA were switched (so that iPLA₂ was bait, and ACaMKII was prey), similar results were obtained (Fig. 3B, right column). These results reflect a specific interaction between iPLA₂ and CaMKII.

View larger version (57K): [in this window] [in a new window]   FIG. 3. iPLA2 interacts with CaMKII in yeast cells. A illustrates binary yeast two-hybrid assays performed using full-length iPLA2 (or CaMKII) as bait and full-length CaMKII (or iPLA2) as prey. B contains schematic structures of wild-type iPLA2 and CaMKII and of constructs that correspond to N- or C-terminal fragments of each protein. An iPLA2 N-terminal fragment that contains the ankyrin repeat domain and a C-terminal fragment that contains the catalytic site are shown, as are a CaMKII N-terminal fragment that contains the catalytic domain and a C-terminal fragment that contains the association domain. C illustrates binary yeast two-hybrid assays involving coexpression of N- or C-terminal fragments of iPLA2 and of CaMKII as bait/prey pairs together (lanes 1–4) or, in control experiments, expression of an N- or C-terminal fragment of one of the proteins alone (lanes 5–8). The blue colonies reflect specific interactions between two proteins that constitute bait-prey partners in the binary yeast two-hybrid assay. The arrow identifies such blue colonies formed by the -galactosidase reaction product after incubation with the chromogenic substrate X-gal.

CaMKII Can Be Expressed from Its DNA at High Levels in a Baculovirus-Sf9 Cell System and Retains Activity after Purification—Sf9 cells have been used to express iPLA₂ (2, 23, 33), and we found that His-tagged ACaMKII can also be expressed at high levels in Sf9 cells infected with baculovirus containing its cDNA. Cytosol from Sf9 cells infected with baculovirus containing DNA encoding His-tagged ACaMKII was loaded onto TALON metal affinity columns, which were then washed to remove nonadsorbed proteins. Interaction of His-tagged ACaMKII with metal ions on the column resin was then disrupted with imidazole-containing buffers, and this caused desorption of His-tagged ACaMKII protein, which was collected in 0.5-ml fractions of column eluant. Proteins in eluant fractions were analyzed by SDS-PAGE and visualized by immunoblotting using a CaMKII antibody to demonstrate expression and purification of His-tagged ACaMKII (Fig. 4A). Purified His-tagged ACaMKII retained catalytic activity reflected by phosphorylation of the synthetic substrate autocamtide-3 in the presence of added Ca²⁺/CaM. In the absence of added Ca²⁺/CaM little activity was detected (Fig. 4B). The intensity of the immunochemical signal for CaMKII in the eluant fractions (Fig. 4A) correlated well with CaMKII activity in these fractions (Fig. 4B).

View larger version (40K): [in this window] [in a new window]   FIG. 4. Expression of His-tagged CaMKII in Sf9 cells and its adsorption to and desorption from metal affinity columns. In A, cytosol from Sf9 cells that had been infected with baculovirus containing DNA that encodes His-tagged CaMKII was incubated with TALON metal affinity resin, as described under "Experimental Procedures." The resin was then loaded into a gravity-flow column and washed with buffer, and His-tagged CaMKII was eluted with imidazole-containing buffer and collected in 0.5-ml fractions. Proteins in aliquots of the load (L), wash (W), and elution fractions were analyzed by SDS-PAGE, and immunoblotting was then performed with CaMKII antibody. In B, the protein content of each fraction was measured, and CaMKII activity was determined in the presence (+) or absence (-) of added Ca2+/CaM. When Ca2+/CaM was not added, 1 mM EGTA was added. For each assay, an aliquot of each eluant fraction was mixed with assay buffer, peptide substrate (autocamtide-3), and [-32P]ATP, as described under "Experimental Procedures." Displayed values represent the means, and error bars denote S.E. (n = 6).

ACaMKII and iPLA₂ Interact with Each Other When Coexpressed in Sf9 Cells—

View larger version (29K): [in this window] [in a new window]   FIG. 5. iPLA2 interacts with CaMKII when the two proteins are coexpressed in Sf9 insect cells. In A, Sf9 cells were infected simultaneously with baculovirus containing full-length HisCaMKII and iPLA2 DNAs, cultured, and then homogenized, as described under "Experimental Procedures." Cytosol prepared from homogenates was loaded onto a TALON metal affinity column and washed with buffer. HisCaMKII was eluted with imidazole-containing buffer and collected in 0.5-ml fractions. The proteins in aliquots of load (L), wash (W), and elution fractions were analyzed by SDS-PAGE, and immunoblotting was performed with antibodies to iPLA2 (upper panel) or HisCaMKII (lower panel). In B, an aliquot of load, wash, or elution fractions was added to assay buffer containing 10 mM EGTA, 1 mM ATP, and 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine substrate. Reactions to measure iPLA2 activity were performed and terminated as described under "Experimental Procedures," and released [14C]linoleic acid was isolated by TLC and measured by liquid scintillation spectrometry. Displayed values represent the means, and error bars denote S.E. (n = 6). In C, an aliquot of load, wash, or elution fractions was mixed with assay buffer containing 0.1 mM ATP, 0.75 mM CaCl2, 20 µg/ml calmodulin, 20 µM autocamtide-3, and 2 µCi of [-32P]ATP and incubated at 30 °C for 3 min to determine CaMKII activity. An aliquot of the reaction mixture was applied to phosphocellulose paper, which was then washed. CaMKII activity was calculated from the amount of phosphorylated autocamtide-3, as determined by liquid scintillation spectrometric measurement of 32P content. Displayed values represent the means, and error bars denote S.E. (n = 6).

View larger version (42K): [in this window] [in a new window]   FIG. 6. The stoichiometry of the interaction between iPLA2 and CaMKII. In A, purified, recombinant, His-tagged CaMKII from Sf9 cells was mixed with TALON metal affinity resin, and the resin was then washed. Bound CaMKII was measured with a Coomassie protein assay kit. iPLA2 protein expressed in Sf9 cells was purified as described previously (33), and 850 µg (10 nmol) of the protein was mixed with metal affinity resin to which 570 µg (10 nmol) of His-tagged CaMKII had been adsorbed. The mixture was incubated at room temperature for 30 min with shaking, and the resin was washed and loaded onto a gravity-flow column. Bound proteins were eluted with imidazole-containing buffer and collected in 0.5-ml fractions. Proteins in aliquots of the load (L), wash (W), and elution fractions were analyzed by 10% SDS-PAGE, and immunoblotting was then performed with antibodies specific for iPLA2 (upper panel) or CaMKII (lower panel). In B, 200 µl of metal affinity resin slurry to which 150 µg (2.64 nmol) of His-tagged CaMKII had been adsorbed was mixed with FLAG-tagged iPLA2 in amounts that varied from 0 to 5.28 nmol. The mixture was incubated at 4 °C overnight with shaking, and the resin was then washed to remove noncomplexed proteins. Proteins were eluted from the metal affinity resin and analyzed by 10% SDS-PAGE. Immunoblotting was then performed with primary antibodies specific for iPLA2 (upper panel) or CaMKII (lower panel).

The Stoichiometry of the Interaction between iPLA₂ and CaMKII—

The Calmodulin Antagonist W13 Does Not Prevent the Interaction of CaMKII with iPLA₂—Because both iPLA₂ and CaMKII have calmodulin binding domains, calmodulin might mediate the interaction between these two proteins by forming a ternary complex. To evaluate this possibility, the interaction between iPLA₂ and CaMKII was examined in the presence and absence of added calmodulin. FLAG-tagged iPLA₂ was expressed in Sf9 cells and purified with a FLAG M kit (Sigma). FLAG-tagged iPLA₂ was then mixed with TALON metal affinity resin that had previously been loaded with His-tagged ACaMKII in the presence or absence of calmodulin and then washed. When calmodulin was not added, the calmodulin antagonist W13 was added to block binding of any contaminating calmodulin to the target proteins. Adsorbed proteins were eluted from the metal affinity resin with imidazole-containing buffer, and proteins in eluant fractions were analyzed by SDS-PAGE and immunoblotting with iPLA₂-specific antibody. Fig. 7A illustrates that added calmodulin is not required for the interaction between iPLA₂ and CaMKII and that this interaction is not prevented by the calmodulin antagonist W13. These results are consistent with the findings that the CaM binding site(s) of iPLA₂ reside in its C-terminal domain (2) and that the interaction of iPLA₂ and CaMKII occurs between their N-terminal domains (Fig. 3C).

View larger version (48K): [in this window] [in a new window]   FIG. 7. The interaction between iPLA2 and CaMKII does not require added calmodulin and is not prevented by a calmodulin antagonist or the Ca2+ chelator EGTA. In A, FLAG-tagged iPLA2 expressed in Sf9 cells was purified with a FLAG M kit. Recombinant, purified, His-tagged CaMKII was adsorbed to TALON metal affinity resin. The FLAG-tagged iPLA2 was incubated with the resin to which His-tagged CaMKII had been adsorbed at room temperature for 30 min with shaking in the presence of 0.25 mM Ca2+ and 1 mM CaM (upper left panel) or 0.4 mM calmodulin antagonist W13 (upper right panel). The resin was then washed, and adsorbed proteins were eluted with imidazole-containing buffer. Aliquots of wash and elution fractions were analyzed by SDS-PAGE and immunoblotting with antibody specific for iPLA2 or CaMKII. In B, cytosol was prepared from baculovirus-infected Sf9 cells that expressed FLAG-iPLA2, CaMKII without a FLAG tag, or the control fusion protein N-terminal FLAG-tagged alkaline phosphatase (Flag-BAP). Binary mixtures of cytosols were prepared and incubated with anti-FLAG M2 affinity resin for 2 h at 4 °C in the presence or absence of 10 mM EGTA. Immunoprecipitated material was recovered by centrifugation and washed four times with wash buffer. Samples immunoprecipitated with anti-FLAG affinity resin were eluted with buffer containing FLAG peptide. Proteins in the eluant were analyzed by 10% SDS-PAGE and transferred onto a nylon membrane, and immunoblotting was performed with iPLA2 or CaMKII antibodies.

Chelator EGTA Does Not Prevent the Interaction between iPLA₂ and CaMKII—

The Activities of Both iPLA₂ and CaMKII Increase When the Proteins Associate with Each Other—Because results from yeast two-hybrid assays and protein pull-down experiments indicate that the ACaMKII and iPLA₂ proteins interact with each other, we next determined whether this interaction affects the catalytic activity of either enzyme. PLA₂ activity assays involved measuring radiolabeled free fatty acid release from phospholipid substrates and were performed in buffer supplemented with 10 mM EGTA and 10 mM ATP with no added Ca²⁺. Under these conditions, adding purified, recombinant, His-tagged ACaMKII to purified, recombinant, His-tagged iPLA₂ resulted in a statistically significant increase in PLA₂ activity (Fig. 8A). Results from dose-response studies under conditions where [iPLA₂] was constant and [CaMKII] was varied indicate that the maximal iPLA₂ activity is achieved at a 1:1 molar ratio of the two enzymes (Fig. 8B), which is consistent with the finding in Fig. 6B that iPLA₂ and CaMKII form a complex with 1:1 stoichiometry.

View larger version (33K): [in this window] [in a new window]   FIG. 8. Influence of CaMKII on iPLA2 activity. In A, His-tagged CaMKII and His-tagged iPLA2 were purified with TALON metal affinity columns. Purified, His-tagged CaMKII, His-tagged iPLA2, or both were then added to buffer containing 10 mM ATP, 10 mM EGTA, and the radiolabeled substrate 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine. The iPLA2 activity was then calculated from released [14C]linoleate as in Fig. 5. Values are represented as the mean ± S.E. (n = 4). Statistical significance is denoted by an asterisk (*), which indicates a p value < 0.05. In B, iPLA2 activity assays were performed in the presence of 0.5 nmol of iPLA2 and the indicated amounts of CaMKII. Displayed values represent the means ± S.E. (n = 3).

View larger version (41K): [in this window] [in a new window]   FIG. 9. Influence of iPLA2 on CaMKII activity. CaMKII activity was measured in the presence (+) or absence (-) of iPLA2,Ca2+, and CaM. For each assay, His-tagged CaMKII was mixed with assay buffer containing ATP, autocamtide-3 substrate, and [-32P]ATP. CaMKII activity was calculated from the amount of phosphorylated autocamtide-2 as in Fig. 4. Values are represented as the mean ± S.E. (n = 5). Statistical significance is denoted by an asterisk, which indicates a p value < 0.01 compared with the group to which no iPLA2 was added (-iPLA2).

Arachidonic Acid and 2-Lysophosphatidylcholine Inhibit CaMKII Activity—

View larger version (40K): [in this window] [in a new window]   FIG. 10. Inhibition of CaMKII activity by arachidonic acid (AA) and lysophosphatidylcholine (LPC). CaMKII activity was measured in the presence or absence of arachidonic acid or lysophosphatidylcholine as in Fig. 4. For each assay, two separate measurements were performed simultaneously, one in the presence and the other in the absence of added Ca2+/CaM. Activity values were calculated from the difference between these two measurements and are represented as the mean ± S.E. (n = 3). Statistical significance is denoted by an asterisk (*), which indicates a p value < 0.05 compared with control.

Arachidonic Acid Release from INS-1 Insulinoma Cells Is Suppressed by Inhibitors of CaMKII and iPLA₂—

View larger version (47K): [in this window] [in a new window]   FIG. 11. Arachidonic acid (AA) release from INS-1 cells is suppressed by inhibitors of iPLA2 and CaMKII. INS-1 cells were prelabeled with [3H]arachidonic acid and then washed free of unincorporated radiolabel. The labeled cells were then treated without or with 20 µM BEL or 8 µM KN93. [3H]Arachidonic acid release was then measured as described under "Experimental Procedures." Release values are represented as the mean ± S.E. (n = 3). Statistical significance is denoted by an asterisk (*) or a double asterisk (**), which indicates a p value < 0.05 or 0.01, respectively, compared with control.

CaMKII and iPLA₂ Form a Complex in Insulin-secreting Cells—

View larger version (34K): [in this window] [in a new window]   FIG. 12. Forskolin stimulates complex formation between iPLA2 and CaMKII in INS-1 insulinoma cells. A illustrates coimmunoprecipitation of iPLA2 and CaMKII. In lane 1 of the left panel, control preimmune serum was used for sham immunoprecipitation of INS-1 cell cytosol as a negative control. In lanes 2 and 3 of the left panel, cytosol from INS-1 cells (lane 2) or from INS-1 cells that overexpress iPLA2 (lane 3) were incubated with anti-CaMKII antibody attached to protein A-agarose. The immunoprecipitate was collected by centrifugation, washed, boiled in SDS-PAGE sample loading buffer, and analyzed by SDS-PAGE. After transfer of proteins to nylon membranes, immunoblotting was performed with antibodies against iPLA2 (upper blot) or CaMKII (lower blot). Similar results were obtained from the reverse immunoprecipitation experiment (right panel of A), in which cytosol from INS-1 cells (lane 2) or INS-1 cells that overexpress iPLA2 (lane 3) was immunoprecipitated with iPLA2 antibody-protein A-agarose. In lane 1 of B, control preimmune serum was used in sham immunoprecipitation of INS-1 cell cytosol as a negative control. In lanes 2 and 3 of B, INS-1 cells that overexpress iPLA2 were incubated without (lane 2) or with (lane 3) 4 µM forskolin. The cytosol was then immunoprecipitated with CaMKII antibody-protein A-agarose. After SDS-PAGE analyses of the immunoprecipitates, immunoblotting was performed with iPLA2 antibody (upper blot) or with CaMKII antibody (lower blot).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We first observed the complex between iPLA₂ and CaMKII by using iPLA₂ as bait in yeast two-hybrid screening of a rat brain cDNA library. Formation of a complex between the two enzymes was confirmed in binary yeast two-hybrid assays in which iPLA₂ was bait and CaMKII was prey and in the converse assay configuration in which CaMKII was bait and iPLA₂ was prey. Pull-down assays with recombinant, His-tagged proteins adsorbed to metal affinity matrices also provided direct evidence for the physical association of CaMKII and iPLA₂. These findings clearly demonstrate that iPLA₂ and CaMKII interact with each other. We have demonstrated here that an immunoprecipitatable complex of these two enzymes exists in insulinoma cells and that the amount of the complex increases upon stimulation of intact -cells with forskolin, which is an adenylyl cyclase activator that amplifies insulin secretion and induces subcellular redistribution of iPLA₂ in -cells (22).

We have demonstrated previously that depletion of internal Ca²⁺ stores causes activation of iPLA₂ in -cells (23) and in vascular smooth muscle cells (24). It has been demonstrated recently that iPLA₂ participates in SOC entry from the extracellular space (25, 32), and this process is required for insulin secretion (26–31). Lysophospholipid products of iPLA₂ activate SOC channels that mediate capacitative Ca²⁺ influx (25, 32), and CaMKII also affects Ca²⁺ fluxes by potentiating SOC channel activity (58) and regulating T-type voltage-operated calcium channels (59). Our findings indicate that iPLA₂ interacts with the specific isoform of CaMKII that is expressed in -cells and that this interaction affects activities of both iPLA₂ and CaMKII. This suggests that CaMKII and iPLA₂ form a signaling complex, and this complex represents a potential means to regulate SOC entry.

Such a complex could orchestrate bidirectional signals that result in Ca²⁺ influx into -cells and insulin secretion. Upon complexation with iPLA₂, CaMKII could displace CaM from iPLA₂ (2, 23) and increase iPLA₂ activity by relieving tonic inhibition of the enzyme by CaM (2, 8, 23, 24). Lysophospholipids activate SOC channels (32) and are produced by iPLA₂ action. Both the CaMKII inhibitor KN93 and the iPLA₂ inhibitor BEL inhibit insulin secretion (9, 10, 19–22), and both compounds are also demonstrated here to inhibit arachidonate release from INS-1 insulinoma cells, which supports the possibility that iPLA₂ and CaMKII form a signaling complex in -cells. CaMKII is capable of decoding the frequency of oscillations in intracellular [Ca²⁺] by its autophosphorylation (54, 61). Autophosphorylated CaMKII has 1,000-fold greater affinity for Ca²⁺/CaM than does nonphosphorylated CaMKII (62). CaMKII activity is affected by association with iPLA₂ (Fig. 9) and by products of iPLA₂ action (Fig. 10), including lysophospholipids that also modulate Ca²⁺ channel activities (63, 64). The interaction between CaMKII and iPLA₂ at the -cell plasma membrane could thus affect Ca²⁺ influx and cytosolic [Ca²⁺], which is a key determinant of insulin secretion (26–31).

Alignment of the deduced amino acid sequences of HCaMKII (48) and ACaMKII, which have been cloned from adult human -cells and adult rat islets, respectively, reveals more than 99% sequence conservation, and this indicates that there is little species-to-species variation in pancreatic islet -cell expression of CaMKII isoforms. The expression pattern of CaMKII isoforms does change with development in islets, as reflected by the difference in isoforms expressed in neonatal and adult islets, and there is also tissue-to-tissue heterogeneity in CaMKII isoform expression, as reflected by the different isoforms expressed by islets and brain. The high degree of CaMKII sequence conservation between rat and human islets and the fact that islets express only a single, predominant CaMKII isoform is consistent with the possibility that the islet isoform has a special function in -cells and that iPLA₂ and other proteins that interact with this enzyme modulate that function. It is thus of interest that expression of both iPLA₂ and of CaMKII has recently been found to occur at the same stage of differentiation of pancreatic progenitor cells to endocrine progenitor cells during development (60).

	FOOTNOTES

 
^* 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.

¹ The abbreviations used are: PLA₂, phospholipase A₂; BEL, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one; CaM, calmodulin; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; iPLA₂, calcium-independent phospholipase A₂; Pipes, 1,4-piperazinediethanesulfonic acid; SOC, store-operated calcium channel; X-gal: 5-bromo-4-chloro-3-indolyl--D-galactoside.

	ACKNOWLEDGMENTS

 
We thank Dr. Mary Wohltmann, Sheng Zhang, Wu Jin, and Alan Bohrer for excellent technical assistance, and we are grateful to Denise Kampwerth for secretarial assistance.

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