P85 Subunit Of Phosphatidylinositol 3-kinase Research Articles

Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling

Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as cysteine (Cys) in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed phosphatidylinositol-3 kinase (PI3K)–Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor (EGF) receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K–Akt signaling by MVK reversed EGF-induced negative regulation of autophagy, and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K–Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K–Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.

Astaxanthin Inhibits p70 S6 Kinase 1 Activity to Sensitize Insulin Signaling.

Astaxanthin (AST) is a carotenoid with therapeutic values on hyperglycemia and diabetic complications. The mechanisms of action of AST remain incompletely understood. p70 S6 kinase 1 (S6K1) is a serine/threonine kinase that phosphorylates insulin receptor substrate 1 (IRS-1)S1101 and desensitizes the insulin receptor (IR). Our present study aims to determine if AST improves glucose metabolisms by targeting S6K1. Western blot analysis revealed that AST inhibited the phosphorylation of two S6K1 substrates, S6S235/236 and IRS-1S1101, but enhanced the phosphorylation of AKTT308, AKTS473, and S6K1T389 by feedback activation of the phosphatidylinositol-3 (PI-3) kinase in 3T3-L1 adipocytes and L6 myotubes. In vitro kinase assays revealed that AST inhibited S6K1 activity with an IC50 value of approximately 13.8 μM. AST increased insulin-induced IR tyrosine phosphorylation and IRS-1 binding to the p85 subunit of PI-3 kinase. Confocal microscopy revealed that AST increased the translocation of the glucose transporter 4 (GLUT4) to the plasma membrane in L6 cells. Glucose uptake assays using a fluorescent dye, 2-NBDG (2-N-(Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), revealed that AST increased glucose uptake in 3T3-L1 adipocytes and L6 myotubes under insulin resistance conditions. Our study identifies S6K1 as a previously unrecognized molecular target of AST and provides novel insights into the mechanisms of action of AST on IR sensitization.

Genomic landscape of young ATLL patients identifies frequent targetable CD28 fusions

AbstractAdult T-cell leukemia/lymphoma (ATLL) in Japan presents at a median age of 70 years and only 5% of patients are <50 years of age. We conducted RNA and targeted DNA sequencing of 8 ATLLs from Japanese patients <50 years of age and identified 3 (37.5%) with both CTLA4-CD28 and inducible costimulator (ICOS)-CD28 fusions. Mutations of PLCG1, PRKCB, and STAT3, which were frequent in other ATLL-sequencing studies, were not identified. Differential expression analysis identified the negative checkpoint molecule LAG3 as the most downregulated gene among cases with the fusions. Immunohistochemistry demonstrated expression of CD80 and CD86, the ligands for CTLA4 and CD28, on ATLL cells and tumor-associated macrophages, respectively. Expression of CTLA4-CD28 in Ba/F3 cells conferred cytokine-independent growth when cocultured with Raji cells that express CD80 and CD86. Growth was associated with recruitment of the p85 subunit of phosphatidylinositol 3-kinase to CTLA4-CD28 and phosphorylation of AKT and extracellular signal-regulated kinase. A CTLA4-blocking antibody reduced cytokine-independent growth in a dose-dependent manner. Together, these results suggest that young Japanese ATLL cases have a unique biology dependent on cell-nonautonomous interactions that drive CD28 signaling. Assessment for CD28 fusions and treatment with CTLA4 blockade should be considered in younger patients with relapsed/refractory ATLL.

Involvement of intracellular caveolin-1 distribution in the suppression of antigen-induced mast cell activation by cationic liposomes.

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross-linking of high-affinity immunoglobulin E receptor in a time- and dose-dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca2+ concentration ([Ca2+ ]i ) via the inhibition of store-operated Ca2+ entry. Further, we revealed that the mechanism underlying an impaired [Ca2+ ]i increase is the inhibition of the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Yet, how cationic liposomes inhibit the PI3K-Akt pathway is still unclear. Here, we focused on caveolin-1, a major component of caveolae, which is reported to be involved in the activation of the PI3K-Akt pathway in various cell lines. In this study, we showed that caveolin-1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin-1 to the plasma membrane and the colocalization of caveolin-1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.

Marek's Disease Virus Activates the PI3K/Akt Pathway Through Interaction of Its Protein Meq With the P85 Subunit of PI3K to Promote Viral Replication.

It is known that viruses can active the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in host cells to support cell survival and viral replication; however, the role of PI3K/Akt signaling in the pathogenic mechanisms induced by Marek’s disease virus (MDV) which causes a neoplastic Marek’s disease in poultry, remains unknown. In this study, we showed that MDV activated the PI3K/Akt pathway in chicken embryo fibroblasts (CEFs) at the early phase of infection, whereas treatment with a PI3K inhibitor LY294002 prior to MDV infection decreased viral replication and DNA synthesis. Flow cytometry analysis showed that inhibition of the PI3K/Akt pathway could significantly increase apoptosis in MDV-infected host cells, indicating that activation of PI3K/Akt signaling could facilitate viral replication through support of cell survival during infection. Evaluation of the underlying molecular mechanism by co-immunoprecipitation and laser confocal microscopy revealed that a viral protein Meq interacted with both p85α and p85β regulatory subunits of PI3K and could induce PI3K/Akt signaling in Meq-overexpressing chicken fibroblasts. Our results showed, for the first time, that MDV activated PI3K/Akt signaling in host cells through interaction of its Meq protein with the regulatory p85 subunit of PI3K to delay cell apoptosis and promote viral replication. This study provides clues for further studies of the molecular mechanisms underlying MDV infection and pathogenicity for the host.

Identification of the Binding Sites on Rab5 and p110beta Phosphatidylinositol 3-kinase

Rab5 is a small monomeric GTPase that mediates protein trafficking during endocytosis. Inactivation of Rab5 by GTP hydrolysis causes a conformational change that masks binding sites on its “switch regions” from downstream effectors. The p85 subunit of phosphatidylinositol 3-kinase (PI3K) is a GTPase activating protein (GAP) towards Rab5. Whereas p85 can bind with both Rab5-GTP and Rab5-GDP, the PI3K catalytic subunit p110β binds only Rab5-GTP, suggesting it interacts with the switch regions. Thus, the GAP functions of the catalytic arginine finger (from p85) and switch region stabilization (from p110β) may be provided by both proteins, acting together. To identify the Rab5 residues involved in binding p110β, residues in the Rab5 switch regions were mutated. A stabilized recombinant p110 protein, where the p85-iSH2 domain was fused to p110 (alpha or beta) was used in binding experiments. Eleven Rab5 mutants, including E80R and H83E, showed reduced p110β binding. The Rab5 binding site on p110β was also resolved through mutation of p110β in its Ras binding domain, and includes residues I234, E238 and Y244. This is a second region within p110β important for Rab5 binding. The Rab5-GTP:p110β interaction may be further elucidated through the characterization of these non-binding mutants in cells.

Wu-Mei-Wan Reduces Insulin Resistance via Inhibition of NLRP3 Inflammasome Activation in HepG2 Cells

Wu-Mei-Wan (WMW) is a Chinese herbal formula used to treat type 2 diabetes. In this study, we aimed to explore the effects and mechanisms of WMW on insulin resistance in HepG2 cells. HepG2 cells were pretreated with palmitate (0.25 mM) to impair the insulin signaling pathway. Then, they were treated with different doses of WMW-containing medicated serum and stimulated with 100 nM insulin. Results showed that palmitate could reduce the glucose consumption rate in HepG2 cells and impair insulin signaling related to phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), thereby regulating the downstream signaling pathways. However, medicated serum of WMW restored impaired insulin signaling, upregulated the expression of phospho-IR (pIR), phosphatidylinositol 3-kinase p85 subunit, phosphoprotein kinase B, and glucose transporter 4, and decreased IRS serine phosphorylation. In addition, it decreased the expression of interleukin-1β and tumor necrosis factor-α, which are the key proinflammatory cytokines involved in insulin resistance; besides, it reduced the expression of NLRP3 inflammasome. These results suggested that WMW could alleviate palmitate-induced insulin resistance in HepG2 cells via inhibition of NLRP3 inflammasome and reduction of proinflammatory cytokine production.

Effects of electroacupuncture on insulin resistance and hypothalamic insulin signal molecule in rats with diet-induced obesity

To observe the effects of electroacupuncture (EA) on body weight, insulin resistance (IR) and hypothalamic insulin signal molecule in rats with diet-induced obesity (DIO), and to explore the action mechanism of EA on DIO. Fifty SD male rats were randomly divided into a low fat diet (LFD) group (10 rats) and a high fat diet (HFD) group (40 rats). Rats were fed with LFD and HFD, respectively, and the DIO model was established in the HFD group. After the model was established, the rats were randomly divided intoa model group, an EA group and a medication group, ten rats in each one. The rats in the EA group were treated with EA at "Housanli" (ST 36) and "Quchi" (LI 11) for 20 min, once a day for totally 28 days. The rats in the medication group were treated with intragastric administration of orlistat, once a day for 28 days. The rats in the LFD group and model group received no treatment. After treatment, HE staining method was applied to observe the morphological changes of liver; the biochemistry technique and radioimmunoassay method were applied to detect the fasting plasma glucose (FPG) and fasting insulin (FINS); Western blot method was applied to measure the expression of phosphatidylinositol-3 kinase p85 subunit (PI3K-p85) and insulin receptor substrate 2 (IRS2). Under light microscope, compared with the model group, the fatty degenerative cells were below 1/2 in the EA group and the medication group, accompanied with decreased lipid droplet, mild edema and none inflammatory infiltration. The body weight, FPG, FINS, homeostasis model assessment-insulin resistance index (HOMA-IR) and PI3K-p85 in the EA group were significantly lower than those in the model group (P<0.01, P<0.05), but the expression of IRS2 was not significantly different from the model group (P>0.05). The body weight, HOMA-IR and PI3K-p85 in the medication group were lower than those in the model group (P<0.01, P<0.05), but the expression of IRS2 was higher than that in the model group (P<0.05). The differences of each index were not significant between the EA group and the medication group (all P>0.05). In addition, rats in the medication group showed watery defecation, decreased activity, fatigue mentality and yellow hairs, while rats in the EA group showed normal defecation and vivid hair. EA can decrease the expression of PI3K-p85 to prompt the IR of DIO rats, inhibit the weight body and improve hepatic steatosis, which is probably one of the action mechanisms of EA on DIO. Besides, the adverse effects as the medication group can be avoided.

Dehydroglyasperin D Inhibits the Proliferation of HT-29 Human Colorectal Cancer Cells Through Direct Interaction With Phosphatidylinositol 3-kinase.

Background:Despite recent advances in therapy, colorectal cancer still has a grim prognosis. Although licorice has been used in East Asian traditional medicine, the molecular properties of its constituents including dehydroglyasperin D (DHGA-D) remain unknown. We sought to evaluate the inhibitory effect of DHGA-D on colorectal cancer cell proliferation and identify the primary signaling molecule targeted by DHGA-D.Methods:We evaluated anchorage-dependent and -independent cell growth in HT-29 human colorectal adenocarcinoma cells. The target protein of DHGA-D was identified by Western blot analysis with a specific antibody, and direct interaction between DHGA-D and the target protein was confirmed by kinase and pull-down assays. Cell cycle analysis by flow cytometry and further Western blot analysis was performed to identify the signaling pathway involved.Results:DHGA-D significantly suppressed anchorage-dependent and -independent HT-29 colorectal cancer cell proliferation. DHGA-D directly suppressed phosphatidylinositol 3-kinase (PI3K) activity and subsequent Akt phosphorylation and bound to the p110 subunit of PI3K. DHGA-D also significantly induced G1 cell cycle arrest, together with the suppression of glycogen synthase kinase 3β and retinoblastoma phosphorylation and cyclin D1 expression.Conclusions:DHGA-D has potent anticancer activity and targets PI3K in human colorectal adenocarcinoma HT-29 cells. To our knowledge, this is the first report to detail the molecular basis of DHGA-D in suppressing colorectal cancer cell growth.

New and Unexpected Biological Functions for the Src-Homology 2 Domain-Containing Phosphatase SHP-2 in the Gastrointestinal Tract.

SHP-2 is a tyrosine phosphatase expressed in most embryonic and adult tissues. SHP-2 regulates many cellular functions including growth, differentiation, migration, and survival. Genetic and biochemical evidence show that SHP-2 is required for rat sarcoma viral oncogene/extracellular signal-regulated kinases mitogen-activated protein kinase pathway activation by most tyrosine kinase receptors, as well as by G-protein–coupled and cytokine receptors. In addition, SHP-2 can regulate the Janus kinase/signal transducers and activators of transcription, nuclear factor-κB, phosphatidyl-inositol 3-kinase/Akt, RhoA, Hippo, and Wnt/β-catenin signaling pathways. Emerging evidence has shown that SHP-2 dysfunction represents a key factor in the pathogenesis of gastrointestinal diseases, in particular in chronic inflammation and cancer. Variations within the gene locus encoding SHP-2 have been associated with increased susceptibility to develop ulcerative colitis and gastric atrophy. Furthermore, mice with conditional deletion of SHP-2 in intestinal epithelial cells rapidly develop severe colitis. Similarly, hepatocyte-specific deletion of SHP-2 induces hepatic inflammation, resulting in regenerative hyperplasia and development of tumors in aged mice. However, the SHP-2 gene initially was suggested to be a proto-oncogene because activating mutations of this gene were found in pediatric leukemias and certain forms of liver and colon cancers. Moreover, SHP-2 expression is up-regulated in gastric and hepatocellular cancers. Notably, SHP-2 functions downstream of cytotoxin-associated antigen A (CagA), the major virulence factor of Helicobacter pylori, and is associated with increased risks of gastric cancer. Further compounding this complexity, most recent findings suggest that SHP-2 also coordinates carbohydrate, lipid, and bile acid synthesis in the liver and pancreas. This review aims to summarize current knowledge and recent data regarding the biological functions of SHP-2 in the gastrointestinal tract.

Over-expression of IGF-1 via nanoparticle-mediated Trophoblast specific gene delivery reduces AKT1 expression but induces downstream signaling via GSK-beta

Studies have shown similarities between the histopathological characteristics of NPC and Alzheimer’s disease (AD) including amyloid and tau pathologies. While dysfunction in insulin signaling was widely detected in AD brain, the function of insulin signaling proteins has not been examined in NPC disease. In this study, we have examined the expression and phosphorylation of proteins linked to the insulin signaling pathway in the brain of 9 weeks old NPCnih mice. Our results showed lower expression of insulin receptor substrate 2 (IRS2) in the NPCnih mice, and insulin receptor substrate 1 (IRS1) expression was almost non-detectable in this NPC mouse model. This reduction was associated with the loss of expression for the regulatory p85 subunit of phosphatidylinositol 3-kinase (p85/PI3K). Interestingly, the impairment was observed to link to a greater reduction of Akt phosphorylation at residue T308 than S473. This aberrant Akt phosphorylation could be contributing to lower GSK3β phosphorylation detected in the NPCnih mouse brain. To our knowledge, this is the first report documenting impaired insulin signaling in the brain of a NPC mouse model.

A rapid cytoplasmic mechanism for PI3 kinase regulation by the nuclear thyroid hormone receptor, TRβ, and genetic evidence for its role in the maturation of mouse hippocampal synapses in vivo.

Several rapid physiological effects of thyroid hormone on mammalian cells in vitro have been shown to be mediated by the phosphatidylinositol 3-kinase (PI3K), but the molecular mechanism of PI3K regulation by nuclear zinc finger receptor proteins for thyroid hormone and its relevance to brain development in vivo have not been elucidated. Here we show that, in the absence of hormone, the thyroid hormone receptor TRβ forms a cytoplasmic complex with the p85 subunit of PI3K and the Src family tyrosine kinase, Lyn, which depends on two canonical phosphotyrosine motifs in the second zinc finger of TRβ that are not conserved in TRα. When hormone is added, TRβ dissociates and moves to the nucleus, and phosphatidylinositol (3, 4, 5)-trisphosphate production goes up rapidly. Mutating either tyrosine to a phenylalanine prevents rapid signaling through PI3K but does not prevent the hormone-dependent transcription of genes with a thyroid hormone response element. When the rapid signaling mechanism was blocked chronically throughout development in mice by a targeted point mutation in both alleles of Thrb, circulating hormone levels, TRβ expression, and direct gene regulation by TRβ in the pituitary and liver were all unaffected. However, the mutation significantly impaired maturation and plasticity of the Schaffer collateral synapses on CA1 pyramidal neurons in the postnatal hippocampus. Thus, phosphotyrosine-dependent association of TRβ with PI3K provides a potential mechanism for integrating regulation of development and metabolism by thyroid hormone and receptor tyrosine kinases.

Effect of fructose and 3,5-diiodothyronine (3,5-T(2)) on lipid accumulation and insulin signalling in non-alcoholic fatty liver disease (NAFLD)-like rat primary hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is nowadays considered as one of the most serious pathological conditions affecting the liver. NAFLD is supposed to be initiated by the accumulation of lipids in the liver, which finally results in an impaired hepatic insulin signalling. Many researchers have recently focused their attention on the role played by fructose as a NAFLD-triggering agent, because of the increased diffusion of fructose-sweetened food. However, epidemiological data do not permit to evaluate the role of fructose per se, because these foods are often associated with elevated energy intake and unhealthy lifestyle. In the present work, we analysed the effects of fructose on the accumulation of lipids and insulin signalling in rat primary hepatocytes. Moreover, we investigated the effect of the thyroid hormone metabolite, devoid of thyrotoxic effects, 3,5-diiodothyronine (3,5-T2) over the same parameters. To evaluate the effect on insulin signalling we took into consideration three key proteins, such as p85 subunit of phosphatidylinositol 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), and Akt. Our results show that fructose in vitro, in the range of physiological concentrations, was not able to stimulate either lipid accumulation or to impair insulin signalling in our NAFLD-like rat primary hepatocytes. Our data thus support the idea that fructose per se may exert detrimental effects mainly triggering systemic effects, rather than directly affecting isolated hepatocytes. Moreover, we demonstrated that 3,5-T2, at physiological levels, reduces lipid content and triggers phosphorylation of Akt in an insulin receptor-independent manner, revealing new interesting properties as a biologically active molecule.

Loss of Cbl–PI3K Interaction Enhances Osteoclast Survival due to p21‐Ras Mediated PI3K Activation Independent of Cbl‐b

Cbl family proteins, Cbl and Cbl-b, are E3 ubiquitin ligases and adaptor proteins, which play important roles in bone-resorbing osteoclasts. Loss of Cbl in mice decreases osteoclast migration, resulting in delayed bone development where as absence of Cbl-b decreases bone volume due to hyper-resorptive osteoclasts. A major structural difference between Cbl and Cbl-b is tyrosine 737 (in YEAM motif) only on Cbl, which upon phosphorylation interacts with the p85 subunit of phosphatidylinositol-3 Kinase (PI3K). In contrast to Cbl(-/-) and Cbl-b(-/-) , mice lacking Cbl-PI3K interaction due to a Y737F (tyrosine to phenylalanine, YF) mutation showed enhanced osteoclast survival, but defective bone resorption. To investigate whether Cbl-PI3K interaction contributes to distinct roles of Cbl and Cbl-b in osteoclasts, mice bearing CblY737F mutation in the Cbl-b(-/-) background (YF/YF;Cbl-b(-/-) ) were generated. The differentiation and survival were augmented similarly in YF/YF and YF/YF;Cbl-b(-/-) osteoclasts, associated with enhanced PI3K signaling suggesting an exclusive role of Cbl-PI3K interaction, independent of Cbl-b. In addition to PI3K, the small GTPase Ras also regulates osteoclast survival. In the absence of Cbl-PI3K interaction, increased Ras GTPase activity and Ras-PI3K binding were observed and inhibition of Ras activation attenuated PI3K mediated osteoclast survival. In contrast to differentiation and survival, increased osteoclast activity observed in Cbl-b(-/-) mice persisted even after introduction of the resorption-defective YF mutation in YF/YF;Cbl-b(-/-) mice. Hence, Cbl and Cbl-b play mutually exclusive roles in osteoclasts. Whereas Cbl-PI3K interaction regulates differentiation and survival, bone resorption is predominantly regulated by Cbl-b in osteoclasts.

Jiaotai pill (交泰丸) enhances insulin signaling through phosphatidylinositol 3-kinase pathway in skeletal muscle of diabetic rats

To investigate the effect of Jiaotai Pill (, JTP) at different constitutional proportions on insulin signaling through phosphatidylinositol 3-kinase (PI3K) pathway in the skeletal muscle of diabetic rats. The rat model of type 2 diabetes mellitus (T2DM) was established by intravenous injection of a small dose of streptozotoein plus high fat diet feeding. JTP at the same dosage of cinnamon and the increasing dosage of Coptis chinensis was administered to diabetic rats for nine weeks respectively. Plasma glucose and insulin levels were assayed. The expressions of proteins were determined by Western blot method. All the three formulations of JTP decreased plasma glucose and fasting insulin levels as well as increased the protein expressions of insulin receptor β (InsRβ) subunit, insulin receptor substrate-1 (IRS-1), PI3K p85 subunit and glucose transporter 4 (GLUT4) in skeletal muscle. Meanwhile, JTP increased the tyrosine phosphorylation of InsRβ subunit and IRS-1, and reduced the serine phosphorylation of IRS-1 in skeletal muscle. Interestingly, the effect of JTP on improving insulin sensitivity was not dose-dependent. In contrast, JTP containing the least amount of Coptis chinensis exhibited the best effect. JTP at different constitutional proportions attenuates the development of diabetes in a rat model of T2DM. The mechanism might be associated with enhancing insulin signaling through PI3K pathway in the skeletal muscle.

The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer

The identification of primary molecular targets of cancer-preventive phytochemicals is essential for a comprehensive understanding of their mechanism of action. In the present study, we investigated the chemopreventive effects and molecular targets of acacetin, a flavonoid found in Robinia p seudoacacia, also known as black locust. Acacetin treatment significantly suppressed epidermal growth factor (EGF)-induced cell transformation. Immunoblot analysis revealed that acacetin attenuated EGF-induced phosphorylation of Akt and p70(S6K), which are downstream effectors of phosphatidylinositol 3-kinase (PI3-K). An immunoprecipitation kinase assay of PI3-K and pull-down assay results demonstrated that acacetin substantially inhibits PI3-K activity by direct physical binding. Acacetin exhibited stronger inhibitory effects against anchorage-dependent and -independent cell growth in cells expressing higher PI3-K activity compared with those exhibiting relatively low PI3-K activity. Binding assay data combined with computational modeling suggest that acacetin binds in an adenosine triphosphate (ATP)-competitive manner with the p110α subunit of PI3-K and interacts with Val828, Glu826, Asp911, Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900 residues. Acacetin was also found to significantly reduce SK-MEL-28 tumor growth and Akt phosphorylation in vivo. Taken together, these results indicate that acacetin is an ATP-competitive PI3-K inhibitor and a promising agent for melanoma chemoprevention.

Abstract 2589: The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer.

Abstract Verifying potential target proteins of phytochemicals is important for explaining their health benefits. In the present study, we examined the chemopreventive effect and molecular target of acacetin, which is a flavonoid from Robinia Psuedoacacia also known as Black Locust. Treatment with acacetin significantly inhibits EGF-induced transformation of JB6 P+ cells. Western blot assay results revealed that acacetin suppresses EGF-induced phosphorylation of Akt/p70S6K, which is a well-known downstream signaling molecule of phosphatidylinositol 3-kinase (PI3-K). In vitro kinase and pull-down assay results showed that acacetin significantly inhibits PI3-K activity by direct binding. Additionally, acacetin also attenuated EGF-induced PI3-K activity and binds with PI3-K in JB6 P+ cells. Acacetin showed significant effects against anchorage-dependent and -independent cell growth in cells displaying higher PI3-K activity compared with those exhibiting lower PI3-K activity. Binding assay data and computational modeling showed that acacetin binds in an ATP-competitive manner with the p110 kinase domain of PI3-K and interacts with Val828, Glu826 Asp911 Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900. Acacetin reduced SK-MEL-28 xenograft tumor growth and phosphorylation of Akt in vivo. Overall, these results indicate that acacetin is an ATP competitive PI3-K inhibitor and might be a promising chemopreventive agent targeting PI3-K activity. Citation Format: Jon Eun Kim, Sung Keun Jung, Sanguine Byun, Sung-Young Lee, Myoung Ok Kim, Tae Gyu Lim, Ann M. Bode, Hyong Joo Lee, Ki Won Lee, Zigang Dong. The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2589. doi:10.1158/1538-7445.AM2013-2589

Abstract 551: The requirement of Runx2-dependent Akt phosphorylation in breast cancer cell survival and metastasis.

Abstract Breast cancer is not only most commonly diagnosed in women worldwide but its metastasis shows serious life threatening consequences. The cell survival and mitogenesis via phosphatidyl inositol 3’ kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling pathways are critical for breast cancer cell survival, proliferation, metabolism and metastasis. A de-regulation of osteogenic Runt-related transcription factor-2 (Runx2) is recently implicated in mammary carcinogenesis. We found that Runx2 differentially regulates the serine/threonine kinase Akt phosphorylation (pAkt), a downstream effecter of PI3K signaling, in non-tumorigenic and invasive mammary epithelial cell lines. The decline in pAkt upon Runx2 suppression in invasive MDA-MB-231 cells was completely abrogated by PI3K inhibitor LY294002. In contrast, the MAPK inhibitor, U0126, showed partial restoration of pAkt upon Runx2 depletion suggesting that Runx2-mediated crosstalk stimulates feedback mechanism within the PI3K and MAPK signaling pathways. The expression analysis of kinases and phosphatases functional in pAkt regulation revealed that the decline in pAkt upon Runx2 knockdown in MDA-MB-231 cells is due to the reduction in PI3K p85 subunit and mammalian target of rapamycin complex-2 (mTORC2). Additionally, we find altered expression of FOXO1, a downstream molecule of PI3K/Akt signaling, due to Runx2-mediated regulation of pAkt levels in MCF-10A and MDA-MB-231 cell lines. To examine the phenotypic effects of Runx2 and PI3K/Akt pathway crosstalk, we evaluated the Runx2-depleted MDA-MB-231 cells for survival under stress conditions. These studies revealed an altered morphology coupled with increased rounding and sub-G1 population in response to glucose- and serum-deprivation upon Runx2 knockdown. The increase in sub-G1 population was restored by glucose and serum re-constitution in the culture media. An increase in Annexin V staining, decline in pAkt levels and cleavage of caspase-3 confirmed the induction of apoptotic cell death. To understand the Runx2-mediated regulation of PI3K/Akt signaling pathway in breast cancer metastasis, the control or Runx2-depleted MDA-MB-231 cells were inoculated intra-cardiacally into immunodeficient mice. The tumors at lung and bone metastatic sites were harvested, FACS-sorted and examined for pAkt levels. Interestingly, to this end, the in-vivo metastatic tumor-derived Runx2 knockdown MDA-MB-231 cells showed an increase in pAkt levels in contrast to in-vitro cultured Runx2 knockdown MDA-MB-231 cells. The data suggests that the mechanism of pAkt activation might involve a dynamic requirement of transcriptional control and feedback regulation within PI3K/Akt and MAPK signaling pathways during different phases of tumor growth in primary and metastatic microenvironment. Citation Format: Manish Tandon, Zujian Chen, Jitesh Pratap. The requirement of Runx2-dependent Akt phosphorylation in breast cancer cell survival and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 551. doi:10.1158/1538-7445.AM2013-551

The recruitment and activation of phosphatidylinositol 4-phosphate 5-kinases a critically regulate CD28-dependent signaling responses

The recruitment and activation of phosphatidylinositol 4-phosphate 5-kinases a critically regulate CD28-dependent signaling responses

Varicella-Zoster Virus ORF12 Protein Activates the Phosphatidylinositol 3-Kinase/Akt Pathway To Regulate Cell Cycle Progression

Varicella-zoster virus (VZV) activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and alters cell cycle progression, but the viral protein(s) responsible for these activities is unknown. We previously reported that the VZV open reading frame 12 (ORF12) protein triggers phosphorylation of ERK. Here, we demonstrate that the VZV ORF12 protein also activates the PI3K/Akt pathway to regulate cell cycle progression. Transfection of cells with a plasmid expressing the ORF12 protein induced phosphorylation of Akt, which was dependent on PI3K. Infection of cells with wild-type VZV triggered phosphorylation of Akt, while infection with an ORF12 deletion mutant induced less phosphorylated Akt. The activation of Akt by ORF12 protein was associated with its binding to the p85 subunit of PI3K. Infection of cells with wild-type VZV resulted in increased levels of cyclin B1, cyclin D3, and phosphorylated glycogen synthase kinase 3β (GSK-3β), while infection with the ORF12 deletion mutant induced lower levels of these proteins. Wild-type VZV infection reduced the G(1) phase cell population and increased the M phase cell population, while infection with the ORF12 deletion mutant had a reduced effect on the G(1) and M phase populations. Inhibition of Akt activity with LY294002 reduced the G(1) and M phase differences observed in cells infected with wild-type and ORF12 mutant viruses. In conclusion, we have found that the VZV ORF12 protein activates the PI3K/Akt pathway to regulate cell cycle progression. Since VZV replicates in both dividing (e.g., keratinocytes) and nondividing (neurons) cells, the ability of the VZV ORF12 protein to regulate the cell cycle is likely important for VZV replication in various cell types in the body.