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AS160 (TBC1D4) is a known Akt substrate that is phosphorylated downstream of insulin action and that leads to regulated traffic of GLUT4. As GLUT4 vesicle fusion with the plasma membrane is a highly regulated step in GLUT4 traffic, we investigated whether AS160 and 14-3-3 interactions are involved in this process. Fusion was inhibited by a human truncated AS160 variant that encompasses the first N-terminal phosphotyrosine-binding (PTB) domain, by either of the two N-terminal PTB domains, and by a tandem construct of both PTB domains of rat AS160. We also found that in vitro GLUT4 vesicle fusion was strongly inhibited by the 14-3-3-quenching inhibitors R18 and fusicoccin. To investigate the mode of interaction of AS160 and 14-3-3, we examined insulin-dependent increases in the levels of these proteins on GLUT4 vesicles. 14-3-3 gamma was enriched on insulin-stimulated vesicles, and its binding to AS160 on GLUT4 vesicles was inhibited by the AS160 tandem PTB domain construct. These data suggest a model for PTB domain action on GLUT4 vesicle fusion in which these constructs inhibit insulin-stimulated 14-3-3 gamma interaction with AS160 rather than AS160 phosphorylation.

Type II-secreted phospholipase A(2) (type II-sPLA(2)) is expressed in smooth muscle cells during atherosclerosis or in response to interleukin-1beta. The present study shows that the induction of type II-sPLA(2) gene by interleukin-1beta requires activation of the NFkappaB pathway and cytosolic PLA(2)/PPARgamma pathway, which are both necessary to achieve the transcriptional process. Interleukin-1beta induced type II-sPLA(2) gene dose- and time-dependently and increased the binding of NFkappaB to a specific site of type II-sPLA(2) promoter. This effect was abolished by proteinase inhibitors that block the proteasome machinery and NFkappaB nuclear translocation. Type II-sPLA(2) induction was also obtained by free arachidonic acid and was blocked by either AACOCF(3), a specific cytosolic-PLA(2) inhibitor, PD98059, a mitogen-activated protein kinase kinase inhibitor which prevents cytosolic PLA(2) activation, or nordihydroguaiaretic acid, a lipoxygenase inhibitor, but not by the cyclooxygenase inhibitor indomethacin, suggesting a role for a lipoxygenase product. Type II-sPLA(2) induction was obtained after treatment of the cells by 15-deoxy-Delta(12,14)-dehydroprostaglandin J(2), carbaprostacyclin, and 9-hydroxyoctadecadienoic acid, which are ligands of peroxisome proliferator-activated receptor (PPAR) gamma, whereas PPARalpha ligands were ineffective. Interleukin-1beta as well as PPARgamma-ligands stimulated the activity of a reporter gene containing PPARgamma-binding sites in its promoter. Binding of both NFkappaB and PPARgamma to their promoter is required to stimulate the transcriptional process since inhibitors of each class block interleukin-1beta-induced type II-sPLA(2) gene activation. We therefore suggest that NFkappaB and PPARgamma cooperate at the enhanceosome-coactivator level to turn on transcription of the proinflammatory type II-sPLA(2) gene.

Secretory phospholipases A2(sPLA2s) represent a rapidly expanding family of structurally related enzymes found in mammals as well as in insect and snake venoms. In this report, a cDNA coding for a novel sPLA2 has been isolated from human fetal lung, and its gene has been mapped to chromosome 16p13.1-p12. The mature sPLA2protein has a molecular mass of 13.6 kDa, is acidic (pI 5.3), and made up of 123 amino acids. Key structural features of the sPLA2include: (i) a long prepropeptide ending with an arginine doublet, (ii) 16 cysteines located at positions that are characteristic of both group I and group II sPLA2s, (iii) a C-terminal extension typical of group II sPLA2s, (iv) and the absence of elapid and pancreatic loops that are characteristic of group I sPLA2s. Based on these structural properties, this sPLA2 appears as a first member of a new group of sPLA2s, called group X. A 1.5-kilobase transcript coding for the human group X (hGX) sPLA2 was found in spleen, thymus, and peripheral blood leukocytes, while a less abundant 0.8-kilobase transcript was detected in the pancreas, lung, and colon. When the hGX sPLA2cDNA was expressed in COS cells, sPLA2 activity preferentially accumulated in the culture medium, indicating that hGX sPLA2 is an actively secreted enzyme. It is maximally active at physiological pH and with 10 mm Ca2+. hGX sPLA2 prefers phosphatidylethanolamine and phosphatidylcholine liposomes to those of phosphatidylserine.