Midkine Inhibits Caspase-Dependent Apoptosis via the Activation of Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase in Cultured Neurons

Midkine (MK) is a new member of the heparin-binding neurotrophic factor family. MK plays important roles in development and carcinogenesis and has several important biological effects, including promotion of neurite extension and neuronal survival. However, the mechanism by which MK exerts its neurotrophic actions on neurons has not been elucidated to date. We have established an apoptosis induction system by serum deprivation in primary neuronal cultures isolated from mouse cerebral cortices. Neuronal apoptosis induced by serum deprivation was accompanied by the activation of caspase-3. MK, when added into the culture medium, inhibited the induction of apoptosis and activation of caspase-3 in a dose-dependent manner. Extracellular signal-regulated kinase (ERK) and Akt were not activated by serum deprivation, whereas ERK and Akt were rapidly activated by addition of MK. In addition, the trophic actions of MK of suppressing apoptosis and suppressing the activation of caspase-3 were abolished by concomitant treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and with wort-mannin or LY294002, specific inhibitors of phosphatidyl-inositol 3-kinase (PI 3-kinase). These PI 3-kinase inhibitors also inhibited the activation of ERK in response to MK, demonstrating a link between ERK and the caspase-3 pathway that is modulated by the PI 3-kinase activation. These results indicate that the ERK cascade plays a central role in MK-mediated neuronal survival via inhibition of caspase-3 activation.     

Carbachol Inhibits Insulin-Stimulated Phosphatidylinositol 3-Kinase Activity in SH-SY5Y Neuroblastoma Cells

Abstract: SH-SY5Y human neuroblastoma cells express muscarinic M₃ receptors as well as insulin receptors, thus offering the opportunity to investigate possible cross-talk following activation of two distinct intracellular signal transduction pathways that convert the precursor phosphatidylinositol (PI) to its 3′ phosphate or its 4′ phosphate, respectively. In this study, the effect of carbachol on insulin-stimulated PI 3-kinase (PI3K) activity was examined in SH-SY5Y cells. Insulin addition to the cell medium induced a 10–26-fold increase in anti-phosphotyrosine-immunoprecipitable PI3K activity. Preincubation with 1 mM carbachol inhibited the insulin-stimulated PI3K activity in a time-dependent manner, with half-maximal and maximal inhibition times of 4 and 15 min, respectively. Atropine blocked the inhibitory effect of carbachol. Although carbachol did not change the amount of 85-kDa subunit protein regulatory unit associated with tyrosine-phosphorylated proteins, either in control or in insulin-stimulated cells, it appears to decrease the amount of associated 110-kDa catalytic subunit protein in the latter instance. Because PI3K activity from SH-SY5Y cells has been shown to be inhibited in vitro in the presence of cytidine diphosphodiacylglycerol (CDP-DAG) or phosphatidate (PA), we examined the presence of these lipids in SH-SY5Y cells that had been treated with carbachol. Formation of both lipids was increased in a time-dependent manner following carbachol addition, and their increased levels are proposed to account for the observed in vivo inhibition of PI3K. Addition of the cell-permeable homologue didecanoyl-CDP-DAG to intact cells inhibited insulin-stimulated PI3K activity up to 75%, with an IC₅₀ of 0.5 µM, a result that further supports a proposed lipid-mediated inhibition of PI3K. Exogenously added didecanoyl-PA, however, did not affect PI3K activity. The possibility that stimulation of the PI 4-kinase-mediated signal transduction pathway leads to down-regulation of the PI3K-mediated signal transduction pathway in vivo, via inhibition of PI3K by CDP-DAG or by other consequences of phosphoinositidase C-linked receptor activation, is discussed.     

Activated Phosphatidylinositol 3-Kinase and Akt Kinase Promote Survival of Superior Cervical Neurons

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that over-expression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.     

Focal Adhesion Kinase Knockdown in Carcinoma‐Associated Fibroblasts Inhibits Oral Squamous Cell Carcinoma Metastasis via Downregulating MCP‐1/CCL2 Expression

Carcinoma‐associated fibroblasts (CAFs) have been demonstrated to play an important role in the occurrence and development of oral squamous cell carcinoma (OSCC). The aim of this study is to investigate the influence of CAFs on OSCC cells and to explore the role of focal adhesion kinase (FAK) in this process. The results showed that oral CAFs expressed a higher level of FAK than normal human gingival fibroblasts (HGFs), and the conditioned medium (CM) of CAFs could induce the invasion and migration of SCC‐25, one oral squamous carcinoma cell line. However, knockdown of FAK by small interfering RNA (siRNA) resulted in inhibition of CAF–CM induced cell invasion and migration in SCC‐25, probably by reducing the production of monocyte chemoattractant protein‐1 (MCP‐1/CCL2), one of downstream target chemokines. Therefore, our findings indicated that targeting FAK in CAFs might be a promising strategy for the treatment of OSCC in the future.     

Synergistic Therapeutic Effect of Cisplatin and Phosphatidylinositol 3-Kinase (PI3K) Inhibitors in Cancer Growth and Metastasis of Brca1 Mutant Tumors

Drug resistance and cancer metastasis are two major problems in cancer research. During a course of therapeutic treatment in Brca1-associated tumors, we found that breast cancer stem cells (CSCs) exhibit an intrinsic ability to metastasize and acquire drug resistance through distinct signaling pathways. Microarray analysis indicated that the cytoskeletal remodeling pathway was differentially regulated in CSCs, and this was further evidenced by the inhibitory role of reagents that impair this pathway in the motility of cancer cells. We showed that cisplatin treatment, although initially inhibiting cancer growth, preventing metastasis through blocking cytoskeletal remodeling, and retarding CSC motility, eventually led to drug resistance associated with a marked increase in the number of CSCs. This event was at least partially attributed to the activation of PI3K signaling, and it could be significantly inhibited by co-treatment with rapamycin. These results provide strong evidence that cytoskeletal rearrangement and PI3K/AKT signaling play distinct roles in mediating CSC mobility and viability, respectively, and blocking both pathways synergistically may inhibit primary and metastatic cancer growth.     

磷脂酰肌醇3-激酶γ/δ与炎症相关疾病

磷脂酰肌醇3-激酶(PI3K)是一类脂质与蛋白激酶家族,其主要通过在磷脂酰肌醇的肌醇环三位进行磷酸化产生胞内重要的第二信使——磷脂酰肌醇-3,4,5-三磷酸(phosphatidyl inositol 3,4,5-trisphosphate,PIP3)而发挥作用.磷脂酰肌醇3-激酶γ/δ(PI3Kγ/δ)是I类PI3K家族中的成员,其主要表达于免疫相关细胞中,这2种PI3K亚型参与先天性与获得性免疫应答.因此,PI3Kγ/PI3Kδ被视为因免疫反应调控异常导致的炎症疾病的治疗药物靶点.目前,利用特异性抑制剂靶向干预PI3Kγ和/或PI3Kδ,成为炎症相关疾病治疗的新策略.本文简介了PI3Kγ与PI3Kδ在不同类型免疫细胞中的功能;并就采用小分子特异性抑制剂,靶向抑制PI3Kγ和/或PI3Kδ在各类炎症相关疾病中的治疗作用和效果进行综述.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 T^ts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-x_L or inhibition of Jun kinase activity.     

Knockdown of Angiopoietin-2 Suppresses Metastasis in Human Pancreatic Carcinoma by Reduced Matrix Metalloproteinase-2

Angiopoietin-2 (Ang2) has been shown highly expressed in resected human pancreatic carcinoma samples, and has tightly combination with tumor angiogenesis, but the role in metastasis of it is less clear. We were, therefore, interested in exploring the effects of Ang2 silencing on the invasion and metastasis of pancreatic carcinoma. Lentivirus (LV)-mediated Ang2 small hairpin RNA (LV-RNAi) and mock lentivirus (LV-NC) were transfected into pancreatic carcinoma cell line MIA PaCa-2. Groups were designed in this study: the control group (MIA PaCa-2 cells), the LV-NC group (cells transfected with the LV-NC), the LV-RNAi-KD1 group (cells transfected with LV-RNAi of knock down sequence (1) and the LV-RNAi-KD2 group (cells transfected with LV-RNAi of knock down sequence (2). Boyden chamber transwell assay was used to detect the cell invasion change. The protein levels of Ang2, MMP-2, and MMP-9 gene and mRNA level of MMP-2, MMP-9 were detected by Western blot and real-time polymerase chain reaction, respectively. Orthotopic pancreatic carcinoma xenotransplantation model were successfully built with MIA PaCa-2 cells injection. After treatment with intraperitoneal injection of LV-RNAi-KD2 (LV-RNAi), mice growth, liver function test, tumor volume and peritoneal metastatic numbers were observed and counted. Moreover, expression of Ang2, MMP-2, MMP-9 were measured by immunohistochemistry. Ang2 expression were successfully knocked down in two LV-RNAi groups, especially in the LV-RNAi-KD2group. Compared with the control group and the LV-NC group, the mRNA and protein level of MMP-2 gene were downregulated significantly in LV-RNAi groups, also the invasion cell number decreased in boyden chamber transwell assay after LV-RNAi transfection. Meanwhile, no obvious MMP-9 gene expression changes were found among all the groups. LV-RNAi injection inhibited pancreatic carcinoma metastasis and growth in vivo by downregulating the expression of MMP-2 not MMP-9. Most importantly, LV-mediated gene therapy with Ang2 knockdown exhibited almost no toxicity in vivo. These findings demonstrate that Ang2 gene silencing exert an anti-metastasis effect in vitro and in vivo, and Ang2 targeted gene therapy has the potential to serve as a novel way for pancreatic carcinoma treatment.     

盐霉素抑制人肝细胞癌转移与侵袭的体外与体内实验研究

目的：肝癌的转移与复发是肝癌治疗的一大难题,盐霉素是近年来新发现的具有抗肿瘤作用的抗生素,本文研究了盐霉素在体外及体内对人肝细胞癌转移与侵袭能力的作用及机制。方法：在体外对肝癌细胞株HepG2,SMMC-7721,BEL-7402给予盐霉素处理,体内建立裸鼠肝脏原位肿瘤模型,并给予腹腔注射盐霉素治疗。观察肿瘤细胞的转移侵袭能力以及肝内肿瘤转移灶的情况,进一步测定E．cadherin,Vimentin的表达,来研究盐霉素对肝癌转移及侵袭能力的影响及机制。结果：经盐霉素处理后,肝癌细胞株HepG2,SMMC．7721,BEL．7402的转移及侵袭能力明显下降,肝内转移灶的数目也减少。分子机制检测发现盐霉素处理后E．cadherin表达增高,Vimentin表达下降。结论：盐霉素在体内与体外都抑制了肝癌的转移与侵袭,其机制可能抑制了肿瘤细胞的上皮间质化（EMT）过程。这为控制肝癌的转移和复发提供了新的治疗思路。     

Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells

Background

Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells.

Methodology/Principal Findings

Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol’s action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1.

Conclusion and Implications

This investigation demonstrates that PI3-K/Akt activation is an important signaling in resveratrol-mediated activation of AMPK phosphorylation and SIRT1 expression, and inhibition of phosphorylation of CREB and MAPKs activation, proinflammatory mediators and cytokines production in response to LPS in RAW 264.7 cells.     

14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival

Integrated cascades of protein tyrosine and serine/threonine phosphorylation play essential roles in transducing signals in response to growth factors and cytokines. How adaptor or scaffold proteins assemble signaling complexes through both phosphotyrosine and phosphoserine/threonine residues to regulate specific signaling pathways and biological responses is unclear. We show in multiple cell types that endogenous 14-3-3ζ is phosphorylated on Tyr¹⁷⁹ in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3ζ can function as an intermolecular bridge that couples to phosphoserine residues and also directly binds the SH2 domain of Shc via Tyr¹⁷⁹. The assembly of these 14-3-3:Shc scaffolds is specifically required for the recruitment of a phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN cell survival in response to cytokine. The biological significance of these findings was further demonstrated using primary bone marrow-derived mast cells from 14-3-3ζ^-/- mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3ζ^-/- mice when reconstituted with wild type 14-3-3ζ, but the Akt phosphorylation and survival response was reduced in cells reconstituted with the Y179F mutant. Together, these results show that 14-3-3:Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses.The ability of a cell to respond to extrinsic stimuli critically hinges on its ability to regulate specific intracellular protein-protein interactions in a reversible manner. Such signals are relayed within the cell through the assembly of signaling complexes that are built using protein scaffolds. One important mechanism by which this occurs is via the binding of Src homology 2 (SH2)⁵ or phosphotyrosine-binding (PTB) domains to phosphotyrosine residues (1, 2). Importantly, the ability of individual SH2 or PTB domains to recognize specific phosphotyrosine motifs in different proteins enables the assembly of purpose-built signaling complexes that promote signaling via specific pathways (3). In some cases, signaling proteins not only contain more than one SH2 and/or PTB domain but are also themselves tyrosine-phosphorylated, leading to a network of phosphotyrosine-mediated protein-protein interactions.Although less well studied, phosphoserine/threonine-binding proteins are also important for the assembly of signaling complexes. For example, the 14-3-3 family of proteins is able to bind phosphoserine/threonine residues in a sequence-specific context (RSX(S/T)XP and RXXX(S/T)XP, where (S/T) is phosphoserine/threonine) (4, 5). The 14-3-3 proteins have been proposed to function as “modifiers” or “sequestrators”; however, because of their dimeric structure, they have also been proposed to function as “adaptor” or “scaffold” proteins through their ability to bring together two serine/threonine phosphorylated proteins (4–7). Additionally, a number of phosphoserine/threonine-binding modules such as tryptophan-tryptophan (WW), Forkhead-associated (FHA), Polo box (PBD), and BRCA1 C-terminal (BRCT) domains have been shown to interact with phosphoserine/threonine residues within a sequence-specific context and have also been proposed to be important for the assembly of multi-protein signaling complexes (8).The genes/cassettes encoding each phosphotyrosine- and phosphoserine/threonine-binding protein/module arose as a separate evolutionary event, and the DNA encoding these modules has been subject to frequent duplication and shuffling. For example, the 14-3-3 family of proteins is ubiquitously expressed in mammalian tissues and is composed of seven different isoforms, each encoded by a separate gene (6). In addition, duplication and shuffling of SH2, PTB, WW, FHA, PBD, and BCRT cassettes has led to their wide distribution among signaling proteins. Yet, despite the frequent duplication and shuffling of the DNA encoding these domains throughout evolution, proteins that contain both a phosphotyrosine-binding cassette (e.g. SH2 or PTB) and a phosphoserine/threonine-binding cassette (e.g. 14-3-3, WW, FHA, PBD, and BCRT) have not been identified. This is perhaps surprising given the highly integrated nature of phosphotyrosine and phosphoserine/threonine signaling and would suggest that alternative strategies to regulate integration are at play.We show here that 14-3-3ζ is tyrosine-phosphorylated, enabling it to interact with Shc and provide a scaffold for the assembly of signaling complexes via both phosphoserine/threonine and phosphotyrosine residues. Our results show that Tyr¹⁷⁹ of 14-3-3ζ directly binds to the SH2 domain of Shc and that this interaction is critical for the assembly of a phosphatidylinositol (PI) 3-kinase signaling complex in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Moreover, we show that Tyr¹⁷⁹ of 14-3-3ζ is necessary and sufficient for the ability of GM-CSF to regulate PI 3-kinase and cell survival in the CTL-EN line. Furthermore, reconstitution of primary mast cells derived from 14-3-3ζ^-/- mice with wild type (wt) or mutant 14-3-3ζ demonstrated an important role for Tyr¹⁷⁹ in cytokine-mediated Akt phosphorylation and cell survival. These multivalent 14-3-3:Shc scaffolds provide a novel mechanism by which phosphoserine/threonine and phosphotyrosine pathways can be integrated for the regulation of specific cellular responses.     

Targeted Knockdown of IQGAP1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo

IQGAP1 is a scaffolding protein that can regulate several distinct signaling pathways. The accumulating evidence has demonstrated that IQGAP1 plays an important role in tumorigenesis and tumor progression. However, the function of IQGAP1 in esophageal squamous cell carcinoma (ESCC) has not been thoroughly investigated. In the present study, we showed that IQGAP1 was overexpressed in ESCC tumor tissues, and its overexpression was correlated with the invasion depth of ESCC. Importantly, by using RNA interference (RNAi) technology we successfully silenced IQGAP1 gene in two ESCC cell lines, EC9706 and KYSE150, and for the first time found that suppressing IQGAP1 expression not only obviously reduced the tumor cell growth, migration and invasion in vitro but also markedly inhibited the tumor growth, invasion, lymph node and lung metastasis in xenograft mice. Furthermore, Knockdown of IQGAP1 expression in ESCC cell lines led to a reversion of epithelial to mesenchymal transition (EMT) progress. These results suggest that IQGAP1 plays crucial roles in regulating ESCC occurrence and progression. IQGAP1 silencing may therefore develop into a promising novel anticancer therapy.     

Role of Phosphatidylinositol 3-Kinase in Friend Spleen Focus-Forming Virus-Induced Erythroid Disease

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin (Epo), because of interaction of the viral envelope protein with the erythropoietin receptor and a short form of the receptor tyrosine kinase Stk (sf-Stk), leading to constitutive activation of several signal transduction pathways. Our previous in vitro studies showed that phosphatidylinositol 3-kinase (PI3-kinase) is activated in SFFV-infected cells and is important in mediating the biological effects of the virus. To determine the role of PI3-kinase in SFFV-induced disease, mice deficient in the p85α regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85α status determined the extent of erythroid hyperplasia induced by the sf-Stk-dependent viruses SFFV-P (polycythemia-inducing strain of SFFV) and SFFV-A (anemia-inducing strain of SFFV) but not by the sf-Stk-independent SFFV variant BB6. Our data also indicate that p85α status determines the response of mice to stress erythropoiesis, consistent with a previous report showing that SFFV uses a stress erythropoiesis pathway to induce erythroleukemia. We further showed that sf-Stk interacts with p85α and that this interaction depends upon sf-Stk kinase activity and tyrosine 436 in the multifunctional docking site. Pharmacological inhibition of PI3-kinase blocked proliferation of primary erythroleukemia cells from SFFV-infected mice and the erythroleukemia cell lines derived from them. These results indicate that p85α may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia.The Friend spleen focus-forming virus (SFFV) is a highly pathogenic retrovirus that induces rapid erythroblastosis in susceptible strains of mice (for a review, see reference 42). Friend SFFV is a replication-defective virus with deletions in its env gene, giving rise to a unique glycoprotein, SFFV gp55. This unique glycoprotein confers pathogenicity to the virus; a vector encoding SFFV gp55 alone is sufficient to induce erythroblastosis in susceptible strains of mice (49). The Fv-2 gene encodes one of the key susceptibility factors for SFFV-induced erythroid disease (18, 37), as follows: the receptor tyrosine kinase Stk/RON, a member of the Met family of receptor tyrosine kinases (11-12). Susceptibility to SFFV-induced disease is associated with expression of a short form of the receptor tyrosine kinase Stk, termed sf-Stk, that is transcribed from an internal promoter within the Stk gene of Fv-2-susceptible (Fv-2^ss) mice but not Fv-2-resistant (Fv-2^rr) mice (37) and is abundantly expressed in erythroid cells (11). Infection of erythroid cells with the polycythemia-inducing strain of SFFV (SFFV-P) induces erythropoietin (Epo)-independent proliferation and differentiation, whereas erythroid cells infected with the anemia-inducing strain of SFFV (SFFV-A) proliferate in the absence of Epo but still require Epo for differentiation (42). Previous studies demonstrated that this Epo-independent erythroblastosis is due to the cell surface interaction of the SFFV envelope protein with the Epo receptor (EpoR) and sf-Stk (31). While interaction with the EpoR appears to be responsible mainly for the induction of Epo-independent differentiation (52), Epo-independent erythroid cell proliferation depends upon activation of sf-Stk. We recently demonstrated that sf-Stk covalently interacts with SFFV-P gp55 in hematopoietic cells that express the EpoR and that this interaction induces sf-Stk activation (31). Furthermore, exogenous expression of sf-Stk, but not a kinase-inactive mutant of sf-Stk, in bone marrow cells from sf-Stk null mice can restore Epo-independent erythroid colony formation in response to SFFV infection (5, 41). Thus, the SFFV envelope glycoprotein induces Epo-independent proliferation of erythroid cells mainly by activating sf-Stk. While sf-Stk is a key susceptibility factor for erythroblastosis induced by both SFFV-P and SFFV-A (18), it is not required for the induction of erythroblastosis by the SFFV mutant BB6, which encodes an envelope glycoprotein, gp42, that is deleted in the membrane-proximal extracellular domain (19) and does not induce sf-Stk activation (31). gp42 of SFFV-BB6 appears to exert its biological effects on erythroid cells by efficiently interacting with the EpoR (9). Compared with wild-type SFFV, SFFV-BB6 causes a relatively indolent and slowly developing disease in mice (19).A number of signaling pathways normally activated in erythroid cells after erythropoietin (Epo) binds to its cell surface receptor (40) are constitutively activated in erythroid cells infected with SFFV. These include JAK/STAT, Ras/Raf/mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, and the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathways (24, 25, 28-30, 32). SFFV gp55 is thought to activate these pathways by interacting with either the EpoR or sf-Stk (17, 31, 43). In several in vitro systems, class IA PI3-kinase has been shown to be activated by Epo through the EpoR (8, 20, 21) or by SFFV through sf-Stk (5, 14). We and others have shown that the PI3-kinase pathway is important for the induction of Epo independence by SFFV (5, 29). The class IA subclass of PI3-kinase is a heterodimer comprising the p110 (α, β, δ) catalytic unit and one of five regulatory subunits (85α, p55α, p50α, 85β, and 55γ) (15). The first 3 regulatory subunits are all splice variants of the same gene (pik3r1). Deletion of pik3r1, which encodes p85α, p55α, and p50α, is lethal (6, 7), and these regulatory subunits of PI3-kinase are required for normal murine fetal erythropoiesis in mice (10).To determine the role of p85α in SFFV-induced erythroleukemia, we used a distinct nonlethal pik3r1 knockout mouse which lacks only the p85α regulatory subunit of PI3-kinase (45, 47), allowing the study of SFFV-induced erythroleukemia in adult mice. Our results indicate that p85α regulates SFFV-induced erythroid hyperplasia induced in vivo by sf-Stk-dependent, but not sf-Stk-independent, isolates of the virus as well as stress-induced erythropoiesis and suggest that this regulation may occur through the interaction of sf-Stk with p85α.     

Phosphatidylinositol 3-Phosphate 5-Kinase,FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is an important lipid in membrane trafficking in animal and yeast systems; however, its role is still largely obscure in plants. Here, we demonstrate that the phosphatidylinositol 3-phosphate 5-kinase, formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), and its product, PtdIns(3,5)P₂, are essential for the maturation process of endosomes to mediate cortical microtubule association of endosomes, thereby controlling proper PIN-FORMED protein trafficking in young cortical and stele cells of root. We found that FAB1 predominantly localizes on the Sorting Nexin1 (SNX1)-residing late endosomes, and a loss of FAB1 function causes the release of late endosomal proteins, Ara7, and SNX1 from the endosome membrane, indicating that FAB1, or its product PtdIns(3,5)P₂, mediates the maturation process of the late endosomes. We also found that loss of FAB1 function causes the release of endosomes from cortical microtubules and disturbs proper cortical microtubule organization.Phosphoinositides play an important role in various cellular processes, including determination of organelle identity and mediating signal transduction by recruiting effector molecules to various organelles (Balla, 2013). Among those, D3-phosphorylated phosphoinositides, phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂], play essential roles in the endosomal trafficking and the vacuolar sorting. PtdIns3P is produced from phosphatidylinositol by class III PI3-kinase, vacuolar protein sorting34 (VPS34). In animal cells, PtdIns3P predominantly localizes to the early endosomes and controls endosome maturation, recycling, and degradation of cargo proteins coordinated with Rab5 GTPases (Jean and Kiger, 2012). In Arabidopsis (Arabidopsis thaliana), PtdIns3P mainly resides on the late endosomes and the prevacuolar membrane (Vermeer et al., 2006; Simon et al., 2014). Dysfunction of AtVPS34 resulted in a defect in growth (Welters et al., 1994), root hair elongation (Lee et al., 2008a), and pollen development (Lee et al., 2008b), indicating an important role for AtVPS34 and its product PtdIns3P in plant development. VPS34-mediated PtdIns3P synthesis at the endosomes recruits phosphatidylinositol 3-phosphate 5-kinase formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), then FAB1/PIKfyve produces PtdIns(3,5)P₂ from PtdIns3P to mediate late endosome maturation in yeast (Saccharomyces cerevisiae) and animals (Ho et al., 2012; Jean and Kiger, 2012). PtdIns(3,5)P₂ has crucial roles in the maintenance of lysosome/vacuole morphology and acidification, membrane trafficking of proteins, autophagy, and signaling mediation in response to various stresses (Shisheva, 2008).FAB1 was discovered in yeast, where mutations were found to result in the formation of aploid and binucleate cells (hence its name FAB). In addition, a loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth (Yamamoto et al., 1995). In mammalian cells, this kinase is called PIKfyve (FYVE is a PI3P-binding domain). FAB1/PIKfyve forms a protein complex with an adaptor-like protein, Vacuole14 (Bonangelino et al., 1997) and PtdIns(3,5)P₂ 5-phosphatase (Fig. 4; Gary et al., 2002), indicating that the FAB1 complex catalyzes both PtdIns(3,5)P₂ synthesis and turnover simultaneously. In mammalian cells, interference of FAB1/PIKfyve function causes severe defects during embryogenesis, resulting in embryonic lethality in Drosophila spp., Caenorhabditis elegans, and mice (Nicot et al., 2006; Rusten et al., 2006; Ikonomov et al., 2011; Takasuga et al., 2013). Whereas most genomes from human to yeast contain a single-copy gene, the Arabidopsis genome codes for four FAB1 genes (FAB1A–D), of which only FAB1A and FAB1B contain a FYVE domain (Mueller-Roeber and Pical, 2002), and fab1a/fab1b double mutant reveals male gametophyte lethality phenotype in Arabidopsis (Whitley et al., 2009). The mutant pollen shows severe defects in vacuolar reorganization following the first mitotic division of development, suggesting an important role of FAB1 and PtdIns(3,5)P₂ in vacuolar rearrangement for pollen development (Whitley et al., 2009).Open in a separate windowFigure 4.Localization of endosomal markers upon down-regulation of FAB1A/B or inhibition of PtdIns(3,5)P₂ synthesis in young root cortical cells. Localization of mRFP-SYP43, mRFP-vesicle-associated membrane protein (VAMP727), mRFP-ARA7, and SNX1-mRFP without estradiol (A, E, I, and M) or with estradiol (B, F, J, and N) in the FAB1A/B-amiRNA line, or wild-type (WT) plants without YM201636 (C, G, K, and O) or with YM201636 (D, H, L, and P). Bar = 10 μm. Measurement of fluorescent dot structures (Q). Data represent fluorescent dots per cell (mean ± sd). *, P < 0.001 (Student’s t test).We previously developed a transgenic Arabidopsis line that is able to conditionally down-regulate FAB1A and FAB1B expression simultaneously, and demonstrated that a loss of FAB1 function causes various abnormal phenotypes, including growth inhibition, hypersensitivity to exogenous auxin, disturbance of root gravitropism, and floral organ abnormalities (Hirano et al., 2011). In addition, we found that down-regulation of FAB1A/B expression impaired endomembrane homeostasis, including endocytosis, vacuole formation, and vacuolar acidification, likely causing pleiotropic developmental phenotypes that mostly related to the auxin signaling in Arabidopsis (Hirano et al., 2011; Hirano and Sato, 2011). In plants, auxin is a crucial phytohormone that has a wide variety of physiological roles associated with growth, development, and tropic responses (Zhao, 2010). The polar cell-to-cell transport of auxin is mediated by auxin transporters localized on the plasma membrane (PM), such as PIN-FORMED (PIN) proteins (Vieten et al., 2007; Feraru and Friml, 2008). PINs are used as model molecules for polarity establishment on the PM in Arabidopsis. The establishment of PIN polarity is accomplished by the recycling of PINs between the PM and endosomal compartments comprising the trans-Golgi network/early endosomes (TGN/EEs) and the late endosomes (LEs)/prevacuolar compartments. The PIN-recycling pathway is mediated by multiple endosomal regulatory proteins, such as Rab family GTPases and Sorting Nexin (SNX; Jaillais et al., 2006; Park and Jürgens, 2011).Rab proteins function as molecular switches to regulate the tethering and fusion step of transport vesicles to target membranes. Rab5 members of the Rab GTPases have various functions in the endocytic pathway in eukaryotes. The maturation of the early-to-late endosomes is regulated by Rab5-to-Rab7 conversion, which is regulated by the Mon1/Sand-1-Ccz1 complex (Nordmann et al., 2010; Poteryaev et al., 2010). In plants, Rab5-family proteins, Ara6 and Ara7, and Rha1 play important roles in Rab5-mediated endosomal trafficking including the vacuolar trafficking pathway, thereby regulating of the polar transport of auxin and responses to environmental conditions (Ebine et al., 2011; Inoue et al., 2013).SNXs are composed of two conserved domains: the PHOX domain, involved in the interaction with the phosphoinositides, PtdIns3P and PtdIns (3,5)P₂, in the endosomal membrane in animals (Cozier et al., 2002), and the BAR domain, mediating dimerization and binding to curved membranes (Peter et al., 2004). Loss of SNX function disrupts the stable association of the retromer subcomplex, VPS26-Vps29-Vps35, with endosomal membranes, and thus results in retromer dysfunction, indicating that SNXs have a crucial role in the assembly and maintenance of the core retromer function (Teasdale et al., 2001; Cullen and Korswagen, 2012). The first plant SNX was identified as a protein that interacts with various receptor kinases in Brassica oleracea (Vanoosthuyse et al., 2003), and then three SNX genes (SNX1, SNX2a, and SNX2b) were identified in Arabidopsis. The snx1 null mutant exhibits a semidwarf phenotype with other subtle developmental defects (Pourcher et al., 2010). SNX1 is localized to the late endosome and is involved in PIN2 recycling between endosomes and the PM (Jaillais et al., 2006). SNX1 has been reported to interact with cortical microtubules via the microtubule-associated protein Cytoplasmic Linker Associated Protein (CLASP), and the clasp1 null mutant displays aberrant SNX1 endosomes and enhanced PIN2 degradation in the lytic vacuoles, suggesting that an association of SNX1 endosomes and CLASP is important for recycling of PIN transporters (Ambrose et al., 2013).Although many analyses of FAB1/PIKfyve, Rab5 family GTPases, SNXs, and microtubles have been reported, and there are significant similarities in endosomal trafficking, a functional relationship between them is still largely obscure.In this study, we demonstrate that FAB1 produced PtdIns(3,5)P₂ in Arabidopsis, and knockdown of FAB1 expression or inhibition of FAB1 activity with a FAB1/PIKfyve inhibitor, YM201636, decreased PtdIns(3,5)P₂ content. We also found that FAB1 and its product PtdIns(3,5)P₂ mediate the late endosome maturation by recruiting endosomal effector molecules, Ara7 and SNX1, onto endosomes to establish endosome-cortical microtubule interaction. Subsequently, the basal polarity of PIN2 in young cortical cells and PIN1 in stele cells is achieved.     

PI3K调控水稻种子萌发的机理研究

PI3K在植物的正常生长和胁迫应答过程中发挥着很重要的功能.在本文中,我们证实了在种子萌发过程中,PI3K促进了种子的萌发.通过RT-PCR的分析,我们发现,PI3K在整个过程中,伴随着吸胀时间的延长,表达量逐渐上升.而PI3K抑制剂的处理,证实了萌发过程中,PI3K促萌发的效应.DCF检测ROS的产生发现,在萌发的过程中,PBK促进了ROS的生成.PI3K产物PI3P亚细胞定位的分析发现,PI3P主要定位于膜结构区域.总之,本文借助于光学分子成像技术发现PI3K在种子萌发过程中的重要作用,提出PI3K通过调控ROS生成促进种子萌发的重要机理.     

Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface.A unique lipid composition and lipid-protein interactions appear to exist at the transient membrane domain that defines the division site in bacterial cells (1). Using the cardiolipin (CL)⁴-specific fluorescent dye 10-N-nonyl acridine orange (NAO), we previously found CL-enriched membrane domains located at cell poles and near potential division sites in Escherichia coli (2). Subsequently others reported similar CL domains in Bacillus subtilis (3) and Pseudomonas putida (4). In addition, cell pole and division site enrichment in CL in E. coli was confirmed by lipid analysis of minicells spontaneously budded off from the cell poles of a ΔminCDE mutant (5). We suggested that formation of CL domains at cell pole/division sites plays an important role in selection and recognition of the division site by amphitropic cell cycle and cell division proteins, such as DnaA (initiation of DNA replication at oriC), MinD (a part of MinCDE system preventing positioning of the divisome at cell poles in E. coli), and FtsA (bacterial actin, which is a linker protein for cytoskeletal protein FtsZ (bacterial tubulin), responsible for targeting the Z-ring to the mid-cell membrane domain). They interact directly with membrane phospholipids through specific amphipathic motifs enriched in basic amino acids, which confers the preference for anionic lipids (for references see Ref. 1). In E. coli the ATP-bound form of MinD recruits an inhibitor of Z-ring formation, MinC, to the membrane, whereas the topological regulator, MinE, induces hydrolysis of ATP bound to MinD resulting in release of MinD, and consequently MinC, from the membrane into the cytoplasm. As a result, all three proteins oscillate between the cell poles maintaining the maximum concentration of the inhibitor MinC at the cell poles and its minimum concentration at the cell center. Pole-to-pole oscillation of Min proteins occurs by dynamic redistribution of the proteins within a helical oligomeric structure that winds around the cell (for recent review and references see Ref. 6).Our previous study of a mutant lacking phosphatidylethanolamine (PE) and containing highly elevated levels of phosphatidylglycerol (PG) and CL demonstrated a strong inhibition of cell division and aggregation of MinD and FtsZ/FtsA proteins at domains enriched in CL (7, 8). To further investigate the role of lipids in the process of cell division, we chose an E. coli mutant with an opposite extreme in phospholipid composition to PE-lacking mutants, namely a ΔpgsA mutant (pgsA encodes phosphatidylglycerol phosphate synthase, which catalyzes the committed step to PG and CL synthesis (9)). This mutant is devoid of PG and CL (contribute ∼20 mole % of phospholipids in wild type) and contains higher levels of PE (∼90 mole % versus 80 mole % in wild type) (10, 11). Interestingly, the ΔpgsA null mutant accumulates elevated amounts of the phospholipid precursors, phosphatidic acid (PA) (∼4 mole %) and CDP-diacylglycerol (∼3 mole %), which are also anionic lipids that were proposed to fulfill the structural and functional roles of PG and CL (10, 11). These results suggest that a minimum of 5–10% anionic lipid is required to support viability. Another minor anionic phospholipid, N-acylphosphatidylethanolamine was suggested to be present in wild-type E. coli (12), which, if proven true, might be also elevated in this mutant. Finally, if anionic lipids are essential for cell division, then we would expect these normally minor lipids to segregate into similar anionic lipid domains, as does CL.In this report we identified N-acyl-PE in E. coli and, along with PA, its enrichment in polar/septal membrane domains of the ΔpgsA mutant UE54 (11) lacking PG and CL. Thus E. coli has a mechanism for preferential segregation of anionic phospholipids to the polar/septal regions where several amphitropic proteins, which show preference for interaction with anionic phospholipids in vitro, are functionally located.