IGF2BP1: A post-transcriptional “driver” of tumor cell migration

The oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA binding protein 1) controls the cytoplasmic fate of specific target mRNAs including ACTB and CD44. During neural development, IGF2BPs promote neurite protrusion and the migration of neuronal crest cells. In tumor-derived cells, IGF2BP1 enhances the formation of lamellipodia and invadopodia. Accordingly, the de novo synthesis of IGF2BP1 observed in primary malignancies was reported to correlate with increased metastasis and an overall poor prognosis. However, if and how the protein enhances metastasis remains controversial. In recent studies, we reveal that IGF2BP1 promotes the directed migration of tumor-derived cells in vitro by controlling the expression of MAPK4 and PTEN. The IGF2BP1-facilitated inhibition of MAPK4 mRNA translation interferes with MK5-directed phosphorylation of the heat shock protein 27 (HSP27). This limits G-actin sequestering by phosphorylated HSP27, enhances cell adhesion and elevates the velocity of tumor cell migration. Concomitantly, IGF2BP1 promotes the expression of PTEN by interfering with PTEN mRNA turnover. This results in a shift of cellular PtdIns(3,4,5)P₃/PtdIns(4,5)P₂ ratios and enhances RAC1-dependent cell polarization which finally promotes the directionality of tumor cell migration. These findings identify IGF2BP1 as a potent oncogenic factor that regulates the adhesion, migration and invasiveness of tumor cells by modulating intracellular signaling.     

Targeting phospshatidylinositol 3-kinase signaling with novel phosphatidylinositol 3,4,5-triphosphate antagonists

The critical role of phopshatidylinositol-3-kinase (PtdIns3K) signaling in the regulation of a wide range of cellular functions, including cell survival and proliferation, autophagy, metabolism and cell migration, is well recognized. Activation of PtdIns3K leads to the generation of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P₃). PtdIns(3,4,5)P₃ activates a complex signaling network controlling these diverse cellular functions through binding to Pleckstrin Homology (PH) domains of the effector proteins. We have recently described a new structural class of nonphosphoinositide small molecule inhibitors targeting binding of PtdIns(3,4,5) P₃ to PH domain targets. Using an in vitro PtdIns(3,4,5)P₃-PH domain binding assay, we identified two distinct PtdIns(3,4,5)P₃ antagonists, PIT-1 and PIT-2. Further cellular analysis revealed that both PITs inhibit PtdIns(3,4,5) P₃-dependent signaling mediated by Akt kinase, leading to the induction of apoptosis, metabolic stress and autophagy. An improved PIT-1 analog, DM-PIT-1, displays significant anticancer activity in the mouse syngeneic 4T1 breast cancer model in vivo. Discovery of PITs as well as other PtdIns(3,4,5)P₃ antagonists recently described by other laboratories suggest the possibility of targeting a key universal PtdIns(3,4,5)P₃/PH domain binding step in the PtdIns3K pathway using heterologous small molecule modulators.     

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin‐dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P₂ as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G‐actin unlike PIPKs from other organisms. PtdIns(4,5)P₂, the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP‐tagged PH‐domain from PLCδ, which specifically binds PtdIns(4,5)P₂ in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.     

The inositol polyphosphate 5-phosphatase, PIPP, Is a novel regulator of phosphoinositide 3-kinase-dependent neurite elongation

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P₃), which localizes and facilitates Akt activation and stimulates GSK-3β inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P₃ signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P₂) and/or PtdIns(3,4,5)P₃. We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P₃ forming PtdIns(3,4)P₂, decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3β, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P₃ at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3β signaling.     

Cellular phosphatase activity of C1-Ten/Tensin2 is controlled by Phosphatidylinositol-3,4,5-triphosphate binding through the C1-Ten/Tensin2 SH2 domain

Regulation of tyrosine phosphorylation on insulin receptor substrate-1 (IRS-1) is essential for insulin signaling. The protein tyrosine phosphatase (PTP) C1-Ten/Tensin2 has been implicated in the regulation of IRS-1, but the molecular basis of this dephosphorylation is not fully understood. Here, we demonstrate that the cellular phosphatase activity of C1-Ten/Tensin2 on IRS-1 is mediated by the binding of the C1-Ten/Tensin2 Src-homology 2 (SH2) domain to phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃). We show that the role of C1-Ten/Tensin2 is dependent on insulin-induced phosphoinositide 3-kinase activity. The C1-Ten/Tensin2 SH2 domain showed strong preference and high affinity for PtdIns(3,4,5)P₃. Using site-directed mutagenesis, we identified three basic residues in the C1-Ten/Tensin2 SH2 domain that were critical for PtdIns(3,4,5)P₃ binding but were not involved in phosphotyrosine binding and PTP activity. Using a PtdIns(3,4,5)P₃ binding-deficient mutant, we showed that the specific binding of the C1-Ten/Tensin2 SH2 domain to PtdIns(3,4,5)P₃ allowed C1-Ten/Tensin2 to function as a PTP in cells. Collectively, our findings suggest that the interaction between the C1-Ten/Tensin2 SH2 domain and PtdIns(3,4,5)P₃ produces a negative feedback loop of insulin signaling through IRS-1.     

Exclusion of exogenous phosphatidylinositol-3,4,5-trisphosphate from neutrophil-polarizing pseudopodia: stabilization of the uropod and cell polarity

Although there is accumulating evidence that the generation and localization of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃) have important functions in neutrophil polarization and chemotaxis, the mechanism of this linkage has yet to be established. Here, using exogenous fluorescent PtdIns(3,4,5)P₃ introduced into the inner leaflet of the neutrophil plasma membrane by a cationic carrier, we show that: first, PtdIns(3,4,5)P₃ uniformly delivered to the neutrophil plasma membrane is excluded from newly forming pseudopodia; second, PtdIns(3,4,5)P₃ translocates to and is immobilized at the pole opposite a stable polarizing pseudopod; third, asymmetric delivery of PtdIns(3,4,5)P₃ to the neutrophil triggers the generation of polarizing pseudopodia at the opposite pole; and finally, PtdIns(3,4,5)P₃ triggers repetitive Ca²⁺ signals, the onset of which precedes morphological polarization. These data suggest that translocation and immobilization of PtdIns(3,4,5)P₃ or a 3,x-phosphorylated metabolite in the uropod functions as an important polarization cue that defines neutrophil polarity and stabilizes the generation of pseudopodia at the opposite pole.     

Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300–400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC₅₀ values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.     

Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress

Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite''s defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite''s cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica''s functions and indicate a new role for arginase in E. histolytica''s resistance to OS.     

Two StAR-related lipid transfer proteins play specific roles in endocytosis,exocytosis, and motility in the parasitic protist Entamoeba histolytica

Lipid transfer proteins (LTPs) are the key contributor of organelle-specific lipid distribution and cellular lipid homeostasis. Here, we report a novel implication of LTPs in phagocytosis, trogocytosis, pinocytosis, biosynthetic secretion, recycling of pinosomes, and motility of the parasitic protist E. histolytica, the etiological agent of human amoebiasis. We show that two StAR-related lipid transfer (START) domain-containing LTPs (named as EhLTP1 and 3) are involved in these biological pathways in an LTP-specific manner. Our findings provide novel implications of LTPs, which are relevant to the elucidation of pathophysiology of the diseases caused by parasitic protists.     

Signaling via Class IA Phosphoinositide 3-Kinases (PI3K) in Human,Breast-Derived Cell Lines

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ) and p50–101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P₃) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P₃-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN^−/− cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN^−/− MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110β function in the absence of PTEN.     

Regulation of the Src Homology 2 Domain-containing Inositol 5��-Phosphatase (SHIP1) by the Cyclic AMP-dependent Protein Kinase

Many agents that activate hematopoietic cells use phos pha tidyl ino si tol 3,4,5-trisphosphate (PtdIns 3,4,5-P₃) to initiate signaling cascades. The SH2 domain-containing inositol 5′ phosphatase, SHIP1, regulates hematopoietic cell function by opposing the action of phos pha tidyl ino si tol 3-kinase and reducing the levels of PtdIns 3,4,5-P₃. Activation of the cyclic AMP-de pend ent protein kinase (PKA) also opposes many of the pro-inflammatory responses of hematopoietic cells. We tested to see whether the activity of SHIP1 was regulated via phos pho ryl a tion with PKA. We prepared pure recombinant SHIP1 from HEK-293 cells and found it can be rapidly phos pho ryl a ted by PKA to a stoichiometry of 0.6 mol of PO₄/mol of SHIP1. In ³²P-labeled HEK-293 cells transfected with SHIP1, stimulation with Sp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate (Sp-cAMPS) or activation of the β-adrenergic receptor increased the phos pho ryl a tion state of SHIP1. Inhibition of protein phosphatase activity with okadaic acid also increased the phos pho ryl a tion of SHIP1. Phosphorylation of SHIP1 in vitro or in cells by PKA increased the 5′ phosphatase activity of SHIP1 by 2–3-fold. Elevation of Ca²⁺ in DT40 cells in response to B cell receptor cross-linking, an indicator of PtdIns 3,4,5-P₃ levels, was markedly blunted by pretreatment with Sp-cAMPS. This effect was absent in SHIP^−/− DT40 cells showing that the effect of Sp-cAMPS in DT40 cells is SHIP1-de pend ent. Sp-cAMPS also blunted the ability of the B cell receptor to increase the phos pho ryl a tion of Akt in DT40 and A20 cells. Overall, activation of G protein-coupled receptors that raise cyclic AMP cause SHIP1 to be phos pho ryl a ted and stimulate its inositol phosphatase activity. These results outline a novel mechanism of SHIP1 regulation.Activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase)² is central to regulation of multiple cell functions including cell shape changes, cell migration, cell activation, and proliferation (1). PtdIns 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate in the inner leaflet of the plasma membrane to generate phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P₃) (2). PtdIns 3,4,5-P₃ then activates downstream signaling pathways by interacting with pleckstrin homology domain-containing proteins, such as phosphoinositide-dependent kinase 1 and the serine-threonine kinase Akt (3). The finding of abnormal activation of the PtdIns 3-kinase pathway in cancer cells has led to interest in the development of inhibitors for PtdIns 3-kinase (4).The level of PtdIns 3,4,5-P₃ is stimulated by multiple members of the PtdIns 3-kinase family (2) and is opposed by two phosphatidylinositol phosphatases: the Src homology 2 (SH2) domain-containing inositol 5′ phosphatase (SHIP) and the 3′ inositol phosphatase, phosphatase and tensin homolog (PTEN) (5). PTEN removes phosphate from the 3′ position in the inositol ring of PtdIns 3,4,5-P₃ and converts it to phosphatidylinositol 4,5-bisphosphate (6). PTEN has a C2 domain, a PDZ-binding motif, and a N-terminal phosphatidylinositol 4,5-bisphosphate binding motif essential for translocation to the membrane and interaction with other regulatory proteins (7). There are serine and threonine residues in PTEN that have been found to be phosphorylated, but their role in regulating the activity of the enzyme is not clear (8). Mutations in the PTEN protein have been observed in many tumors, suggesting a role for this enzyme in cancer (9).In contrast, SHIP dephosphorylates the 5′ position on the inositol ring and produces phosphatidylinositol 3,4-bisphosphate (10). There are three isoforms of SHIP: the 145-kDa hematopoietic cell restricted SHIP (also known as SHIP1); the 104-kDa stem cell-restricted SHIP, sSHIP; and the more widely expressed 150-kDa SHIP2 (11). SHIP1 is the major inositol phosphatase regulating PtdIns 3,4,5-P₃ in monocytes, macrophages, B cells, and T cells (11). SHIP1 has three known structural features: the N-terminal SH2 domain, the central inositol 5′ phosphatase domain, and two NPXY sequences in the C-terminal region. The currently accepted model for regulation of PtdIns 3,4,5-P₃ levels by SHIP1 envisions translocation of SHIP1 from the cytosol to the membrane. Upon stimulation by growth factors, cytokine receptors, or immunoreceptors, SHIP1 is recruited via its N-terminal SH2 domain to phosphorylated tyrosine residues in receptor kinases and degrades the elevated levels of PtdIns 3,4,5-P₃ near the activated receptor (12). During this translocation process, SHIP1 is not thought to change its 5′ phosphatase activity (13). Although it is known that SHIP1 can be phosphorylated on tyrosine residues by the lyn cytoplasmic kinase (12) or following the activation of the T cell receptor (14), neither event appears to influence the 5′ phosphatase activity. To date, direct regulation of SHIP1 activity by serine/threonine kinases has not been studied.Activation of G protein-coupled receptors that raise cAMP (i.e. β-adrenergic receptors or adenosine A_2a receptors) is known to blunt the pro-inflammatory responses generated by receptors that raise the level of PtdIns 3,4,5-P₃ (15). Therefore, we investigated the possibility that phosphorylation of SHIP1 by cyclic AMP-dependent protein kinase (PKA) might regulate the activity of SHIP1. We found that SHIP1 can be phosphorylated by PKA both in vitro and in cells leading to a stimulation of SHIP1 activity. Activation of PKA in DT40 and A20 cells blunted indicators of the PtdIns 3,4,5-P₃ response to B cell receptor stimulation. These results indicate that SHIP1 activity can be regulated both in vitro and in cells by activation of the cyclic AMP-dependent protein kinase and highlight a new mode of SHIP regulation by G protein-coupled receptors.     

The localisation of PtdIns3P in Drosophila fat responds to nutrients but not insulin: a role for Class III but not Class II phosphoinositide 3-kinases

PtdIns3P and PtdIns(3,4,5)P₃ are regulated differently in fat body in response to nutritional status and insulin signalling. In feeding larvae PtdIns(3,4,5)P₃ is upregulated at the cell membrane where it is generated in response to insulin signalling. In contrast PtdIns3P is down regulated in the fat body of well-fed larvae but on starvation it accumulates in punctate vesicles throughout the cytoplasm and on refeeding it relocalises to vesicles at the periphery of the cell. Both responses are independent of insulin signalling and on the presence of glutamine which has previously been linked to nutritional sensing. We find that both Class II and Class III PI3Ks are capable of generating PtdIns3P in vivo but the source of PtdIns3P in the fat body and the response to nutritional status can be exclusively accounted for by Class III PI3K activity.     

SNAP-Tag Technology Optimized for Use in Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite responsible for invasive intestinal and extraintestinal amebiasis. The pathology of amebiasis is still poorly understood, which can be largely attributed to lack of molecular tools. Here we present the optimization of SNAP-tag technology via codon optimization specific for E. histolytica. The resultant SNAP protein is highly expressed in amebic trophozoites, and shows proper localization when tagged with an endoplasmic reticulum retention signal. We further demonstrate the capabilities of this system using super resolution microscopy, done for the first time in E. histolytica.     

SHIP-2 and PTEN are expressed and active in vascular smooth muscle cell nuclei,but only SHIP-2 is associated with nuclear speckles

Recently, the control of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3)-dependant signaling by phosphatases has emerged, but there is a shortage of information on intranuclear PtdIns(3,4,5)P3 phosphatases. Therefore, we investigated the dephosphorylation of [32P]PtdIns(3,4,5)P3 specifically labeled on the D-3 position of the inositol ring in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). In vitro PtdIns(3,4,5)P3 phosphatase assays revealed the production of both [32P]PtdIns(3,4)P2 and inorganic phosphate, demonstrating the presence of PtdIns(3,4,5)P3 5- and 3-phosphatase activities inside the VSMC nucleus, respectively. Both activities presented the same potency in cellular lysates, whereas the nuclear PtdIns(3,4,5)P3 5-phosphatase activity appeared to be the most efficient. Immunoblot experiments showed for the first time the expression of the 5-phosphatase SHIP-2 (src homology 2 domain-containing inositol phosphatase) as well as the 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) in VSMC nuclei. In addition, immunoprecipitations from nuclear fractions indicated a [32P]PtdIns(3,4,5)P3 dephosphorylation by both SHIP-2 and PTEN. Moreover, confocal microscopy analyses demonstrated that SHIP-2 but not PTEN colocalized with a speckle-specific component, the SC35 splicing factor. These results suggest that SHIP-2 may be the primary enzyme for metabolizing PtdIns(3,4,5)P3 into PtdIns(3,4)P2 within the nucleus, thus producing another second messenger, whereas PTEN could down-regulate nuclear phosphoinositide 3-kinase signaling. Finally, intranuclear PtdIns(3,4,5)P3 phosphatases might be involved in the control of VSMC proliferation and the pathogenesis of vascular proliferative disorders.     

Eukaryotic translation initiation factor 5A and its posttranslational modifications play an important role in proliferation and potentially in differentiation of the human enteric protozoan parasite Entamoeba histolytica

The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein and is essential in all eukaryotes. However, the specific roles of eIF5A in translation and in other biological processes remain elusive. In the present study, we described the role of eIF5A, its posttranslational modifications (PTM), and the biosynthetic pathway needed for the PTM in Entamoeba histolytica, the protozoan parasite responsible for amoebic dysentery and liver abscess in humans. E. histolytica encodes two isotypes of eIF5A and two isotypes of enzymes, deoxyhypusine synthase (DHS), responsible for their PTM. Both of the two eIF5A isotypes are functional, whereas only one DHS (EhDHS1, but not EhDHS2), is catalytically active. The DHS activity increased ~2000-fold when EhDHS1 was co-expressed with EhDHS2 in Escherichia coli, suggesting that the formation of a heteromeric complex is needed for full enzymatic activity. Both EhDHS1 and 2 genes were required for in vitro growth of E. histolytica trophozoites, indicated by small antisense RNA-mediated gene silencing. In trophozoites, only eIF5A2, but not eIF5A1, gene was actively transcribed. Gene silencing of eIF5A2 caused compensatory induction of expression of eIF5A1 gene, suggesting interchangeable role of the two eIF5A isotypes and also reinforcing the importance of eIF5As for parasite proliferation and survival. Furthermore, using a sibling species, Entamoeba invadens, we found that eIF5A1 gene was upregulated during excystation, while eIF5A2 was downregulated, suggesting that eIF5A1 gene plays an important role during differentiation. Taken together, these results have underscored the essentiality of eIF5A and DHS, for proliferation and potentially in the differentiation of this parasite, and suggest that the hypusination associated pathway represents a novel rational target for drug development against amebiasis.     

Tensin2 reduces intracellular phosphatidylinositol 3,4,5-trisphosphate levels at the plasma membrane

Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P₃ levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P₃ and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.     

IGF2BP1

The oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA binding protein 1) controls the cytoplasmic fate of specific target mRNAs including ACTB and CD44. During neural development, IGF2BPs promote neurite protrusion and the migration of neuronal crest cells. In tumor-derived cells, IGF2BP1 enhances the formation of lamellipodia and invadopodia. Accordingly, the de novo synthesis of IGF2BP1 observed in primary malignancies was reported to correlate with increased metastasis and an overall poor prognosis. However, if and how the protein enhances metastasis remains controversial. In recent studies, we reveal that IGF2BP1 promotes the directed migration of tumor-derived cells in vitro by controlling the expression of MAPK4 and PTEN. The IGF2BP1-facilitated inhibition of MAPK4 mRNA translation interferes with MK5-directed phosphorylation of the heat shock protein 27 (HSP27). This limits G-actin sequestering by phosphorylated HSP27, enhances cell adhesion and elevates the velocity of tumor cell migration. Concomitantly, IGF2BP1 promotes the expression of PTEN by interfering with PTEN mRNA turnover. This results in a shift of cellular PtdIns(3,4,5)P₃/PtdIns(4,5)P₂ ratios and enhances RAC1-dependent cell polarization which finally promotes the directionality of tumor cell migration. These findings identify IGF2BP1 as a potent oncogenic factor that regulates the adhesion, migration and invasiveness of tumor cells by modulating intracellular signaling.     

Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity

The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P₃] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110β at several points during the cell cycle. We studied the potential connections between p110α and p110β activation, and we show that cell stimulation promotes p110α and p110β association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P₃ levels.