Kazuaki Tatei

Identification of 9-hydroxyoctadecadienoic acid and other oxidized free fatty acids as ligands of the G protein-coupled receptor G2A.

G2A is a G protein-coupled receptor that is predominantly expressed in lymphoid tissues and macrophages. G2A can be induced by diverse stimuli to cause cell cycle arrest in the G2/M phase in pro-B and T cells. G2A is also expressed in macrophages within atherosclerotic lesions, suggesting G2A involvement in atherosclerosis. Recently, G2A was discovered to possess proton-sensing ability. In this paper, we report another function of G2A, that is, as a receptor for 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized free fatty acids. G2A, expressed in CHO-K1 or HEK293 cells, showed 9-HODE-induced intracellular calcium mobilization, inositol phosphate accumulation, inhibition of cAMP accumulation, [35S]guanosine 5′-3-O-(thio)triphosphate binding, and MAP kinase activation. Furthermore, G2A was activated by various oxidized derivatives of linoleic and arachidonic acids, but it was weakly activated by cholesteryl-9-HODE. Oxidized phosphatidylcholine (1-palmitoyl-2-linoleoyl) when hydrolyzed with phospholipase A2 also evoked intracellular calcium mobilization in G2A-expressing cells. These results indicate that G2A is activated by oxidized free fatty acids produced by oxidation and subsequent hydrolysis of phosphatidylcholine or cholesteryl linoleate. Thus, G2A might have a biological role in diverse pathological conditions including atherosclerosis.

CDK5-dependent Phosphorylation of the Rho Family GTPase TC10α Regulates Insulin-stimulated GLUT4 Translocation

Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10α on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10α was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10α) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10α but not T197A-TC10α and in parallel, T197D-TC10α but not T197A-TC10α depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10α in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10α through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10α cycling between lipid raft and non-lipid raft compartments.

Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca(2+) for its maximum activity. The K(M) values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 microM, and the V(max) values were 27.3 and 1.62 micromol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.

Protein purification and cloning of diacylglycerol lipase from rat brain.

Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia.

Identification and Analysis of Two Splice Variants of Human G2A Generated by Alternative Splicing

G2A is a G protein-coupled receptor that can be induced by various stressors. G2A is reported to have proton-sensing activity that mediates intracellular inositol phosphate (IP) accumulation with decreasing pH. We previously showed that G2A is also activated by some oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). In this study, we identified a novel alternative splice variant of G2A (G2A-b) that has a partially different N terminus compared with the G2A originally reported (G2A-a). The two splice variants of G2A show similar tissue distributions, but G2A-b is expressed more abundantly. There was no difference between the two variants in 9-HODE-induced cellular responses, such as intracellular calcium mobilization and GDP/GTP exchange of Gα protein, and in proton-sensitive IP accumulation. However, G2A-b showed a higher basal activity in terms of IP accumulation. Mutagenesis study revealed that the difference in the basal activity is attributable to the K7 residue that exists only in G2A-a. We further demonstrated that an R42A mutation largely impaired both the basal and proton-sensing activities, but did not affect the 9-HODE-induced intracellular calcium increase. Taken together, we found an additional novel G2A variant (G2A-b) that is the major transcript with functional response to ligand stimulation as well as G2A-a, and succeeded in discriminating proton-sensing and oxidized fatty acid-sensing activities of G2A.

Expression pattern of class I phosphoinositide 3-kinase and distribution of its product, phosphatidylinositol-3, 4, 5-trisphosphate, during Drosophila embryogenesis

The class I phosphoinositide 3-kinase (PI3K) can be activated by a large variety of extracellular stimuli and is responsible for generating phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P(3)) from phosphatidylinositol-4,5-bisphosphate at the plasma membrane. The expression pattern of the class I PI3K and distribution of PI(3,4,5)P(3), visualized by its specific binding protein, GRP1-PH, were examined during Drosophila embryogenesis. We found that the RNA of Pi3K21B, encoding the Drosophila p60 regulatory subunit of the class I PI3Ks, was expressed maternally and expressed primarily in pole cells after cellularization until completion of germ band elongation. The RNA of Pi3K92E, encoding the Drosophila p110 catalytic subunit of the class I PI3Ks, was also expressed maternally. During gastrulation, its transcript level became lower and was slightly enriched in invaginating cells. Both Pi3K21B and Pi3K92E were expressed ubiquitously after germ band elongation and persisted during germ band shortening. PI(3,4,5)P(3) was distributed at the apical region of the invaginating cells during gastrulation. These findings suggest a possible involvement of class I PI3K and PI(3,4,5)P(3) in the regulation of invagination during gastrulation.