Phosphatidylinositide 3-kinase Activity Research Articles

Alkaline intracellular pH (pHi) increases PI3K activity to promote mTORC1 and mTORC2 signaling and function during growth factor limitation

The conserved protein kinase mTOR (mechanistic target of rapamycin) responds to diverse environmental cues to control cell metabolism and promote cell growth, proliferation, and survival as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. Our prior work demonstrated that an alkaline intracellular pH (pHi) increases mTORC2 activity and cell survival in complete media in part by activating AMP-activated protein kinase, a kinase best known to sense energetic stress. It is important to note that an alkaline pHi represents an underappreciated hallmark of cancer cells that promotes their oncogenic behaviors. In addition, mechanisms that control mTORC1 and mTORC2 signaling and function remain incompletely defined, particularly in response to stress conditions. Here, we demonstrate that an alkaline pHi increases phosphatidylinositide 3-kinase (PI3K) activity to promote mTORC1 and mTORC2 signaling in the absence of serum growth factors. Alkaline pHi increases mTORC1 activity through PI3K-Akt signaling, which mediates inhibitory phosphorylation of the upstream proteins tuberous sclerosis complex 2 and proline-rich Akt substrate of 40 kDa and dissociates tuberous sclerosis complex from lysosomal membranes, thus enabling Rheb-mediated activation of mTORC1. Thus, alkaline pHi mimics growth factor-PI3K signaling. Functionally, we also demonstrate that an alkaline pHi increases cap-dependent protein synthesis through inhibitory phosphorylation of eIF4E binding protein 1 and suppresses apoptosis in a PI3K- and mTOR-dependent manner. We speculate that an alkaline pHi promotes a low basal level of cell metabolism (e.g., protein synthesis) that enables cancer cells within growing tumors to proliferate and survive despite limiting growth factors and nutrients, in part through elevated PI3K-mTORC1 and/or PI3K-mTORC2 signaling.

Abstract A15: The selective role of PIK3CB/p110β in temozolomide resistance in glioblastoma

Abstract Background: The divergent roles of phosphatidylinositide 3-kinase (PI3K) family genes in cancer pathogenesis and progression have been highly studied. PI3K genes are divided into three classes (I, II, and III). Class IA PI3K includes three highly homologous catalytic subunits p110α, β, or δ, encoded by the genes PIK3CA, B, and D, respectively. Studies in glioblastoma (GBM), the most lethal and most common brain cancer, have shown PI3K to promote resistance to the front-line chemotherapeutic drug, temozolomide (TMZ) (1). We recently investigated these isoforms in GBM and found that levels of PIK3CB/p110β, but not other PI3K isoforms, present prognostic significance for GBM recurrence and that this isoform selectively activates GBM cell survival pathways (2). Our previous work also shows that a gap-junction protein, Connexin 43 (Cx43), promotes TMZ resistance in GBM by activating PI3K signaling (3). Given that PIK3CB/p110β is the most important isoform in GBM PI3K signaling, it is therefore critical to investigate whether PI3K isoforms have divergent assignments in Cx43-mediated TMZ resistance. Understanding the role of PI3K isoforms in drug resistance will foster the rational design of more effective treatments for GBM. Hypothesis: Based upon the results described above, we hypothesize that PIK3CB/p110β is selectively important for Cx43-mediated TMZ resistance in GBM. Approaches: The expression and activity of PI3K genes and Cx43 are manipulated by overexpression of active PI3K mutants, short hairpin RNA (shRNA)-mediated knockdown, a peptide compound called αCT1, or specific chemical inhibitors. Activity of signaling is assessed by immunoblotting. Cx43 channel activity is monitored by ATP release. Protein-protein interactions are measured using fluorescence microscopy and immunoprecipitation. Cell viability is determined using the MTS viability assay. Results: We find that (1) shRNA knockdown of Cx43 inactivates PI3K signaling in GBM cells, while constitutive activation of PI3K diminishes TMZ sensitization by the Cx43 peptide inhibitor, αCT1; (2) GBM patients with high levels of Cx43 also express more p110β, active AKT, and display TMZ resistance; (3) Cx43 activates PI3K independent of Cx43-formed channels; (4) p110β, but not p110α or p110δ, interacts with Cx43; and (5) selective inhibition of p110β circumvents TMZ resistance together with αCT1. Conclusion/Impact: We conclude that PIK3CB/p110β is selectively important for Cx43-mediated temozolomide resistance, presenting the possibility of selectively targeting PIK3CB/p110β as a new and effective approach to overcoming TMZ resistance in GBM.

Abstract A18: Selectively targeting PI3K isoforms to treat glioblastoma

Abstract The phosphatidylinositide 3-kinase (PI3K) family consists of four catalytic isoforms (PIK3CA, B, D, and G that encode p110alpha, beta, delta, and gamma, respectively), which play deviating roles in the formation and progression of different types of cancer such as breast and colon cancers. However, whether these PI3K isoforms function differently or equivalently in glioblastoma has not yet been explored. Glioblastoma is the most common and aggressive malignant glioma and patients often succumb to tumor recurrence, a progressive disease state that significantly contributes to the poor prognosis of glioblastoma. PI3K signaling is hyperactivated and mutated in glioblastoma; however, targeting PI3K to treat this cancer has not yet been successful. Given that PI3K isoforms are functionally divergent, it is very likely that selectively targeting PI3K catalytic isoforms is a more effective and discriminating approach to treating GBM. This possibility is supported by the following lines of evidence. Firstly, among four PI3K catalytic isoforms, PIK3CB is highly expressed in glioblastoma and high levels of PIK3CB exhibit the strongest correlation with the incidence, risk (time to develop recurrence), and survival of glioblastoma recurrence based upon expression data of PI3K isoforms and patient data from The Cancer Genome Atlas. Secondly, PI3K is often hyperactivated in glioblastoma cells with high levels of p110beta, but not other p110s, and depletion of p110beta inactivates, whereas knockdown of other p110s has no effect on, PI3K activity as well as glioblastoma cell survival. Thirdly, the selective activation of p110beta may be due primarily to a gap junction protein connexin 43 because this cell surface protein activates p110beta in p110beta-high glioblastoma cells through a selective interaction between connexin 43 and p110beta. Lastly, glioblastoma cells display differential responses to p110 isoform-selective inhibitors. Only p110beta selective inhibitors effectively activate apoptosis in, suppress the viability of, and impede the xenograft growth of p110beta hyperactivated glioblastoma cells. More importantly, p110beta inhibitors are much less toxic to normal astrocytes than other p110 inhibitors. These findings have revealed molecular details underlying the divergent roles of PI3K catalytic isoforms in glioblastoma, which will foster a rational design of more effective PI3K-based therapies for this deadly disease. Citation Format: Kevin J. Pridham, Lamvy Le, Sujuan Guo, Robin T. Varghese, Yanping Liang, Cara M. Rodgers, Gary R. Simonds, Deborah F. Kelly, Zhi Sheng. Selectively targeting PI3K isoforms to treat glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A18.

Inhibition of PI3K by copanlisib exerts potent antitumor effects on Merkel cell carcinoma cell lines and mouse xenografts

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer with steadily increasing incidence and poor prognosis. Despite recent success with immunotherapy, 50% of patients still succumb to their diseases. To date, there is no Food and Drug Administration-approved targeted therapy for advanced MCC. Aberrant activation of phosphatidylinositide-3-kinase (PI3K)/AKT/mTOR pathway is frequently detected in MCC, making it an attractive therapeutic target. We previously found PI3K pathway activation in human MCC cell lines and tumors and demonstrated complete clinical response in a Stage IV MCC patient treated with PI3K inhibitor idelalisib. Here, we found that both PI3K-α and -δ isoforms are abundantly expressed in our MCC cell lines and clinical samples; we therefore examined antitumor efficacy across a panel of five PI3K inhibitors with distinctive isoform-specificities, including idelalisib (PI3K-δ), copanlisib (PI3K-α/δ), duvelisib (PI3K-γ/δ), alpelisib (PI3K-α), and AZD8186 (PI3K-β/δ). Of these, copanlisib exerts the most potent antitumor effects, markedly inhibiting cell proliferation, survival, and tumor growth by suppressing PI3K/mTOR/Akt activities in mouse models generated from MCC cell xenografts and patient-derived tumor xenografts. These results provide compelling preclinical evidence for application of copanlisib in advanced MCC with aberrant PI3K activation for which immunotherapy is insufficient, or patients who are unsuitable for immunotherapy.

PI3K/Akt-Beclin1 signaling pathway positively regulates phagocytosis and negatively mediates NF-κB-dependent inflammation in Staphylococcus aureus-infected macrophages

Although autophagy and phagocytosis are involved in the regulation of host inflammatory response to bacterial infection in macrophages, the underlying mechanisms have not been completely elucidated. In the present study, we found that infecting RAW264.7 macrophages with Staphylococcus aureus (S. aureus) activated multiple signaling pathways including phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt), nuclear factor-κB (NF-κB), and Rac1, as well as triggered autophagy. LY294002, a specific PI3K activity inhibitor, significantly decreased autophagy and phagocytosis of macrophages upon S. aureus infection. Similarly, knockdown of Beclin1 by specific siRNA significantly inhibited autophagy and phagocytosis of S. aureus-infected macrophages. Additionally, we showed that although administration of Beclin1 siRNA had no effects on phosphorylation of Akt (p-Akt), inhibition of PI3K activity by LY294002 significantly decreased the expression of Beclin1, suggesting that Beclin1 is a downstream molecular of PI3K. Furthermore, inhibition of autophagy significantly increased the production of NF-κB-dependent TNFα/IL-1β in S. aureus-infected macrophages. Collectively, these findings demonstrated, for the first time, that the PI3K/Akt-Beclin1 signaling pathway positively regulates phagocytosis and negatively mediates NF-κB-dependent inflammation in S. aureus-infected macrophages.

EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation

EGFR activates phosphatidylinositide 3-kinase (PI3K), but the mechanism underlying this activation is not completely understood. We demonstrated here that EGFR activation resulted in lysine acetyltransferase 5 (KAT5)-mediated K395 acetylation of the platelet isoform of phosphofructokinase 1 (PFKP) and subsequent translocation of PFKP to the plasma membrane, where the PFKP was phosphorylated at Y64 by EGFR. Phosphorylated PFKP binds to the N-terminal SH2 domain of p85α, which is distinct from binding of Gab1 to the C-terminal SH2 domain of p85α, and recruited p85α to the plasma membrane resulting in PI3K activation. PI3K-dependent AKT activation results in enhanced phosphofructokinase 2 (PFK2) phosphorylation and production of fructose-2,6-bisphosphate, which inturn promotes PFK1 activation. PFKP Y64 phosphorylation-enhanced PI3K/AKT-dependent PFK1 activation and GLUT1 expression promoted the Warburg effect, tumor cell proliferation, and brain tumorigenesis. These findings underscore the instrumental role of PFKP in PI3K activation and enhanced glycolysis through PI3K/AKT-dependent positive-feedback regulation.

Electrotaxis of cardiac progenitor cells, cardiac fibroblasts, and induced pluripotent stem cell-derived cardiac progenitor cells requires serum and is directed via PI3′K pathways

The limited regenerative capacity of cardiac tissue has long been an obstacle to treating damaged myocardium. Cell-based therapy offers an enormous potential to the current treatment paradigms. However, the efficacy of regenerative therapies remains limited by inefficient delivery and engraftment. Electrotaxis (electrically guided cell movement) has been clinically used to improve recovery in a number of tissues but has not been investigated for treating myocardial damage. The purpose of this study was to test the electrotactic behaviors of several types of cardiac cells. Cardiac progenitor cells (CPCs), cardiac fibroblasts (CFs), and human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPCs) were used. CPCs and CFs electrotax toward the anode of a direct current electric field, whereas hiPSC-CPCs electrotax toward the cathode. The voltage-dependent electrotaxis of CPCs and CFs requires the presence of serum in the media. Addition of soluble vascular cell adhesion molecule to serum-free media restores directed migration. We provide evidence that CPC and CF electrotaxis is mediated through phosphatidylinositide 3-kinase signaling. In addition, very late antigen-4, an integrin and growth factor receptor, is required for electrotaxis and localizes to the anodal edge of CPCs in response to direct current electric field. The hiPSC-derived CPCs do not express very late antigen-4, migrate toward the cathode in a voltage-dependent manner, and, similar to CPCs and CFs, require media serum and phosphatidylinositide 3-kinase activity for electrotaxis. The electrotactic behaviors of these therapeutic cardiac cells may be used to improve cell-based therapy for recovering function in damaged myocardium.

Insensitivity of PI3K/Akt/GSK3 signaling in peripheral blood mononuclear cells of age-related macular degeneration patients.

Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C (IL-17RC), a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration (AMD), was associated with altered activation of phosphatidylinositide 3-kinase (PI3K), Akt, and glycogen synthase kinase 3 (GSK3). We wondered whether or not altered PI3K, Akt, and GSK3 activities could be detected in peripheral blood mononuclear cells (PBMC) obtained from AMD patients. In the patients' PBMC, absent or reduced serine-phosphorylation of GSK3α or GSK3β was observed, which was accompanied with increased phosphorylation of GSK3 substrates (e.g. CCAAT enhancer binding protein α, insulin receptor substrate 1, and TAU), indicative of enhanced GSK3 activation. In addition, decreased protein mass of PI3K85α and tyrosine-phosphorylation of PI3K50α was present in PBMC of the AMD patients, suggesting impaired PI3K activation. Moreover, abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients. These data demonstrate that despite the presence of high levels of IL-17RC, Wnt-3a and vascular endothelial growth factor, the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients. Thus, measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.

The memory-enhancing effect of erucic acid on scopolamine-induced cognitive impairment in mice

Erucic acid is a monounsaturated omega-9 fatty acid isolated from the seed of Raphanus sativus L. that is known to normalize the accumulation of very long chain fatty acids in the brains of patients suffering from X-linked adrenoleukodystrophy. Here, we investigated whether erucic acid enhanced cognitive function or ameliorated scopolamine-induced memory impairment using the passive avoidance, Y-maze and Morris water maze tasks. Erucic acid (3mg/kg, p.o.) enhanced memory performance in normal naïve mice. In addition, erucic acid (3mg/kg, p.o.) ameliorated scopolamine-induced memory impairment, as assessed via the behavioral tasks. We then investigated the underlying mechanism of the memory-enhancing effect of erucic acid. The administration of erucic acid increased the phosphorylation levels of phosphatidylinositide 3-kinase (PI3K), protein kinase C zeta (PKCζ), extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB) and additional protein kinase B (Akt) in the hippocampus. These results suggest that erucic acid has an ameliorative effect in mice with scopolamine-induced memory deficits and that the effect of erucic acid is partially due to the activation of PI3K–PKCζ–ERK–CREB signaling as well as an increase in phosphorylated Akt in the hippocampus. Therefore, erucic acid may be a novel therapeutic agent for diseases associated with cognitive deficits, such as Alzheimer's disease.

Insulin dependent apolipoprotein B degradation and phosphatidylinositide 3-kinase activation with microsomal translocation are restored in McArdle RH7777 cells following serum deprivation

Previous studies in rat hepatocytes demonstrated that insulin-dependent apolipoprotein (apo) B degradation (IDAD) is lost when cells are maintained for 3 d under enriched culture conditions. Loss of IDAD correlates with increased expression of protein tyrosine phosphatase 1B (PTP1B) known to be associated with resistance to insulin signaling in the liver. McArdle RH7777 hepatoma (McA) cells cultured in serum containing medium are resistant to IDAD; demonstrate a 30% increase in apo B secretion, and express increased levels of PTP1B protein and mRNA. In addition, insulin-stimulated Class I phosphatidylinositide 3-kinase (PI3K) activity of anti-pY immunoprecipitates is severely blunted. IDAD resistance in McA cells correlates with diminished translocation of insulin-stimulated pY-IRS1 to intracellular membranes. Incubation of McA cells with RK682, a protein tyrosine phosphatase inhibitor, is sufficient to restore IDAD in resistant McA cells. Overall, results further support the importance of Class I PI3K activity in IDAD, and suggest that loss of this activity is sufficient to cause resistance. Although other factors are involved in downstream events including sortilin binding to apo B, autophagy, and lysosomal degradation, loss of signal generation and reduced localization of Class I PI3K to intracellular membranes plays a significant role in IDAD resistance.

Podocalyxin promotes cisplatin chemoresistance in osteosarcoma cells through phosphatidylinositide 3-kinase signaling

Osteosarcoma (OS) is the most common type of primary bone malignancy. The use of multiagent, intensive chemotherapy has markedly improved the long‑term survival rate of patients with OS. However, chemoresistance continues to be the principal reason for poor survival and disease recurrence in patients with OS. Innate or acquired resistance to cisplatin, which is one of the most effective drugs against OS, is common. Understanding the molecular basis underlying cisplatin chemoresistance in OS cells may serve as a basis for the identification of novel therapeutic targets and biomarkers. High expression levels of podocalyxin (PCX) have been shown to be correlated with poor outcome in various types of cancer. A recent study suggested that PCX may contribute to cancer chemoresistance. The present study aimed to explore the role of PCX in OS by determining its effects on cisplatin chemoresistance in OS cells. Stable overexpression and knockdown of PCX were performed in MG‑63 and U2OS human OS cell lines. Overexpression of PCX in the two cell lines significantly increased the half maximal inhibitory concentration (IC50) of cisplatin, cell colony formation, phosphatidylinositide 3‑kinase (PI3K) activity and Akt phosphorylation at serine 473, and decreased cisplatin‑induced cell apoptosis. Furthermore, the effects of PCX were largely attenuated by treatment with the selective PI3K inhibitor BKM120. Conversely, knockdown of PCX expression markedly decreased the IC50 of cisplatin, cell colony formation, PI3K activity and Akt phosphorylation at serine 473, and increased cisplatin‑induced cell apoptosis. In conclusion, the present study was the first, to the best of our knowledge, to provide evidence that PCX promotes cisplatin chemoresistance in OS cells through a PI3K‑dependent mechanism. The results of the present study provided novel insight not only into the functional role of PCX in cancer, but also into the molecular mechanisms underlying OS chemoresistance.

Dibenzoylmethane, hydroxydibenzoylmethane and hydroxymethyldibenzoylmethane inhibit phorbol-12-myristate13-acetate‑induced breast carcinoma cell invasion.

Dibenzoylmethane (DB), a minor constituent of the root extract of licorice, belongs to the flavonoid family. Hydroxydibenzoylmethane (HDB) and hydroxymethyldibenzoylmethane (HMDB) have an identical structure to DB, but also possess a hydroxyl group and a hydroxyl and methyl group bonded to aromatic rings, respectively. They inhibit cellular proliferation and induce apoptosis in a variety of types of cancer cell, however, the antimetastatic effects of DB, HDB and HMDB on human breast carcinoma cells remain to be elucidated. The present study aimed to clarify the molecular mechanisms underlying the effects of DB and its analogues on phorbol‑12‑myristate 13‑acetate (PMA)‑induced MCF‑7 cell metastasis. The results revealed that DB, HDB and HMDB inhibited cell migration and invasion. In addition, PMA‑mediated MCF‑7 cell invasion was inhibited by DB, HDB and HMDB by inhibiting the expression of matrix metalloproteinase (MMP)‑9. Rottlerin, a protein kinaseC (PKC)δ inhibitor and LY294002, a phosphatidylinositide 3‑kinase (PI3K) inhibitor, reduced the PMA‑mediated expression of MMP‑9 and cell invasion. Furthermore, DB, HDB and HMDB prevented the activation of PKCδ and PI3K by inhibiting their phosphorylation. The present study was the first, to the best of our knowledge, to demonstrate the antimetastatic potential of DB, HDB and HDMB, which decreased cancer cell invasion through the PI3K/PKCδ‑mediated MMP‑9 pathway.

Decoding insulin resistance and metabolic syndrome for promising therapeutic intervention

Decoding insulin resistance and metabolic syndrome for promising therapeutic intervention

Signaling steps in the induction of genomic damage by insulin in colon and kidney cells

Diabetes mellitus (DM), a disease with almost 350 million people affected worldwide, will be the seventh leading cause of death by 2030. Diabetic patients develop various types of complications, among them an increased rate of malignancies. Studies reported the strong correlation between DM and several cancer types, of which colon and kidney cancers are the most common. Hyperinsulinemia, the high insulin blood level characteristic of early diabetes type 2, was identified as a risk factor for cancer development. In previous studies, we showed that an elevated insulin level can induce oxidative stress, resulting in DNA damage in colon cells in vitro and in kidney cells in vitro and in vivo. In the present study, we elucidate the signaling pathway of insulin-mediated genotoxicity, which is effective through oxidative stress induction in colon and kidney. The signaling mechanism is starting by phosphorylation of the insulin and insulin-like growth factor-1 receptors, followed by activation of phosphatidylinositide 3-kinase (PI3K), which in turn activates AKT. Subsequently, mitochondria and nicotinamide adenine dinucleotide phosphate oxidase (NADPH) isoforms (Nox1 and Nox4 in colon and kidney, respectively) are activated for reactive oxygen species (ROS) production, and the resulting excess ROS can attack the DNA, causing DNA oxidation. We conclude that hyperinsulinemia represents an important risk factor for cancer initiation or progression as well as a target for cancer prevention in diabetic patients.

Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms.

Insulin resistance is a major underlying mechanism responsible for the 'metabolic syndrome', which is also known as insulin resistance syndrome. The incidence of the metabolic syndrome is increasing at an alarming rate, becoming a major public and clinical problem worldwide. The metabolic syndrome is represented by a group of interrelated disorders, including obesity, hyperglycemia, hyperlipidemia, and hypertension. It is also a significant risk factor for cardiovascular disease and increased morbidity and mortality. Animal studies have demonstrated that insulin and its signaling cascade normally control cell growth, metabolism, and survival through the activation of MAPKs and activation of phosphatidylinositide-3-kinase (PI3K), in which the activation of PI3K associated with insulin receptor substrate 1 (IRS1) and IRS2 and subsequent Akt→Foxo1 phosphorylation cascade has a central role in the control of nutrient homeostasis and organ survival. The inactivation of Akt and activation of Foxo1, through the suppression IRS1 and IRS2 in different organs following hyperinsulinemia, metabolic inflammation, and overnutrition, may act as the underlying mechanisms for the metabolic syndrome in humans. Targeting the IRS→Akt→Foxo1 signaling cascade will probably provide a strategy for therapeutic intervention in the treatment of type 2 diabetes and its complications. This review discusses the basis of insulin signaling, insulin resistance in different mouse models, and how a deficiency of insulin signaling components in different organs contributes to the features of the metabolic syndrome. Emphasis is placed on the role of IRS1, IRS2, and associated signaling pathways that are coupled to Akt and the forkhead/winged helix transcription factor Foxo1.

Cell Signaling Associated with Na+/K+-ATPase: Activation of Phosphatidylinositide 3-Kinase IA/Akt by Ouabain Is Independent of Src

Exposure of intact cells to selective inhibitors of Na+/K+-ATPase such as ouabain activates several growth-related cell signaling pathways. It has been suggested that the initial event of these pathways is the binding of ouabain to a preexisting complex of Src with Na+/K+-ATPase of the plasma membrane. The aim of this work was to evaluate the role of Src in the ouabain-induced activation of phosphatidylinositide 3-kinase 1A (PI3K1A) and its downstream consequences. When fibroblasts devoid of Src (SYF cells) and controls (Src++ cells) were exposed to ouabain, PI3K1A, Akt, and proliferative growth were similarly stimulated in both cell lines. Ouabain-induced activation of Akt was not prevented by the Src inhibitor PP2. In contrast, ERK1/2 were not activated by ouabain in SYF cells but were stimulated in Src++ cells; this was prevented by PP2. In isolated adult mouse cardiac myocytes, where ouabain induces hypertrophic growth, PP2 also did not prevent ouabain-induced activation of Akt and the resulting hypertrophy. Ouabain-induced increases in the levels of co-immunoprecipitation of the α-subunit of Na+/K+-ATPase with the p85 subunit of PI3K1A were noted in SYF cells, Src++ cells, and adult cardiac myocytes. In conjunction with previous findings, the results presented here indicate that (a) if there is a preformed complex of Src and Na+/K+-ATPase, it is irrelevant to ouabain-induced activation of the PI3K1A/Akt pathway through Na+/K+-ATPase and (b) a more likely, but not established, mechanism of linkage of Na+/K+-ATPase to PI3K1A is the ouabain-induced interaction of a proline-rich domain of the α-subunit of Na+/K+-ATPase with the SH3 domain of the p85 subunit of PI3K1A.

Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2

Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K–AKT and ERK signaling pathways

The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K–AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K–AKT and ERK signaling pathways which are critical in neurite development.

Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from lipopolysaccharide-stimulated myeloid cells

Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated, receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumor necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumor necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, the findings of this study support a novel function for factor Xa as an endogenous, receptor-associated protein-sensitive, protease-activated receptor 2-dependent regulator of myeloid cell pro-inflammatory cytokine production.

Differential BDNF signaling in dentate gyrus and perirhinal cortex during consolidation of recognition memory in the rat

Consolidation of long-term memory is dependent on synthesis of new proteins in the hippocampus and associated cortical regions. The neurotrophin brain-derived neurotrophic factor (BDNF) is tightly regulated by activity-dependent cellular processes and is strongly linked with mechanisms underlying learning and memory. BDNF activation of tyrosine receptor kinase (TrkB) stimulates intracellular signaling cascades implicated in plasticity, including the extracellular-signal related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositide-3-kinase (PI3K)/Akt pathway. Here, we investigate the role of BDNF, ERK/MAPK, and PI3K/AKT signaling cascade in recognition memory in the rat. We report that recognition memory was associated with increased release of BDNF in the dentate gyrus and perirhinal cortex. This was associated with significant increases in p44ERK activation and c-fos expression in the dentate gyrus and PI3K activation and c-fos expression in the perirhinal cortex. Furthermore, both recognition memory and the associated cell signaling events in dentate gyrus and perirhinal cortex were blocked by intraperitoneal injection of the Trk receptor inhibitor tyrphostin AG879. These data are consistent with the hypothesis that BDNF-stimulated intracellular signaling plays a role in consolidation of recognition memory in the rat.