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Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology is maintained strictly for their proper functioning. The roles of lipids are not only to generate the membrane, but also to provide the specific domains for signal transduction, or to transmit signals as second messengers. By using a liquid chromatography-electrospray ionization mass spectrometry (LC-MS)/MS method, we here analyzed sphingolipids in MDCK cysts under various conditions. Our result showed that, compared to the three-dimensional cyst, the two-dimensional MDCK sheet is relatively enriched in sphingolipids. During cystogenesis, the contents of sphingomyelin (SM) and lactocylceramide (LacCer)-but, none those of ceramide, hexocylceramide, or GM3-are altered depending on their acyl chains. While the total SM is decreased more efficiently by SMS-1 knockdown than by SMS-2 knockdown, depletion of SMS-2, but not SMS-1, inhibits cyst growth. Finally upon the switching on of activated K-Ras expression which induces luminal cell filling, ceramide and LacCer are increased. Our parallel examinations of the microarray data for mRNA of sphingolipid metabolic enzymes failed to fully explain the remodelling of the sphingolipids of MDCK cysts. However, these results should be useful to investigate the cell-type- and structure-specific lipid metabolism.

Differential changes in various sphingolipids between TNF-induced necroptosis and apoptosis were investigated using liquid chromatography-tandem mass spectrometry. A marked increase in d18:1/16:0 ceramide was detected in U937 cells treated with TNF in the presence of Z-VAD-fmk (VAD). The level of d18:1/16:0 ceramide in necroptosis was almost twice as high as that in apoptosis after 4h, while an increase in PI-positive cells was observed only in necroptosis within 4h. Necroptosis-resistant U937 (UNR) sublines were established to more clearly discriminate between necroptosis and apoptosis. All three UNR sublines were almost completely resistant to the treatment with TNF/VAD, but were as sensitive to TNF-induced apoptosis as parental cells. The expression of RIP3, a pivotal kinase in necroptosis, was lost in all three UNR sublines. In contrast with the large increase in ceramide levels in TNF/VAD-treated parental cells, they were only slightly increased in UNR cells. Although intracellular levels of reactive oxygen species (ROS) were elevated in both necroptosis and apoptosis, the treatment with butylated hydroxyanisole, an antioxidant, significantly inhibited increases in ceramide levels and PI-positive cells only in necroptosis. These results implicate that the ROS-induced large increase in ceramide levels may play a role in plasma membrane permeabilization in TNF-induced necroptosis.

Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.

Sphingolipids accumulate in plasma membrane microdomain sites, such as caveolae or lipid rafts. Such microdomains are considered to be important nexuses for signal transduction, although changes in the microdomain lipid components brought about by signaling are poorly understood. Here, we applied a cationic colloidal silica bead method to analyze plasma membrane lipids from monolayer cells cultured in a 10 cm dish. The detergent-resistant fraction from the silica bead-coated membrane was analyzed by LC-MS/MS to evaluate the microdomain lipids. This method revealed that glycosphingolipids composed the microdomains as a substitute for sphingomyelin (SM) in mouse embryonic fibroblasts (tMEFs) from an SM synthase 1/2 double KO (DKO) mouse. The rate of formation of the detergent-resistant region was unchanged compared with that of WT-tMEFs. C2-ceramide (Cer) stimulation caused greater elevations in diacylglycerol and phosphatidic acid levels than in Cer levels within the microdomains of WT-tMEFs. We also found that lipid changes in the microdomains of SM-deficient DKO-tMEFs caused by serum stimulation occurred in the same manner as that of WT-tMEFs. This practical method for analyzing membrane lipids will facilitate future comprehensive analyses of membrane microdomain-associated responses.

Sphingomyelin synthases (SMSs) are enzymes converting ceramide to sphingomyelin. The behavioral phenotype attributed to their disruption has not been well described. We examined learning ability and hippocampal synaptic plasticity in mice deficient in SMS2 (SMS2 KO). In context-dependent fear learning and novel object recognition test, no difference in learning ability was detected in SMS2 KO and wild-type (WT) mice. By contrast, achievement of the Morris water maze (MWM) test was deteriorated in SMS2 KO mice. In the hippocampal CA1, while the basic synaptic transmission was normal, both short- and long-term synaptic plasticity was moderately suppressed. We interpret that the MWM test taking place in wet environment may represent learning paradigm under more stressful condition than those performed in dry conditions, and that the learning ability of SMS2 KO mice failed to manifest itself fully in stressful situations. In agreement, forced swimming induced depression-like behavior more easily in SMS2 KO mice. Mass spectrometry suggested a slightly altered species distribution of ceramide in the hippocampus of SMS2 KO mice. These findings support the proposal that altered synthesis of ceramide, which is the substrate of SMS2 and therefore expected to be modified in SMS2 KO mice, is associated with depression-like tendency in animal models and depressive disorder in humans.

Arachidonic acid (AA) is remarkably enriched in phosphatidylinositol (PI). Studies using knockout mice of lysophosphatidylinositol acyltransferase 1, which selectively incorporates AA into PI, reveal that AA-containing PI plays a crucial role in cortical lamination and neuronal migration during brain development., Dietary arachidonic acid (AA) has roles in growth, neuronal development, and cognitive function in infants. AA is remarkably enriched in phosphatidylinositol (PI), an important constituent of biological membranes in mammals; however, the physiological significance of AA-containing PI remains unknown. In an RNA interference–based genetic screen using Caenorhabditis elegans, we recently cloned mboa-7 as an acyltransferase that selectively incorporates AA into PI. Here we show that lysophosphatidylinositol acyltransferase 1 (LPIAT1, also known as MBOAT7), the closest mammalian homologue, plays a crucial role in brain development in mice. Lpiat1−/− mice show almost no LPIAT activity with arachidonoyl-CoA as an acyl donor and show reduced AA contents in PI and PI phosphates. Lpiat1−/− mice die within a month and show atrophy of the cerebral cortex and hippocampus. Immunohistochemical analysis reveals disordered cortical lamination and delayed neuronal migration in the cortex of E18.5 Lpiat1−/− mice. LPIAT1 deficiency also causes disordered neuronal processes in the cortex and reduced neurite outgrowth in vitro. Taken together, these results demonstrate that AA-containing PI/PI phosphates play an important role in normal cortical lamination during brain development in mice.

Phosphatidylinositol phosphates (PtdInsPs) are present within the nucleus, as well as in the membrane. In this mass spectrometry study, different acyl-containing species of endonuclear PtdInsPs were analyzed in order to clearly understand the role of individual molecular species. A (34:1) acyl-containing phosphatidylinositol bisphosphate [PtdInsP2(34:1)] and PtdInsP2(36:1) were preferentially detected in envelope-less nuclei prepared from various cultured human cells, while PtdInsP2(38:4) was not a major component within these nuclei. A significant amount of PtdInsP2(34:0) was detected in the HeLa cell nucleus, but not in the A431 and THP-1 cell nuclei. During the cell cycle in HeLa cells, PtdInsP2(34:0) levels increased in the early G1 phase, and then gradually decreased through S phase, while PtdInsP2(34:1) levels tended to decrease only in late G1 phase and PtdInsP2(38:4) did not change significantly. Thus, individual PtdInsP2 species apparently play different roles in nuclear events based on individual regulation of endonuclear levels. The non-arachidonate-containing species were also detected in normal human blood and fluids, suggesting that these minor species may have unique functions in the human body. The techniques used in this study will be applied to clinical studies on a PtdInsPs metabolism. Copyright © 2010 John Wiley & Sons, Ltd.

In the studies of lipid metabolomics, liquid chromatography electrospray ionization mass spectrometry (LC/MS) is a robust and popular technique. Although effective reverse-phase LC methods enabling the separation of phospholipid molecular species have been developed, there are still problems with the separation of phosphatidic acid (PA) and phosphatidylserine (PS). These acidic phospholipids often elute as extensively broad peaks, causing inferior separation, detection, and quantification-a severe limitation of the method. In this study, we have developed reverse-phase LC conditions that reduce the undesired peak tailings in the elution profiles of both PA and PS, by using a starting mobile phase containing a low concentration of phosphoric acid (5 microM) and a high percentage of water (40%). Our method sensitively analyzes PA, PS, and their lysoforms, as well as the other phospholipids within a biological sample, in a single chromatographic step by an LC/MS method and, thus, is suitable for lipidomics.

The elucidation of lipid dynamics on the cytosolic and lumenal sides of a single endomembrane has been challenging in living cells because of the lack of appropriate methods. Diacylglycerol (DAG) is a lipid second messenger that is produced by enzymes that reside on both the cytosolic and lumenal sides of the endomembrane. In the present study, we attempted to observe both the cytosolic and lumenal DAG dynamics at endomembranes including the Golgi apparatus and the endoplasmic reticulum in Madin-Darby canine kidney (MDCK) cells. We developed a Förster resonance energy transfer (FRET)–based probe to detect DAG at the luminal side (lumenal DAG) of endomembranes. In combination with the FRET-based cytosolic DAG probe that has already been established, it was found that lumenal DAG is generated in a calcium-dependent manner by thapsigargin, which increases cytosolic calcium concentrations. In contrast, DAG production at the cytosolic side of endomembranes did not occur under the same experimental conditions. The thapsigargin-induced DAG generation was abolished by treatment with an inhibitor of sphingomyelin synthase (SMS) and phosphatidylcholine-specific phospholipase C (PC-PLC), which produce lumenal DAG. Thus, we have established a successful method for monitoring both cytosolic and lumenal DAG dynamics at the endomembrane in living cells.

Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I–IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 Å resolution. EGFR domains I–III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF•EGFR complex dimerizes through a direct receptor•receptor interaction, in which a protruding β-hairpin arm of each domain II holds the body of the other. The unique “receptor-mediated dimerization” was verified by EGFR mutagenesis.

We previously reported that IL-2 deprivation induced acid sphingomyelinase-mediated (ASM-mediated) ceramide elevation and apoptosis in an NK/T lymphoma cell line KHYG-1. However, the molecular mechanism of ASM–ceramide-mediated apoptosis during IL-2 deprivation is poorly understood. Here, we showed that IL-2 deprivation induces caspase-dependent apoptosis characterized by phosphatidylserine externalization, caspase-8, -9, and -3 cleavage, and degradation of X-linked inhibitor of apoptosis protein (XIAP). IL-2 re-supplementation rescued apoptosis via inhibition of XIAP degradation without affecting caspase cleavage. However, IL-2 deprivation induced ceramide elevation via ASM in lysosomes and activated lysosomal cathepsin B (CTSB) but not cathepsin D. A CTSB inhibitor CA-074 Me and knockdown of CTSB inhibited ceramide-mediated XIAP degradation and apoptosis. Inhibition of ceramide accumulation in lysosomes using an ASM inhibitor, desipramine, decreased cytosolic activation of CTSB by inhibiting its transfer into cytosol from the lysosome. Knockdown of ASM also inhibited XIAP degradation and apoptosis. Furthermore, cell permeable N-acetyl sphingosine (C2-ceramide), which increases mainly endogenous d18:1/16:0 and d18:1/24:1 ceramide-like IL-2 deprivation, induced caspase-dependent apoptosis with XIAP degradation through CTSB. These findings suggest that lysosomal ceramide produced by ASM mediates XIAP degradation by activation of cytosolic CTSB and caspase-dependent apoptosis. The ASM–ceramide–CTSB signaling axis is a novel pathway of ceramide-mediated apoptosis in IL-2-deprived NK/T lymphoma cells.

In order to relate barrier function to stratum corneum structure and the thermal transitions of corneum lipids, samples from hairless rat skin were investigated by using ESR and drug penetration techniques. The phase transition of stratum corneum lipids was estimated using a deeper probe (16-doxyl-stearic acid) inserted in the lipid bilayers and measuring the rotational correlation time, tau(c). Results of ESR study showed that stratum corneum lipids underwent thermal transitions at 39.3 +/- 1.6 degrees C and 63.6 +/- 2.6 degrees C roughly similar to the data obtained by differential scanning calorimetry measurements. Cholesterol oxidase treatment decreased the fluidity of the lipids at lower temperatures. The treatment of stratum corneum with laurocapram (1%) and isopropyl myristate (IPM, 2%) little changed both phase transition temperatures, although the treatment highly increased the molecular motion of the lipids. The flux (J(s)) of lipophilic drugs (beta-estradiol, indomethacin and betahistine) through the skin was enhanced with increasing temperatures, with an increase in the diffusion constant within skin and a decrease in the lag time. There was a good relationship between log J(s) or log permeability coefficient (K(p)) and 1/tau(c) in the temperature range of 45 to 64 degrees C. The calculated activation energy (delta E) for diffusion of these drugs across skin was 17-40 kcal/mol. Judging from our data, stratum corneum lipids of rat probably exist as the gel, crystalline state below 39 degrees C, the mesomorphic state between 39 and 64 degrees C and the fluid, liquid-crystalline state at temperatures of 64 degrees C or above. These results are in line with the permeability of these lipophilic drugs through the intercellular lipids disordered is highly increased.

Phosphatidylinositol phosphates (PtdInsPs) are present within the nucleus, as well as in the membrane. In this mass spectrometry study, different acyl-containing species of endonuclear PtdInsPs were analyzed in order to clearly understand the role of individual molecular species. A (34:1) acyl-containing phosphatidylinositol bisphosphate [PtdInsP(2)(34:1)] and PtdInsP(2)(36:1) were preferentially detected in envelope-less nuclei prepared from various cultured human cells, while PtdInsP(2)(38:4) was not a major component within these nuclei. A significant amount of PtdInsP(2)(34:0) was detected in the HeLa cell nucleus, but not in the A431 and THP-1 cell nuclei. During the cell cycle in HeLa cells, PtdInsP(2)(34:0) levels increased in the early G1 phase, and then gradually decreased through S phase, while PtdInsP(2)(34:1) levels tended to decrease only in late G1 phase and PtdInsP(2)(38:4) did not change significantly. Thus, individual PtdInsP(2) species apparently play different roles in nuclear events based on individual regulation of endonuclear levels. The non-arachidonate-containing species were also detected in normal human blood and fluids, suggesting that these minor species may have unique functions in the human body. The techniques used in this study will be applied to clinical studies on a PtdInsPs metabolism.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) play various roles in cell–cell and cell–environment interactions. GPI is synthesized in the endoplasmic reticulum (ER) from phosphatidylinositol (PI) through step-wise reactions including transfers of monosaccharides and preassembled GPI is transferred en bloc to proteins. Cellular PI contains mostly diacyl glycerol and unsaturated fatty acid in the sn-2 position, whereas mammalian GPI-APs have mainly 1-alkyl-2-acyl PI and almost exclusively stearic acid, a saturated chain, at the sn-2 position. The latter characteristic is the result of fatty acid remodeling occurring in the Golgi, generating GPI-anchors compatible with raft membrane. The former characteristic is the result of diacyl to alkyl-acyl change occurring in the third GPI intermediate, glucosaminyl-inositolacylated-PI (GlcN-acyl-PI). Here we investigated the origin of the sn-1 alkyl-chain in GPI-APs. Using cell lines defective in the peroxisomal alkyl-phospholipid biosynthetic pathway, we demonstrated that generation of alkyl-containing GPI is dependent upon the peroxisomal pathway. We further demonstrated that in cells defective in the peroxisome pathway, the chain composition of the diacyl glycerol moiety in GlcN-acyl-PI is different from those in the first intermediate N-acetylglucosaminyl-PI and cellular PI, indicating that not only diacyl to alkyl-acyl change but also diacyl to diacyl change occurs in GlcN-acyl-PI. We therefore propose a biosynthetic step within GlcN-acyl-PI in which the diacyl glycerol (or diacyl phosphatidic acid) part is replaced by diradyl glycerol (or diradyl phosphatidic acid). These results highlight cooperation of three organelles, the ER, the Golgi, and the peroxisome, in the generation of the lipid portion of GPI-APs.

In this chapter we are going to mention about three different approaches in lipidomics and how to effectively profile or calculate the amounts of phospholipids from major molecular species up to minor ones. 1) Precise identification and profiling of individual molecular species of phospholipids by data-dependent LC-ESIMS/MS combination with "Lipid Search". We have been using this method as a global analysis of phospholipid. We usually applied this method at least once for new biological samples. We constructed an automated search engine, "Lipid Search", for identification and profiling of phospholipids. Once after applying this analysis, a specified retention time can be obtained for each elution peak of individual phospholipid molecular species. Thus, reproducible identification results can be effectively obtained by our search engine from the data obtained by single LC or combination of LC with specified head group survey by using precursor ion scanning or neutral loss scanning. 2) An effective analytical method of LC-ESIMS for the identification of acidic phospholipids such as phosphatidic acid and phosphatidylserine. This is an approach of how to obtain sharp chromatographic peaks for acidic lipids such as phosphatidic acid and phosphatidylserine that are normally detected as broad elution peaks. With this improvement very small amount of molecular species in minor acidic phospholipids were effectively obtained. 3) Identification and profiling of molecular species in focused phospholipids. Third one is a combination analysis of focused methods such as precursor ion scanning or neutral loss scanning and high efficient LC separation. As reported previously, different combinations of fatty acids on sn-1 and sn-2 can be mostly detected as separate peaks by reverse phase LC-ESIMS. Detection limit of precursor ion scanning or neutral loss scanning is more than ten times higher than that of the method without LC separation, because of decreased ion suppression. We will mention about application of this methods for focused analysis on phosphatidylethanolamine-plasmalogens.

In studies on lipid metabolomics, liquid chromatography/ mass spectrometry (LC/MS) is a robust and popular technique. Although effective reversed-phase (RP) LC/ MS methods enabling the separation of phospholipid molecular species have been developed, RPLC methods to analyze phosphatidylinositol phosphates (PIPs) have not been reported. In this study, we developed conditions suitable for PIP analysis. Coupled with (diethylamino)ethyl (DEAE)-cellulose pretreatment, at least 1 pmol each of phosphatidylinositol monophosphates (PIP1), bisphosphates (PIP2), and triphosphates standards per approximately 6 x 10(6) cultured cells could be measured. Using these methods, we detected elevated concentrations of more than 12 PIP1 species in epidermal growth factor (EGF)-stimulated A431 cells, a human epidermoid carcinoma cell line. The PIP2 species detected were not elevated after stimulation. We also detected EGF-induced increases in the levels of several phosphatidic acid species using another set of methods. Our method sensitively determined PIPs within a biological sample and is thus suitable for analysis of phoisphoiniositide metabolism.

Lysophosphatidic acid (LPA) is a simple phospholipid but has numerous biological effects through a series of G-protein-coupled receptors specific to LPA. In general, LPA is short-lived when applied in vivo, which hinders most pharmacological experiments. In our continuing study to identify stable LPA analogues capable of in vivo applications, we identified here lysophosphatidylmethanol (LPM) as a stable and pan-LPA receptor agonist. A synthetic LPM activated all five LPA receptors (LPA(1-5)), and stimulates both cell proliferation and LPA-receptor-dependent cell motility. In addition, LPM showed a hypertensive effect in rodent when applied in vivo. We found that, when fetal calf serum was incubated in the presence of methanol, formation of LPM occurred rapidly, whereas it was completely blocked by depletion of autotaxin (ATX), a plasma enzyme that converts lysophosphatidylcholine (LPC) to LPA. When recombinant ATX was incubated with LPC in the presence of methanol, both LPM and LPA were produced with a ratio of 1:10, showing that ATX has transphosphatidylation activity in addition to its lysophospholipase D activity. Administration of methanol in mice resulted in the formation of several micromoles of LPM in plasma, which is much higher than that of LPA. The present study identified LPM as a novel and stable lysophospholipid mediator with LPA-like activities and ATX as a potential synthetic enzyme for LPM.

In this chapter we are going to mention about three different approaches in lipidomics and how to effectively profile or calculate the amounts of phospholipids from major molecular species up to minor ones. 1) Precise identification and profiling of individual molecular species of phospholipids by data-dependent LC-ESIMS/MS combination with "Lipid Search". We have been using this method as a global analysis of phospholipid. We usually applied this method at least once for new biological samples. We constructed an automated search engine, "Lipid Search", for identification and profiling of phospholipids. Once after applying this analysis, a specified retention time can be obtained for each elution peak of individual phospholipid molecular species. Thus, reproducible identification results can be effectively obtained by our search engine from the data obtained by single LC or combination of LC with specified head group survey by using precursor ion scanning or neutral loss scanning. 2) An effective analytical method of LC-ESIMS for the identification of acidic phospholipids such as phosphatidic acid and phosphatidylserine. This is an approach of how to obtain sharp chromatographic peaks for acidic lipids such as phosphatidic acid and phosphatidylserine that are normally detected as broad elution peaks. With this improvement very small amount of molecular species in minor acidic phospholipids were effectively obtained. 3) Identification and profiling of molecular species in focused phospholipids. Third one is a combination analysis of focused methods such as precursor ion scanning or neutral loss scanning and high efficient LC separation. As reported previously, different combinations of fatty acids on sn-1 and sn-2 can be mostly detected as separate peaks by reverse phase LC-ESIMS. Detection limit of precursor ion scanning or neutral loss scanning is more than ten times higher than that of the method without LC separation, because of decreased ion suppression. We will mention about application of this methods for focused analysis on phosphatidylethanolamine-plasmalogens.

The arylhydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that regulates the transcription of genes encoding xenobiotic metabolizing enzymes and also mediates most of the toxic effects caused by dioxins and polycyclic aromatic hydrocarbons. The cytosolic AhR complex exists as a transcriptionally cryptic complex, consisting of the 90 kDa heat shock protein (HSP90) and the hepatitis B virus X-associated protein 2 (XAP2). The posttranslational modifications, especially phosphorylation, of the cytosolic AhR-HSP90-XAP2 complex are poorly understood, although the phosphorylation of a transcriptionally active heterodimer of AhR and an AhR nuclear translocator is critically involved in AhR function. To reveal the phosphorylation status involved in AhR function, we used mass spectrometry to determine the site-specific phosphorylation of the steady-state cytosolic AhR complex, prepared from Chinese hamster ovary cells stably expressing mouse AhR. We identified phosphorylations of the HSP90 subunits within the AhR complex at Ser225 and Ser254 of HSP90beta and Ser230 of HSP90alpha. By site-directed mutagenesis, these serine residues were substituted with alanine and glutamic acid to elucidate the role of the HSP90beta serine phosphorylations in the AhR function. Immunoprecipitation assays using COS7 transfectants showed that the replacement of Ser225 and Ser254 by Ala, S225/254A, increased the binding affinity for AhR, as compared with the Glu replacement. In a ligand-induced AhR transcription activity assay using Hepa1 transfectants, the S255/254A mutant exhibited more potent transcription activity than the S225/254E mutant, which had activity similar to that of wild-type HSP90beta. These results suggest that the phosphorylations in the charged linker region of the HSP90 molecule modulate the formation of the functional cytosolic AhR complex.