Conceived and designed the experiments: YS JC DRW. Performed the experiments: YS ZL JS RRP EMT YI BA SB YN BK. Analyzed the data: YS JS EMT YI BK. Contributed reagents/materials/analysis tools: YS MI. Wrote the paper: YS DRW.
The authors have declared that no competing interests exist.
Neuregulin1 (NRG1)-ErbB signaling has been implicated in the pathogenesis of cancer and schizophrenia. We have previously reported that NRG1-stimulated migration of B lymphoblasts is PI3K-AKT1dependent and impaired in patients with schizophrenia and significantly linked to the catechol-o-methyltransferase (COMT) Val108/158Met functional polymorphism.
We have now examined AKT1 activation in NRG1-stimulated B lymphoblasts and other cell models and explored a functional relationship between COMT and AKT1. NRG1-induced AKT1 phosphorylation was significantly diminished in Val carriers compared to Met carriers in both normal subjects and in patients. Further, there was a significant epistatic interaction between a putatively functional coding SNP in AKT1 (
Our findings implicate genetic and functional interactions between COMT and AKT1 and may provide novel insights into pathogenesis of schizophrenia and other ErbB-associated human diseases such as cancer.
Neuregulin1 (NRG1)-ErbB signaling has been implicated in the pathogenesis of cancer and schizophrenia. In recent work using a B lymphoblast cell model, we showed that these cells express a functional ErbB signaling pathway, and that NRG1 promotes their adhesion and migration
COMT catalyzes the transfer of the methyl group of S-adenosylmethionine (SAM) to a hydroxyl group on a variety of catechols, including the catecholamine neurotransmitters and carcinogenic catechol estrogen compounds
AKT1 is a serine/threonine kinase that functions as a central element in the cell survival pathway and is activated in many cancers
Despite the overlap of COMT and AKT1 in associations with cancer and schizophrenia, molecular and cell biological interactions of COMT and AKT1 have not been explored. In the present study, using NRG1-ErbB signaling in the B lymphoblast cell system and in other cell models, we show evidence of epistatic effects between these genes in that COMT Val/Met affects AKT1 translocation and activation by competing for methyl donor availability required for PS synthesis. Our findings provide a potential mechanism for the impact of COMT on NRG1-induced adhesion and migration and also possibly for the association of these genes with schizophrenia and cancer.
We previously demonstrated an association between COMT Val/Met (
(A) COMT Val/Meet genotype and NRG1-stimulated Ser-473 phosphorylation of AKT1. NRG1-stumulated increases in AKT1 phosphorylation in B lymphoblasts were significantly decreased in Val homozygotes (n = 18), compared with Met homozygotes (n = 12) (p = 0.0024, t-test). The Y-axis shows the peak fold increases in the phosphorylated-AKT1/total AKT1 ratio during one hour stimulation. (B) Disease and and NRG1-stimulated phosphorylation of AKT1. There was no difference in NRG1-stumulated increases in AKT1 phosphorylation in B lymphoblasts between controls (n = 16) and patients with schizophrenia (n = 14). (C) COMT Val homozygotes had significantly lower NRG1-stimulated phosphorylation of AKT1 in both controls (P = 0.029) and patients with schizophrenia (p = 0.042, t-test).
To confirm whether a functional polymorphism within the AKT1 gene is associated with the expression and function of AKT1 and might interact epistatically with COMT, we genotyped 64 B lymphoblast lines for the coding SNP
(Upper panel; A, B and C) Effects of AKT1
In contrast to the association with protein levels,
The COMT Val/Met polymorphism is associated with variable enzyme activity: the Val allele encodes an enzyme with higher activity than the Met form
SH-SY5Y cells were transfected with COMT-GFP or with the control GFP vector. After 48 hrs, cells were analyzed for enzyme activity and expression of COMT and tested for Ser-473 phosphorylation of AKT1 in response to NRG1α. (A) GFP-detection after transfection of GFP-tagged COMT or control GFP vector showed 70–80% transfection efficiency. (B) Expression of the transfected COMT. Western blotting showed expression of a 60-kDa band, representing GFP-tagged human MB-COMT in cells transfected with COMT-GFP (left three lanes). Endogenous 30-kDa MB- and 25-kDa S- COMT proteins were expressed under both transfection conditions. (C) Effects of COMT transfection on COMT enzyme activities. The COMT enzyme assay indicated a significant five-fold increase in COMT activity (dpm per mg total protein) in cells transfected with COMT-GFP compared to cells transfected with control vector. *p<0.0001, 3 samples per group. The specificity of the assay was confirmed by a complete attenuation of the enzyme activity by 100 µM of the specific COMT inhibitor tolcapone. (D) Effects of COMT transfection on NRG1α-stimulated Ser-473 phosphorylation of AKT1 in SH-SY5Y cells. At 48 hrs after transfection, the transfected cells were stimulated with NRG1α (100 ng/ml) for the time points indicated. Protein isolated from the cells was analyzed by Western blotting. To quantify the level of phosphorylation, the immunoblots were stained with antibodies specific to phosphorylated AKT1- at Ser-473 and were then stripped and reprobed with antibodies to total AKT1. The bar graphs represent changes in the ratio of phosphorylated form to total AKT1 (means±SEM, from 3 transfection experiments). A repeated measure ANOVA showed a significant main effect of COMT transfection on AKT1 phosphorylation (P = 0.0017).
SH-SY5Y cells express ErbB2, 3 and 4 receptors and activate typical tyrosine receptor signaling cascades in response to NRG1-stimulation, such as a PIP3-AKT1 signaling cascade (unpublished observations). Consistent with these observations, NRG1 increased AKT1phosphorylation that persisted for at least 60 min in SH-SY5Y cells transfected with a control vector containing GFP only. In contrast, NRG1-stimulated phosphorylation of AKT1 was significantly reduced in COMT transfected cells (
PIP3-triggered translocation of AKT1 from the cytoplasm to the plasma membrane is a prerequisite for its phosphorylation and activation
(A) There was a positive correlation between NRG1-stimulated Ser-473 phosphorylation of AKT1 and NRG1-simulated PHD-AKT translocation. B lymphoblasts from control subjects were transfected with pEYFP-ph-AKT. At 24 hrs after transfection, PHD-AKT1 translocation was stimulated with NRG1 for 15 min and measured under the fluorescence microscope. Translocation indexes from 8 control subjects were positively correlated with NRG1-stimulated peak fold increases in Ser-473 phosphorylation of AKT1 (phophorylated-AKT1/total AKT1 ratio) that were separately obtained from the same individuals (r = .832, p = 0.0075). (B) Representative fluorescence microscopic images of PHD-AKT1 fluorescence. Single cell images of PHD-AKT1fluorescence before and after NRG1 stimulation in typical COMT Val/Val and Met/Met homozygotes are shown. The bar graph represents PHD-AKT1 tranlocation index (means±SEM). *p = 0.029, individuals with Val/Val genotype (n = 5) vs. Met/Met genotype (n = 5). (C) There was a correlation between COMT enzyme activity and phosphatidylserine (PS) synthesis ability. Y-axis indicates relative COMT activity (dpm per mg total protein) in the B lymphoblasts. X-axis indicates PS synthesis ability (the ratio of total PS fluorescence intensity of B lymphoblast after exposure to serum free medium for 24 hrs/PS fluorescence intensity after culturing in normal culture media containing 15% FBS, which reflects a cell's ability to maintain PS levels) (see
Although PS has no direct effect on AKT1 activity
Another potential mechanism for the poor NRG1-stimulated phosphorylation of AKT1associated wtih COMT Val genotype or high enzyme activity might be reduced PIP3 generation. However, NRG1-stimulated PIP3 generation was not diminished (Supplementary Information,
To further determine if effects of COMT Val/Met genotype on NRG1-stimulated translocation of PHD-AKT1 in B lymphoblasts is due to COMT enzyme activity, we performed an AKT1 translocation experiment using the COMT-transfected SH-SY5Y cell model. pDsRed-ph-AKT was used for this experiment since COMT was tagged with GFP. Our examinations of fluorescence signals confirmed that there was no overlapping signal from green (GFP) and red (pDsRed) fluorescence, indicating that the localization of red fluorescence reflected accurately the localization of PHD-AKT1 expressed in the transfected cells. Approximately 48 hours after double-transfection with either pDsRed-ph-AKT plus pAcGFP-N1-COMT or pDsRed-ph-AKT plus control vector, the cells were stimulated with NRG1 and terminated by fixation buffer at different time points. This time-course study indicated that stimulation with NRG1 produced PHD-AKT1 localization, which was observed as fluorescence distribution patterns of multiple spots, clusters or broad membranous distribution (
(A) Effects of high COMT activity on PHD-AKT1translocation. SH-SY5Y cells were double-transfected with either pDsRed-ph-AKT plus COMT or pDsRed-ph-AKT plus control vector. After 48 hrs, cells were analyzed for PHD-AKT1 translocation in response to NRG1α. The bar graph represents percent positive cells indicating PHD-AKT1 translocation (means±S.E.) from four transfection experiments. Two-way ANOVA, interaction between NRG1α treatment and COMT transfection, *p<0.05, **p = 0.0074. Lower inset shows representative fluorescence microscopic images of PHD-AKT1 fluorescence in a single cell. From left, untranslocated homogenous distribution of PHD-AKT1, translocated PHD-AKT1 as spots, clusters and broad membranous distributions. (B) Effects of high COMT activity on NRG1-stimulated PIP3 generation. SY5Y cells were transfected with either COMT or control vector. The cells were analyzed for NRG1-stimulated PIP3 generation using flow cytometry. Top panel indicates a summation of positive and negative changes in PIP3 during a 30 min observation period and lower panel indicates peak folds of PIP3 during a 30 min period.
We then studied NRG1-stimulated PIP3 generation to determine if the poor NRG1-stimulated translocation and phosphorylation of AKT1 by the COMT transfection is due to reduced PIP3 generation. However, there was no difference in NRG1-stimulated PIP3 generation between the COMT- and control vector-transfected cells in two measures from three independent transfection experiments: summation of changes (P = 0.5782) and peak folds (p = 0.6134) (
PS synthesis is regulated by constitutively active methylation of phospholipids
(A) Effects of SAM treatment on NRG1-stimulated Ser-473 phosphorylation of AKT1 in SH-SY5Y cells overexpressing COMT. At 48 hrs after transfection with COMT-GFP, cells were treated with either 1 mM SAM or vehicle for 60 minutes and stimulated with NRG1α (100 ng/ml). The immunoblot shows a representative time course of Ser-473 phosphorylation of AKT1and expression of total AKT1 after NRG1α. The bar graph represents the ratio of phosphorylated- to total AKT1 at 60 min after the stimulation (means±SEM, from 3 transfection experiments). p = 0.0413, vehicle vs. SAM treatment. (B) Effects of COMT transfection on total PS amount in SH-SY5Ycells. SH-SY5Y cells were transfected with COMT-GFP or with control GFP vector. After 48 hrs, cells were and treated with either 1 mM SAM or vehicle control for 60 min and analyzed to estimate total PS amount of cells by flow cytometry. A graph indicates PS fluorescence intensity (geometric mean) from two independent transfection experiments. A repeated measure of ANOVA showed significantly lower PS fluorescence intensity in COMT-transfected cells than that in cells transfected with control vector (*P = 0.0087) and a significant reversal of the transfection effect by SAM treatment (*p = 0.0004).
Because the neuroblastoma line SH-SY5Y is dopaminergic and these cells express dopamine receptors, it is conceivable that the effect of COMT transfection on PS might be mediated by dopamine in these cells. Therefore, we also tested HEK293 cells, which normally do not express dopamine receptors
If the decrease in PS synthesis is the cause, at least in part, for the poor translocation and phosphorylation of AKT1, the effect of COMT Val/Met genotype or enzyme activity may not be limited to NRG1-ErbB signaling. To test this hypothesis, we studied whether COMT transfection affects ligand-stimulated phosphorylation of AKT1 induced via other signaling pathways, using SH-SY5Y cells. We used BDNF to stimulate the tyrosine kinase receptor trkB, and SDF1 and ACEA to stimulate the G-protein coupled receptors, CXCR4 and the cannabinoid (CB) receptor, respectively. We also tested the β-isoform of NRG1 to confirm that the effect of COMT transfection on NRG1-ErbB-mediated phosphorylation of AKT1is not specific to the α-isoform. Although the inhibition in ACEA or BDNF-stimulated AKT1phosphorylation by COMT overexpression was small, COMT over-expression significantly inhibited SDF1-stimulated phosphorylation of AKT1 (p<0.0005, t-test) (
At 48 hrs after transfection, the transfected SH-SY5Y cells were stimulated with NRG1β (100 ng/ml), cannabinoid CB1 agonist ACEA (14 nM), SDF1β (50 ng/ml) or BDNF (50 ng/ml). The bar graphs represent maximum changes in the ratio of phosphorylated- to total AKT1 during the 60-minutes observation period (means±SEM, from 3–4 transfection experiments per ligand).
Finally, we attempted to determine if high COMT enzyme activity produces a negative effect on cell migration. Since SH-SY5Y cells also migrate in response to NRG1 in a PI3K/AKT1-dependent manner, the SH-SY5Y-COMT transfection system is suited for this experiment. As expected in untransfected SH-SY5Y cells, our migration assay using a transwell chamber showed a positive NRG1-stimulated migration in control-vector transfected cells (
SH-SY5Y cells were transfected with COMT-GFP or control vectors (empty GFP) and cultured in media containing 2%FBS. After 48 hrs, cells were tested for NRG1-stimulated migration. A graph indicates the chemotaxis index (mean± S.E.) from three transfection experiments (n = 4–5 per group). ANOVA showed a significant a SAM treatment effect (p = 0.0193) and transfection-treatment interaction (P = 0.0144).
In the present study, we have found that the valine allele of COMT is associated with diminished NRG1-induced AKT1 phosphorylation in B lymphoblasts from both controls and patients, and showed that COMT overexpression in SH-SY5Y cells led to impaired AKT1 phosphorylation and migration in response to NRG1. These results suggest that the relatively poorer NRG1-induced adhesion and migratory response seen in Val homozygote lymphoblasts
As with other chemokine-stimulated migratory responses, activation of AKT1is essential for NRG1-stimulated cell adhesion and migration in B lymphoblasts, evidenced by the severe attenuation of adhesion and migration by PI3K or AKT1 inhibition in these cells
Identifying the mechanism of how COMT activity inhibits the function of AKT1, and therefore, NRG-dependent adhesion and migration is challenging. Considering the well characterized function of COMT as an enzyme involved in catechol inactivation, it was conceivable that the effect of COMT on migration and adhesion could be related to a dopamine- or catecholic estrogen-mediated mechanism, since these substrates for COMT can be derived from fetal bovine serum or might be produced by B lymphoblasts in culture. However, this mechanism is unlikely to be responsible, since in B lymphoblasts the production of dopamine is low and the expression of dopamine receptors is scarce (unpublished observation), and because the migration assay was performed in serum-free media. Rather, our earlier results suggested another mechanism, an indirect effect of COMT on methylation of other critical molecules involved in phospholipids to explain the inverse relationship between COMT and AKT1 activation.
It has been suggested that an increase in COMT-mediated methylation decreases the SAM pool and increases SAH, which acts as a feedback inhibitor of SAM-dependent methylation processes
After confirming that PEMT, PSS1 and PSS2, which mediate the synthesis of PS
If the reduction in PS synthesis and AKT1 activation is related to COMT-mediated SAM consumption, we would expect that these effects of COMT on AKT1 would not be limited only to NRG1-ErbB signaling. Consistent with this prediction, studies using SH-SY5Y and HEK293 cells demonstrated that COMT overexpression inhibited phosphorylation of AKT1 in other relevant signaling pathways such as SDF1-CXCR4, and similar to our findings for NRG1, this inhibition was overcome by SAM augmentation.
Interestingly, in addition to a single effect of COMT genotype on AKT1, we also found evidence of an interaction between COMT Val/Met and a SNP in AKT1 that is associated with variation in AKT1 protein expression. Thus, the minor A allele of
Since COMT, AKT1, and ErbB-signaling are each implicated in both cancer and schizophrenia, NRG1-ErbB signaling in B lymphoblasts provides a biologically plausible research tool for elucidating cellular mechanisms relevant to both cancer biology and neurobiology. Using this system, we demonstrated epistatic effects of COMT Val/Met and AKT1
The B lymphoblast cell lines used for this study are derived from the same subjects described in our previous reports
Genotypes at
B lymphocytes in a mononuclear cell preparation from the subjects were transformed by infection with EBV and maintained as previously described
Immunoblot and analysis were performed as previously described
Relative PS levels at the single cell level were assessed by flow cytometry after staining both extracellular and intrallular PS with anti-PS antibody (4B6) (Abcam Inc., Cambridge, MA) using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA). Relative PIP3 levels were assessed using anti-PIP3 antibody (Echelon Biosciences Inc., Salt Lake City, UT). Briefly, cells were fixed with Phosflow Fix Buffer I (BD Bioscience) for 10 min at 37°C. Cells were then washed with Phosflow Perm/Wash Buffer I (BD Bioscience), cells permeabilized in Phosflow Perm/Wash Buffer I were stained with anti-phosphatidylserine antibody (4B6) or biotin-conjugated anti-PIP3 antibody (Echelon Biosciences Inc.) for 1 hrs at room temperature. After washing twice with Phosflow Perm/Wash Buffer I, cells were incubated with phycoerythrin-conjugated goat anti-mouse IgG antibody (BD Bioscience) to detect 4B6 or phycoerythrin-conjugated avidin (BD Bioscience) to detect biotin-conjugated anti-PIP3 antibody. After washing with Phosflow Perm/Wash Buffer I, cells were analyzed using FACScan (BD Bioscience). CellQuest software (BD Bioscience) was used to acquire and quantify the fluorescence signal intensities. To assess the cells' PS synthesis ability, we obtained a ratio of geometric mean fluorescence (GMF) of PS fluorescence in B lymphoblast after exposure to serum free medium for 24 hrs, over GMF of PS after culturing in normal culture media containing 15% FBS. To measure NRG1-stimulated PIP3 production, cells were stimulated with either NRG1α (100 ng/ml) or vehicle in a 5% CO2 incubator at 37°C. The reaction was terminated at 0, 5, 10, 15 and 30 min by fixing cells with Phosflow Fix Buffer I (BD Bioscience) for 10 min at 37°C. Summation of positive and negative changes in GMF of PIP3 fluorescence over baseline during a 30 min observation period was calculated to estimate the extent of NRG1-stimulated PIP3 production.
Transfection of pAcGFP-N1-COMT (Supplementary Information,
Transfection of pDsRed-ph-AKT or pEYFP-ph-AKT (Supplementary Information,
PHD-AKT1 translocation was also examined in COMT-transfected SH-SY5Y cells in order to determine the effects of high COMT enzyme activity on AKT1 translocation ability. For this purpose, the cells were transfected with a combination of two plasmids, either pDsRed-ph-AKT plus pAcGFP-N1-COMT or pDsRed-ph-AKT plus control vector. Then AKT1 the translocation assay was performed 48 hrs after the transfection. The transfected cells were stimulated with NRG1α (25 ng/ml) for 10 min in an incubator (95% air/5% CO at 37 C), fixed with Phosflow Fix Buffer (BD Bioscience) and examined under the fluorescence microscope. In contrast to B lymphoblasts, SH-SY5Y cells indicated distinctive patterns of PHD-AKT1 translocation, which were recognized as multiple spots, clusters or broad membranous. The changes in proportion of cells with these patterns before and after NRG1 treatment were measured and analyzed.
The migration assay was carried out using a transwell chamber with an 8-µm pore size (Costar #3422, Corning, NY). Briefly, cells were suspended at 4×105 cells/ml in serum-free RPMI 1640 with or without 1 mM S-(5′-Adenosyl)-L-methionine chloride (A7007 Sigma-Aldrich, St. Louis, MO), Then, 100 µl of the cell suspension (40,000 cells) were applied to the upper wells of the transwell chamber and incubated in an incubator (95% air/5% CO2 at 37°C) for 1 hr. Serum-free RPMI 1640 with or without NRG1 (0.5 ml/well) was then added to the lower wells. After 4 h in an incubator (95% air/5% CO2 at 37°C), cells attached to the lower side of the membrane were detached by dissociation buffer (Trevigen, Gaithersburg, MD), lysed with 0.1% Triton X-100 and measured using CyQUANT GR double-stranded DNA detecting reagent (Molecular Probes, Eugene, Oreg). The results were expressed as a chemotaxis index calculated by the following formula: chemotaxis index = migration in response to NRG1/migration in response to vehicle control (baseline count). All assays were done in quaduplicate.
Case-control comparisons for allele frequencies were carried out by chi-square test and Fisher's exact test. The standard measure of linkage disequilibrium (LD), denoted as D' or R2, was estimated with Haploview 4.0 in the web
The genomic structure and locations of SNPs in the human AKT1 gene. Numbers in parentheses indicate allele frequencies. Numbers underlined indicate chromosomal positions. The LD map of the AKT1 region in this study (64 Caucasians) was obtained through Haploview v 4.0. Pairwise LD is indicated by the numbers of either D' (left) or r-square (right) and depth of red color.
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Effects of COMT Val/Met and AKT1 rs1130233 G/A (G/G = circle, G/A = triangle) genotypes on NRG1-stimulated PIP3 production. Methods to estimate NRG1-stimulated PIP3 production are described in the
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Effects of COMT transfection on total PS amount in HEK293 cells. HEK293 cells were transfected with COMT-GFP or with control GFP vector and cultured in media containing 2%FBS. After 48 hrs, cells were analyzed to estimate the total amount of PS by flow cytometry. (A) A two-parameter histogram represents the expression of PS and transfected GFP. (B) An overlay histogram represents both external and internal expression of PS in COMT-transfected cells (filled in black) and control vector-transfected cells (unfilled) in the gated region (squares in the two-parameter histograms). (C) The ratio of the proportion of cells in the upper right quadrant compared to that in the lower right quadrant in COMT-transfected cells was significantly lower than in cells transfected with the control vector (P = 0.0191). (D) PS fluorescence intensity in the gated region (squares in the two-parameters histogram) was significantly decreased in cells transfected with COMT compared with cells transfected with control vector (p = 0.0319).
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Effects of COMT transfection, S-adenosylhomocysteine hydrolase (SAHH) transfection and SAM treatment on total PS amount in HEK293 cells. HEK293 cells were double-transfected with COMT-GFP or SAHH-XL5 and their control vectors (empty GFP or empty XL5, respectively) and cultured in media containing 2%FBS. After 48 hrs, cells were treated with 1 mM SAM or vehicle for 60 min and analyzed to estimate the total amount of PS by flow cytometry. A graph indicates the ratio of the proportion of cells in the upper right quadrant (high PS) to the lower right quadrant (low PS) in transfected cells. A higher ratio corresponds to higher total PS. ANOVA showed a significant COMT transfection effect (p = 0.0017) and a SAM treatment effect (p = 0.0192), while there was no effect of SAHH transfection (p = 0.5124) on total PS.
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Expression of PSS1, PSS2 and PEMT in B lymphoblasts. Phospholipids biosynthetic pathways. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N- methyltransferase; PS, phosphatidylserine; PSS, phosphatidylserine synthase. Agarose gel electrophoresis of PCR products following RT-PCR for PSS1, PSS2 and PEMT shows expression of these transcripts in B lymphoblasts. The amplicons from human brain tissue in the first left lane serve as positive controls for these amplifications. RT-PCR. Total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI) and reverse transcription performed to generate the first strand of cDNA using a cDNA synthesis kit (Promega). Synthesized cDNA was then amplified by PCR using following primer sets. The sequences of primers and the annealing temperature for PSS1, PSS2 and PEMT amplifications were:
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Effects of disease and AKT1 rs1130233 on AKT1 transcripts. It should be noted that, although there were no differences in AKT1 protein levels or NRG1-stimulated phosphorylation between lymphoblasts from patients and controls, two-way ANOVA showed significant effects of disease on AKT1 mRNA levels (p = 0.0263), but no effects of AKT1 rs1130233 (P = 0.1514) or no interactions between disease and rs1130233 (p = 0.4275). It is unknown whether or not this elevation reflects compensatory mechanism(s) of unknown defects related to AKT1. * Y-axis, AKT1 mRNA, qPCR results normalized to the geometric mean of three house keeping genes. Real-time quantitative PCR. Real-time RT-PCR reactions for AKT1 mRNA expression were performed using the TaqMan gene expression assay system. First-strand cDNA equivalent to 90 ng of total RNA was prepared in a final volume of 10 µl of Master Mix (Eurogentec, San Diego, CA) with a validated Taqman primer/probe mixture specific to the AKT1 gene (Hs00920503_m1) (Applied Biosystems, Foster City, CA). Real-time RT-PCRs for three housekeeping genes, GUSB, B2M, and ACTB were also performed using TaqMan primer-probes Hs99999908_m1, Hs99999907_m1 and Hs99999903_m1, respectively, to obtain a geometric mean normalization factor (NF3). Real-time RT-PCR reactions were performed in triplicate in a 384-well plate using an ABI Prism 7900 HT sequence detection system. All PCR cycles comprised 2 min polymerase activation at 50°C followed by 10 min denaturation at 95°C and 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Standard curves were established using a pooled cDNA stock from 60 individuals. The cycle threshold (Ct) was determined using SDS 2.0 software (Applied Biosystems). Relative mRNA level of the AKT1 gene in each sample was expressed as a Ct ratio to NF3 (the geometric mean of Ct values from three reference genes) according to the normalization method previously described by Vandesompele et al 45. 45. Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome biology 3, RESEARCH0034 (2002).
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A hypothetical mechanisms for COMT-mediated effects on biological functions. The high enzyme activity COMT Val, whose effects can be enhanced by high concentrations of substrates, reduces the SAM pool and increases SAH, which acts as an inhibitor for SAM-dependent methyltransferases. In this manner, PEMT might be affected by COMT activity. A reduction of PEMT activity could change the status of phospholipid synthesis and result in reductions in the PS component of the plasma membrane, which could affect pleckstrin homology domain-mediated binding of protein kinases such as AKT1. Therefore, high COMT could affect not only dopamine or estrogen metabolism, but also other functions mediated by SAM-dependent methyltransferases. Reduced AKT1 responses are expected in individuals with COMT Val than Met homozygotes, as demonstrated in this study. COMT-mediated effects would also be expected to be more robust when there are deficits in essential cofactors such as cholin, B6, B12 and folate and/or when COMT enzyme substrates e.g., high catechols, steroids and related drugs are high.
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Supplementary Information
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We thank Dr. Thomas F. Franke of the New York University School of Medicine for critical review of the manuscript. We dedicate this paper to the memory of Dr. John W. Daly, former chief of Laboratory of Bioorganic Chemistry, the National Institute of Diabetes and Digestive and Kidney Diseases.