Dawn E. Tucker

Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2α in regulating interfacial kinetics and binding and cellular function

Group IVA cytosolic phospholipase A2 (cPLA2α) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA2α (IMLF-/-) were reconstituted with wild type and cPLA2α mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP2) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA2α translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA2α to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA2α to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF-/- expressing phosphorylation site (S505A) and PIP2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA2α, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA2α reduced activation by PIP2 but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA2α hydrolytic activity.

Function, Activity, and Membrane Targeting of Cytosolic Phospholipase A2ζ in Mouse Lung Fibroblasts

Group IVA cytosolic phospholipase A2 (cPLA2α) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA2α–/– lung fibroblasts stimulated with A23187 or serum. cPLA2α+/+ fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA2α–/– fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA2 α–/– fibroblasts was inhibited by the cPLA2α inhibitors pyrrolidine-2 (IC50, 0.03 μm) and Wyeth-1 (IC50, 0.1 μm), implicating another C2 domain-containing group IV PLA2. cPLA2 α–/– fibroblasts contain cPLA2β and cPLA2ζ but not cPLA2ϵ or cPLA2δ. Purified cPLA2ζ exhibited much higher lysophospholipase and PLA2 activity than cPLA2β and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA2β. In contrast to cPLA2β, cPLA2ζ expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA2ζ exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA2α and has been identified as the PLA2 responsible for calcium-induced fatty acid release and prostaglandin E2 production from cPLA2 α–/– lung fibroblasts. In response to ionomycin, EGFP-cPLA2ζ translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA2α translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.

Group IVC cytosolic phospholipase A2γ is farnesylated and palmitoylated in mammalian cells

Cytosolic phospholipase A2γ (cPLA2γ) is a member of the group IV family of intracellular phospholipase A2 enzymes, but unlike the well-studied cPLA2α, it is constitutively bound to membrane and is calcium independent. cPLA2γ contains a C-terminal CaaX sequence and is radiolabeled by mevalonic acid when expressed in cPLA2α-deficient immortalized lung fibroblasts (IMLF−/−). The radiolabel associated with cPLA2γ was identified as the farnesyl group. The protein farnesyltransferase inhibitor BMS-214662 prevented the incorporation of [3H]mevalonic acid into cPLA2γ and partially suppressed serum-stimulated arachidonic acid release from IMLF−/− and undifferentiated human skeletal muscle (SkMc) cells overexpressing cPLA2γ, but not from cells overexpressing cPLA2α. However, BMS-214662 did not alter the amount of cPLA2γ associated with membrane. These results were consistent in COS cells expressing the C538S cPLA2γ prenylation mutant. cPLA2γ also contains a classic myristoylation site and several potential palmitoylation sites and was found to be acylated with oleic and palmitic acids but not myristoylated. Immunofluorescence microscopy revealed that cPLA2γ is associated with mitochondria in IMLF−/−, SkMc cells, and COS cells.

Activation of Cytosolic Phospholipase A2α in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2α). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (ΔhlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and ΔhlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and ΔhlyLM correlates with diminished MAPK activation. WTLM but not ΔhlyLM increases intracellular calcium, which is implicated in regulation of cPLA2α. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2α+/+ but not cPLA2α-/- macrophages in response to WTLM and ΔhlyLM. Tumor necrosis factor (TNF)-α production is significantly lower in cPLA2α+/+ than in cPLA2α-/- macrophages infected with WTLM and ΔhlyLM. Treatment of infected cPLA2α+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFα production to the level produced by cPLA2α-/- macrophages implicating prostaglandins in TNFα down-regulation. Therefore activation of cPLA2α in macrophages may impact immune responses to L. monocytogenes.

Functional characterization of mutations in inherited human cPLA2 deficiency

Group IVA cytosolic phospholipase A(2) (cPLA(2)α) catalyzes the first step in the arachidonic acid cascade leading to the synthesis of important lipid mediators, the prostaglandins and leukotrienes. We previously described a patient deficient in cPLA(2)α activity, which was associated with mutations in both alleles encoding the enzyme. In this paper, we describe the biochemical characterization of each of these mutations. Using saturating concentrations of calcium, we showed that the R485H mutant was nearly devoid of any catalytic activity, that the S111P mutation did not affect the enzyme activity, and that the known K651R polymorphism was associated with activity slightly higher than that of the wild type. Using MDCK cells, we showed that translocation to the Golgi in response to serum activation was impaired for the S111P mutant but not for the other mutants. Using immortalized mouse lung fibroblasts lacking endogenous cPLA(2)α activity, we showed that both mutations S111P and R485H/K651R caused a profound defect in the enzyme catalytic activity in response to cell stimulation with serum. Taken together, our results show that the S111P mutation hampers calcium binding and membrane translocation without affecting the catalytic activity, and that the mutation R485H does not affect membrane translocation but blocks catalytic activity that leads to inactivation of the enzyme. Interestingly, our results show that the common K651R polymorphism confers slightly higher activity to the enzyme, suggesting a role of this residue in favoring a catalytically active conformation of cPLA(2)α. Our results define how the mutations negatively influence cPLA(2)α function and explain the inability of the proband to release arachidonic acid for eicosanoid production.

Regulation of cytosolic phospholipase A2α by hsp90 and a p54 kinase in okadaic acid-stimulated macrophages

In resident mouse peritoneal macrophages, group IVA cytosolic phospholipase A2 (cPLA2α) mediates arachidonic acid (AA) release and eicosanoid production in response to diverse agonists such as A23187, phorbol myristate acetate, zymosan, and the enterotoxin, okadaic acid (OA). cPLA2α is regulated by phosphorylation and by calcium that binds to the C2 domain and induces translocation from the cytosol to membranes. In contrast, OA activates cPLA2α‐induced AA release and translocation to the Golgi in macrophages without an apparent increase in calcium. Inhibitors of heat shock protein 90 (hsp90), geldanamycin, and herbimycin blocked AA release in response to OA but not to A23187, PMA, or zymosan. OA, but not the other agonists, induced activation of a cytosolic serine/threonine 54‐kDa kinase (p54), which phosphorylated cPLA2α in in‐gel kinase assays and was associated with cPLA2α in immunoprecipitates. Activation of the p54 kinase was inhibited by geldanamycin. The kinase coimmunoprecipitated with hsp90 in unstimulated macrophages, and OA induced its loss from hsp90, concomitant with its association with cPLA2α. The results demonstrate a role for hsp90 in regulating cPLA2α‐mediated AA release that involves association of a p54 kinase with cPLA2α upon OA stimulation.

Activation of group IVC phospholipase A2 by polycyclic aromatic hydrocarbons induces apoptosis of human coronary artery endothelial cells

Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A2 (PLA2) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA2 in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA2 inhibited both 3H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA2. Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA2.

Harnessing Teams and Technology to Improve Outcomes in Infants With Single Ventricle

Infants with single ventricle require staged cardiac surgery, with stage I typically performed shortly after birth, stage II at 4 to 6 months of age, and stage III at 3 to 5 years of age. There is a high risk of interstage mortality and morbidity after infants are discharged from the hospital between stages I and II. Traditional home monitoring requires caregivers to record measurements of weight and oxygen saturation into a binder and requires families to assume a surveillance role. We have developed a tablet PC-based solution that provides secure and nearly instantaneous transfer of patient information to a cloud-based server, with the capacity for instant alerts to be sent to the caregiver team. The cloud-based IT infrastructure lends itself well to being able to be scaled to multiple sites while maintaining strict control over the privacy of each site. All transmitted data are transferred to the electronic medical record daily. The system conforms to recently released Food and Drug Administration regulation that pertains to mobile health technologies and devices. Since this platform was developed in March 2014, 30 patients have been monitored. There have been no interstage deaths. The experience of care providers has been unanimously positive. The addition of video has added to the use of the monitoring program. Of 30 families, 23 expressed a preference for the tablet PC over the notebook, 3 had no preference, and 4 preferred the notebook to the tablet PC.