Ayako Tsuchiya

PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

Insulin stimulated translocation of the glucose transporter GLUT4 from the cytosol to the plasma membrane in a concentration (1 nM–1 μM)-dependent manner and increased glucose uptake in 3T3-L1 adipocytes. Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2. Insulin increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474, to activate Akt1/2, in the adipocytes. Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1. In the cell-free Akt assay, PI3K phosphorylated Akt1 both at Thr308 and Ser473 and Akt2 at Ser474 alone. In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309. The results of this study indicate that PI3K activates Akt1, independently of PDK1, and Akt2 by cooperating with PDK1 in the insulin signal transduction pathway linked to GLUT4 translocation.

Crosstalk between PI3 Kinase/PDK1/Akt/ Rac1 and Ras/Raf/MEK/ERK Pathways Downstream PDGF Receptor

Background/Aims: Our earlier studies suggested crosstalk between IRS/PI3 kinase/PDK1/Akt/Rac1/ROCK and (Shc2/Grb2/SOS)/Ras/Raf/MEK/ERK pathways downstream PDGF-ββ receptor responsible for chemotaxis and proliferation of malignant mesothelioma cells. The present study was conducted to obtain evidence for this. Methods: To assess activation of Akt, MEK, and ERK, Western blotting was carried out on MSTO-211H malignant mesothelioma cells using antibodies against phospho-Thr308-Akt, phopho-Ser473-Akt, Akt, phospho-MEK, MEK, phopho-ERK1/2, and ERK1/2. To knock-down Akt, PI3 kinase, PDK1, and Rac1, siRNAs silencing each-targeted gene were constructed and transfected into cells. To monitor Rac1 activity, FRET monitoring was carried out on living and fixed cells. Results: ERK was activated under the basal conditions in MSTO-211H cells, and the activation was prevented by inhibitors for PI3 kinase, PDK1, Akt, and Rac1 or by knocking-down PI3 kinase, PDK1, Akt, and Rac1. Akt was also activated under the basal conditions, and the activation was suppressed by a MEK inhibitor and an ERK1/2 inhibitor. In the FRET analysis, Rac1 was activated under the basal conditions, and the activation was inhibited by a MEK inhibitor and an ERK1/2 inhibitor. Conclusion: The results of the present study show that ERK could be activated by PI3 kinase, PDK1, Akt, and Rac1 and that alternatively, Akt and Rac1 could be activated by MEK and ERK in MSTO-211H cells.

Intracellularly transported adenosine induces MCF-7 human breast cancer cells by accumulating AMID in the nucleus

Extracellular adenosine induced apoptosis of MCF-7 human breast cancer cells in a concentration (10μM-10mM)- and treatment time (24-72h)-dependent manner, and the effect was inhibited by the adenosine transporter inhibitor dipyridamole, but not an inhibitor of adenosine kinase, an inhibitor of AMP-activated protein kinase, or inhibitors for A(1), A(2a), A(2b), and A(3) adenosine receptors. No significant activation of caspase-7, -8, or -9 was obtained with adenosine. Adenosine promoted translocation of apoptosis-inducing factor (AIF)-homologous mitochondrion-associated inducer of death (AMID) from the cytosol into the nucleus, although the total amount of AMID was not affected. Adenosine-induced MCF-7 cell death was abrogated by knocking-down AMID. The results of the present study indicate that intracellularly transported adenosine induces MCF-7 cell apoptosis by accumulating AMID in the nucleus in a caspase-independent manner.

Hyperphosphorylation of Tau at Ser396 occurs in the much earlier stage than appearance of learning and memory disorders in 5XFAD mice.

The present study investigated the relation of age-dependent spatial learning/memory impairment and Tau phosphorylation in 5XFAD mice, a model of Alzheimers disease. In the water maze test, the acquisition and retention latencies for 5XFAD mice at 6 months, but not 2 months, of age was significantly longer than those for wild-type mice at the same months of age, without difference in the swim speed and visual acuity between two groups. The level of glycogen synthase kinase 3β (GSK-3β) phosphorylation at Ser9 in the hippocampus for the 5XFAD mice at >4 months of age was significantly lesser than that for wild-type mice at the same months of age, while a robust increase in the Tyr216 phosphorylation of GSK-3β was found both in wild-type and 5XFAD mice at 6 months of age, without no significant difference in the extent between two groups. There was no significant difference in the Tau phosphorylation at Ser202/Thr205 in the hippocampus between two groups, but Ser396 phosphorylation for 5XFAD mice was significantly higher than that for wild-type mice at ages ranging from 2 to 6 months. The results of the present study indicate that Tau hyperphosphorylation in the brain for 5XFAD mice precedes high activation of GSK-3β and occurs in the much earlier stage than appearance of learning and memory disorders.

Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs) interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and protein tyrosine phosphatase 1B (PTP1B) were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K) or 3-phosphoinositide-dependent protein kinase-1 (PDK1). Results: In the cell-free assay, unsaturated FFAs (uFFAs) such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs) such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

Anticancer effect of adenosine on gastric cancer via diverse signaling pathways.

Extracellular adenosine induces apoptosis in a variety of cancer cells via intrinsic and extrinsic pathways. In the former pathway, adenosine uptake into cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Extracellular adenosine also induces apoptosis of gastric cancer cells. Extracellular adenosine is transported into cells through an adenosine transporter and converted to AMP by adenosine kinase. In turn, AMP activates AMP-activated protein kinase (AMPK). AMPK is the factor responsible for caspase-independent apoptosis of GT3-TKB gastric cancer cells. Extracellular adenosine, on the other hand, induces caspase-dependent apoptosis of MKN28 and MKN45 gastric cancer cells by two mechanisms. Firstly, AMP, converted from intracellularly transported adenosine, initiates apoptosis, regardless of AMPK. Secondly, the A3 adenosine receptor, linked to Gi/Gq proteins, mediates apoptosis by activating the Gq protein effector, phospholipase Cγ, to produce inositol 1,4,5-trisphosphate and diacylglycerol, which activate protein kinase C. Consequently, the mechanisms underlying adenosine-induced apoptosis vary, depending upon gastric cancer cell types. Understand the contribution of each downstream target molecule of adenosine to apoptosis induction may aid the establishment of tailor-made chemotherapy for gastric cancer.

Indomethacin serves as a potential inhibitor of protein phosphatases.

Background/Aims: We have shown that indomethacin has the potential to activate Ca2+/ calmodulin-dependent protein kinase II (CaMKII), regardless of cyclooxygenase (COX) inhibition. To understand the underlying mechanism, the present study investigated the effect of indomethacin on protein phosphatases such as protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and protein tyrosine phosphatase 1B (PTP1B). Methods: Activity of CaMKII was assayed in cultured rat hippocampal neurons and under the cell-free conditions. Activities of protein phosphatases were monitored under the cell-free conditions. Indomethacin binding assay was carried out using a fluorescein-conjugated indomethacin. Results: Indomethacin enhanced CaMKII activity in cultured rat hippocampal neurons, that is abolished the CaMKII inhibitor KN-93. In the cell-free assay, no CaMKII activation was obtained with indomethacin, but indomethacin otherwise inhibited PP1 in a concentration (10 µM-1 mM)-dependent manner, the maximum reaching 70% of basal levels. This indicates that indomethacin indirectly activates CaMKII due to PP1 inhibition. Likewise, indomethacin still inhibited PP2A and PTP1B in a concentration (10 µM-1 mM)-dependent manner, reaching 80 and 10% of basal levels at 1 mM, respectively. In the indomethacin binding assay, indomethacin bound to all the investigated protein phosphatases. Conclusion: The results of the present study indicate that indomethacin inhibits PP1, PP2A, and PTP1B, possibly through its direct binding and that the inhibitory effect of indomethacin on PP1 could cause indirect CaMKII activation. This may represent the novel indomethacin action.

Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aβ-Induced GSK-3β Activation and Tau Phosphorylation

Glycogen synthase kinase-3β (GSK-3β) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer’s disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3β by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3β. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3β than each independent treatment, to suppress amyloid β (Aβ)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy.

Diacylglycerol promotes GLUT4 translocation to the cell surface in a PKCε-dependent and PKCλ/ι and -ζ-independent manner.

AIM Emerging evidence has pointed to the participation of protein kinase C (PKC) in insulin-regulated trafficking of the glucose transporter GLUT4. The present study investigated the effect of the PKC activator diacylglycerol (DAG) on GLUT4 trafficking and glucose uptake. MAIN METHODS 3T3L1-GLUT4myc fibroblast cells expressing GLUT4myc were differentiated into adipocytes. Western blotting, glucose assay, and real-time RT-PCR were carried out in 3T3L1-GLUT4myc adipocytes. PKCλ/ι, -ζ, -ε, and -γ were knocked-down by transfecting each siRNA. Activity of PKC isozymes was assayed under the cell-free conditions. KEY FINDINGS Insulin increased cell surface localization of GLUT4 in 3T3L1-GLUT4myc adipocytes, and a similar effect was obtained with 1,2-dioleoyl-sn-glycerol (DO-DAG), 1-oleoyl-2-acetyl-sn-glycerol (OA-DAG), or 1,2-dipalmitoyl-sn-glycerol (DP-DAG). Like insulin, DO-DAG stimulated glucose uptake into adipocytes, but no significant synergistic increase in the glucose uptake was found with co-treatment with insulin and DO-DAG. Insulin activated Akt in adipocytes, but no Akt activation was induced by any investigated DAG. In the cell-free PKC assay, DAGs examined here activated PKCα, -βI, -βII, -γ, -δ, and -ε, but the atypical PKC isozymes PKCλ/ι and -ζ were not activated. Insulin-induced GLUT4 translocation to the cell surface was inhibited by knocking-down PKCλ/ι and -ζ, but not PKCγ or -ε. In contrast, DO-DAG-induced GLUT4 translocation to the cell surface was clearly prevented by knocking-down PKCε. SIGNIFICANCE The results of the present study indicate that DAG stimulates GLUT4 translocation to the cell surface by activating PKCε, regardless of PKCλ/-ι and -ζ.

PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling

Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). Methods: Activity of protein tyrosine phosphatase 1B (PTP1B) was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET) analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM)-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF)-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1) inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK)/IRS-1/PI3K/Akt/Rac1 axis.