Dissertations / Theses on the topic 'Inositol polyphosphate phosphatases'

Inositol hexakisphosphate (InsP6) is the main storage form of phosphorous in animal feeds. Phytases are enzymes (myo-inositol hexakisphosphate phosphohydrolases) that break down InsP6 by hydrolysis to release inorganic phosphate. Non-ruminant animals do not produce phytases needed to digest dietary InsP6, instead relying on enzymes produced by their gut microbiota. A similar situation is found in humans where several members of the commensal bacteria have been found to produce multiple inositol polyphosphate phosphatases (Minpp) which display phytase activity. In this study, high resolution X-ray crystal structures of Minpps from two human commensal gut bacteria, Bacteroides thetaiotaomicron (BtMinpp) and Bifidobacterium longum (BlMinpp), were solved and refined. High performance liquid chromatography was employed to analyse the products of InsP6 hydrolysis, revealing that Minpps attack InsP6 with high positional promiscuity, unlike bacterial and fungal phytases which display high catalytic specificity. Site-directed mutagenesis was employed to further investigate the catalytic promiscuity of BtMinpp, mutagenizing its active site to mimic that of PhyA, a specific 3- phytase from Aspergillus niger. Further experiments introduced active site residues from human Minpp. The results of these studies reveal that by altering key active site residues, the positional specificity and the ratios of the InsP5 products generated by BtMinpp action can be altered, opening the possibility of engineering catalytic flexibility into phytases used as commercial animal feed additives. Disulfide bridges were engineered into BtMinpp with the aim of enhancing its thermostability – an attractive characteristic for animal feed enzymes. Sites for potential disulfide bridges were identified and a one such mutant was produced. However, the engineered protein did not show a significant enhancement in thermostability. The results of experiments described in this thesis provide novel insights into the hydrolysis of InsP6 by Minpps that suggest a role as precursors for a new generation of phytases for the animal feed industry.

Phytases are enzymes that degrade phytate, the main storage form of phosphorus in plants. Animal feed industries use plant-based feed with abundant phytate content. Without the supplementation with phytases, the animals would not be able to access the phosphorus in the form of phosphate stored in the phytate molecule. This thesis describes research carried out to characterise a group of bacterial phytases from Bacteroides thetaiotaomicron, Bifidobacterium infantis and Bifidobacterium pseudocatenulatum with comparisons to an existing commercial phytase, Quantum Blue. The enzymatic properties, product profiles, binding and thermostability were examined and the structure of the binding site at various stages of the catalytic cycle was investigated with the aid of active site mutagenesis. X-ray structure of the active site mutant helped elucidate the structure of the intermediate and product-bound forms of the Bifidobacterium infantis phytase. Further mutagenesis experiments examined the function of disulphide bridges in the enzyme. The experimental results described in this work provide novel insights into the hydrolysis of phytate by bacterial phytases, the conformational changes during the catalytic cycle and the contribution of disulphide links to thermostability of the enzyme. These results lay the foundations of the work toward optimisation of phytases for use by the industry of the animal feed supplements.

The complete role of inositol signaling in plants and humans is still elusive. The plant Arabidopsis thaliana contains fifteen predicted inositol polyphosphate 5- phosphatases (5PTases, E.C. 3.1.3.36) that have the potential to remove a 5-phosphate from various inositol second messenger substrates. To examine the substrate specificity of one of these Arabidopsis thaliana 5PTases (At5PTases), recombinant At5PTase1 was obtained from a Drosophila melanogaster expression system and analyzed biochemically. This analysis revealed that At5PTase1 has the ability to catalyze the hydrolysis of four potential inositol second messenger substrates. To determine whether At5PTase1 can be used to alter the signal transduction pathway of the major drought-sensing hormone abscisic acid (ABA), plants ectopically expressing At5PTase1 under the control of a constitutive promoter were characterized. This characterization revealed that plants ectopically expressing At5PTase1 had an altered response to ABA. These plants have stomata that are insensitive to ABA, and have lower basal and ABA-induced inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] levels. In addition, At5PTase1 mRNA and protein levels are transiently regulated by ABA. These data strongly suggest that At5PTase1 can act as a signal terminator of ABA signal transduction. Like the Arabidopsis At5PTase1, a human 5PTase, Ocrl, has the ability to catalyze the hydrolysis of a 5-phosphate from several inositol-containing substrates. The loss of functional Ocrl protein results in a rare genetic disorder known as Lowe oculocerebrorenal syndrome. To gather information concerning the specificity determinants of the Ocrl protein, a structure-function analysis of Ocrl was conducted using a vibrational technique, difference Fourier transform infrared (FT-IR) spectroscopy. Upon the introduction of Ins(1,4,5)P₃ substrate, structural changes in carboxylic acid and histidine residues were observed. The net result of changes in these residues indicates that upon Ins(1,4,5)P₃ introduction, a carboxylic acid-containing residue is protonated, and a histidine residue is deprotonated. This interpretation supports the idea that the deprotonation of the histidine residue is concomitant with the coordination of a divalent cation upon Ins(1,4,5)P₃ introduction. This work allows for the proposal of a new model for the role of the active site histidine of OCRL.
Ph. D.

To survive, an organism must constantly adjust its internal state to changes in the environment from which it receives signals. The signals set off a chain of events referred to signal transduction. Signal transduction systems are especially important in multicellular organisms, such as plants and animals, because of the need to coordinate the activities of hundreds to trillions of cells. Plants, in particular, have a special need for perceiving signals from their environment because of their static nature. As in the animal cell, the first steps in perception of a signal include signal interaction with a receptor, signal amplification through second messenger production, and signal termination through second messenger hydrolysis. Myo-inositol polyphosphate 5-phosphatases (5PTases) (EC 3.1.3.56) have unique signal terminating abilities toward the second messenger inositol trisphosphate (Ins (1,4,5)P3, InsP3). In Arabidopsis thaliana there are 15 members of the 5PTase family, the majority of which contain a single 5PTase catalytic domain. Four members of the Arabidopsis 5PTase family, however, have a unique protein domain structure, with additional N-terminal WD40 repeats that are implicated in protein-protein interactions. The research presented here focused on the identification of 5PTase interacting proteins and the characterization of their functional role in Arabidopsis. To accomplish this goal, I examined a 5PTase13-interacting protein, the sucrose (Suc) nonfermenting-1-related kinase, SnRK1.1, an important energy sensor that is highly conserved among eukaryotes. My identification of a 5PTase13:SnRK1.1 complex points to the novel interaction of this metabolic modulator and inositol signaling/metabolism. 5PTase13 , however, plays a regulatory role in other plant specific processes as well, since I also identified the Arabidopsis homolog (Atp80) of the human WDR48 (HsWDR48, Hsp80) as a novel protein interactor of 5PTase13. My results indicate that Atp80 is important for leaf emergence, development and senescence likely via a regulatory interaction with 5PTase13 and PINOID â binding protein (PBP1).
Ph. D.

SHIP2 (SH2 domain-containing inositol polyphosphate 5-phosphatase type 2) est un enzyme de la famille des inositol polyphosphate 5-phosphatases qui déphosphoryle le PtdIns(3,4,5)P3, second messager intervenant dans différentes voies de signalisation cellulaire et impliqué dans de nombreux processus biologiques.

La surexpression de SHIP2 en cellule, de même que son invalidation chez la souris, ont montré un rôle de cet enzyme dans le contrôle négatif de la cascade de signalisation de l’insuline et dans la sensibilité à cette hormone. Par ailleurs, plusieurs études de polymorphismes chez l’homme ont montré une association entre ce gène et le diabète de type2.

La découverte au sein de notre laboratoire de la délétion d’un motif semblable à ceux présents dans les régions déstabilisatrices de type AU-riche dans la région 3’non codante (3’UTR) du gène SHIP2 chez des patients atteints de diabète de type 2, nous a conduit à explorer le rôle de cette région dans le contrôle de l’expression de SHIP2.

Dans ce but, nous avons entrepris d’identifier des protéines capables de lier ce motif AU-riche et d’entraîner l’ARN de SHIP2 vers la dégradation, et ce par deux techniques distinctes :l’une in vivo chez la levure (le triple hybride) et l’autre in vitro, par l’intermédiaire d’une sonde ARN biotinylée. Malheureusement, aucune de ces deux techniques ne nous a permis d’identifier des protéines se liant à l’ARNm de SHIP2. D’autre part, l’analyse de souris génétiquement modifiées présentant dans la région 3’UTR de SHIP2 une mutation similaire à celle observée chez les patients diabétiques n’a pas montré une augmentation significative d’expression de SHIP2 comme on aurait pu s’y attendre.

Malgré les différentes techniques mises en place, nous ne sommes pas parvenus à caractériser le rôle joué par le 3’UTR de SHIP2 sur le contrôle de son expression.

Dans le but de caractériser l’effet d’une surexpression de SHIP2 et de déterminer si une surexpression de ce gène pouvait mimer le phénotype de diabète de type 2 observé au sein de la population, nous avons généré des souris transgéniques d’addition par transgenèse lentivirale.

Deux axes phénotypiques majeurs ont été explorés chez ces souris :le métabolisme du glucose et la prise de poids consécutive à divers régimes alimentaire.

Les souris transgéniques présentent un retard dans la captation du glucose en réponse à une surcharge en glucose, s’accompagnant d’un défaut de sécrétion d’insuline. Par contre, aucune altération de la sensibilité à l’insuline n’est observée suite à une injection de cette hormone. Cette absence d’altération de la sensibilité à l’insuline est également soutenue par le fait qu’aucune altération de la captation de glucose n’est observée chez des souris surexprimant le transgène spécifiquement dans le muscle squelettique.

Les analyses de prise de poids des souris transgéniques ont révélé une résistance à l’obésité des mâles transgéniques lorsqu’ils sont soumis à un régime alimentaire riche en graisse. Par contre, aucune différence n’est observée sous régime alimentaire conventionnel ou faible en graisse. La plus faible prise de poids des souris transgéniques sous régime riche en graisse s’accompagnant d’une plus faible prise de nourriture, un rôle de SHIP2 dans la régulation du comportement alimentaire et de l’appétit n’est pas à exclure.

En conclusion, la surexpression de SHIP2 chez la souris provoque une intolérance au glucose induite, en tout cas en partie, par une plus faible sécrétion d’insuline, ainsi qu’une résistance à l’obésité induite par un régime riche en graisse.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Osteopetrosis is a genetic disorder resulting from excessive amount of bone due to a defect in osteoclasts. Our laboratory characterized the Ostm1 gene that is responsible for the most severe form of osteopetrosis in mice and humans. In addition, our laboratory then identified the Inositol polyphosphate 4- phosphatase type II alpha (Inpp4b alpha) gene in a differential display screen with reduced expression in Ostm1 deficient osteoclasts. Following characterization of the mouse Inpp4b gene, its role in bone biology was investigated. First, Inpp4b alpha co-localized with F-actin in the podosome subunits, which forms the actin ring of the osteoclast cytoskeletal structure that is essential for osteoclast resorption activity. Second, expression of the phosphatase-inactive Inpp4b in RAW 264.7 OCLs resulted in the formation of multiple, small actin rings leading to poor bone resorption. This result suggested that the phosphatase catalytic activity of Inpp4b alpha is crucial for osteoclast actin ring formation and cytoskeletal dynamics in vitro. We then hypothesized that Inpp4b alpha can be an essential phosphatase involved in the osteoclast cytoskeleton and resorption activity in vivo. Toward this aim, we decided to overexpress three forms of Inpp4b in transgenic mice: the native Inpp4b alpha, the Inpp4b alpha phosphatase inactive form (C845A), and the native form missing the lipid binding C2 domain. Specific expression of these transgenes was targeted with the human Ctsk promoter, which is specifically expressed in mature osteoclasts. These mice were also crossed with Inpp4b knockout mice to avoid interference with expression of endogenous Inpp4b and to mimic a knock-in situation. The expression of Inpp4b alpha (C845A) OCLs differentiated in ex vivo presented multiple, small actin rings and disruption in cytoskeletal rearrangement correlating with previous results. The expression of Inpp4b alpha missing the C2 domain had a diffused podosome belt, similar to osteoclasts derived from the Inpp4b knockout mice. These results illustrated that Inpp4b alpha plays a major role in the osteoclast cytoskeletal rearrangements in vivo, and this is directly dependent on the catalytic activity of the Inpp4b phosphatase.
L'ostéopétrose est un désordre génétique résultant d'une accumulation osseuse dûe à un défaut des ostéoclastes. Nôtre laboratoire a caractérisé la mutation du gène Ostm1 en tant que responsable de la forme la plus sévère d'ostéopétrose chez la souris et l'humain. De plus, nôtre laboratoire a parallèlement identifié une diminution de l'expression du gène Inositol polyphosphate 4- phosphatase type II alpha (Inpp4b alpha) dans les ostéoclastes déficients d'Ostm1. À la suite de la caractérisation du gène Inpp4b chez la souris, l'importance de ce gène dans la biologie du tissu osseux a été établie : D'une part, Inpp4b alpha co-localise avec la F-actine qui forme les anneaux d'actine constituant des sous-unités du podosome, une structure responsable de l'activité de résorption des ostéoclastes. D'autre part, l'expression de la forme inactive d'Inpp4b dans la lignée ostéoclastique RAW 264.7 entraîne la formation de petits et multiples anneaux d'actine résultant en une faible résorption osseuse. Ce résultat suggère que l'activité catalytique de la phosphatase Inpp4b alpha soit cruciale pour la formation des anneaux d'actine et de la dynamique du cytosquelette des ostéoclastes in vitro. À la lumière de ces résultats, nous avons émis l'hypothèse qu'Inpp4b alpha est une importante phosphatase impliquée dans le cytosquelette ainsi que dans l'activité de résorption des ostéoclastes in vivo. Pour ce faire, nous avons surexprimé trois formes d'Inpp4b dans des souris transgéniques: la forme native d'Inpp4b alpha, la forme inactive d'Inpp4b alpha (C845A), ainsi que la forme native manquant le domaine de liaison aux lipides C2. La spécificité d'expression de ces transgènes a été ciblée dans les ostéoclastes matures en utilisant le promoteur de Ctsk humain. Ces souris ont été croisées avec des souris Inpp4b knockout afin d'éviter l'interférence avec l'expression d'Inpp4b endogène ainsi que de mimer un knock-in. Les ostéoclastes exprimant la forme inactive d'Inpp4b alpha(C845A) et différentiés ex vivo présentent de petits et multiples anneaux d'actine, ainsi qu'un défaut du réarrangement du cytosquelette tel qu'observé précédemment. Les ostéoclastes démontrant l'expression d'Inpp4b alpha sans domaine C2 présentent une ceinture de podosomes diffuse, comparable à celle observée dans les souris Inpp4b knockout. Ces résultats illustrent l'importance de la phosphatase Inpp4b alpha dans le réarrangement du cytosquelette des ostéoclastes in vivo, et ceci dépend directement de l'activité catalytique d'Inpp4b.

The phosphatidylinositol signaling pathway utilizes inositol-containing second messengers to mediate signaling events. The enzymes that metabolize phosphoinositides can in some cases serve to terminate the signaling actions of phosphoinositides. The inositol polyphosphate 5-phosphatases (5PTases) comprise a large protein family that hydrolyzes 5-phosphates from a variety of inositol phosphate and phosphoinositide substrates. I have examined the substrate specificity of the At5PTase11 protein from the model plant, Arabidopsis thaliana. The At5PTase11 gene (At1g47510) encodes an active 5PTase enzyme that can dephosphorylate the phosphoinositide substrates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. In addition, the At5PTase11 gene is regulated by abscisic acid, jasmonic acid, and auxin, suggesting a role for phosphoinositide action in these signal transduction pathways. To further delineate the function of At5PTase11 in Arabidopsis thaliana, two independent T-DNA insertion mutant lines were isolated (At5ptase11-1 and At5ptase11-2). Analysis of At5ptase11 mutant lines revealed that At5ptase11 mutant seeds germinate slower compared to wild-type seeds. Moreover, At5ptase11 mutant seedlings demonstrated less hypocotyl growth when grown in the dark. These results indicate that At5PTase11 is required for the early stages of seed germination and seedling growth. Since there are 15 predicted 5PTases in Arabidopsis thaliana, a group of 5PTases have been analyzed to identify the 5PTases with similar substrate selectivity. At5PTase1 (At1g34120), At5PTase2 (At4g18010) and At5PTase3 (At1g71710) have been found to hydrolyze all four potential substrates, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], PtdIns(4,5)P2, and PtdIns(3,4,5)P3. At5PTase7 (At2g32010) hydrolyzed PtdIns(4,5)P2, and PtdIns(3,4,5)P3 which is similar to the substrate selectivity of At5PTase11. In addition, At5PTase4 (At3g63240), and At5PTase9 (At2g01900) hydrolyzed only PtdIns(4,5)P2. These results indicate that there are different groups of Arabidopsis thaliana 5PTases based on the substrate selectivity. These results suggest that Arabidopsis thaliana 5PTases with similar substrate selectivity may have overlapping functions. In summary, the findings that At5PTase11 is a phospholipid-specific 5PTase and At5PTase11 functions in the early stages of seed germination and seedling growth indicate that 5PTases play important roles in plant growth and development.
Ph. D.

Research Doctorate - Doctor of Philosophy (PhD)
Colon cancer is one of the most common and deadly malignancies. Despite recent advances in early diagnosis and the development of novel treatment approaches, the overall survival of patients with metastatic colon cancers remains disappointing. Lots of investigations have discovered some pathways important during the initiation and progression of CRC. These include the WNT, RAS−MAPK, PI3K, TGF-β, P53 and DNA mismatch-repair pathways. But Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway is of particular importance in CRC development, progression and resistance to treatment, since many common genetic and epigenetic anomalies in the disease, such as amplification of epidermal growth factor receptor, activating mutations of KRAS and loss of phosphate and tensin homolog deleted on chromosome 10 (PTEN), converge on activation of PI3K signaling. Lipid and protein kinases promote PI3K signaling, whereas lipid and protein phosphatases suppress PI3K signaling (Manning and Toker, 2017; Newton and Trotman, 2014). Activation of PI3K signaling is negatively regulated by three classes of inositol polyphosphate phosphatases. (Rodgers et al., 2017). The inositol polyphosphate 3-phosphatase (3-phosphatase) PTEN dephosphorylates the 3-position of PI(3,4,5)P3 to generate PI(4,5)P2 (Gericke et al., 2013; Kurokawa et al., 2012), whereas 5-phosphatases, such as Src homology 2-containing inositol 5- phosphatase (SHIP) and phosphatidylinositol 4,5-bisphosphate 5-phosphatase (PIB5PA)/proline-rich inositol polyphosphate phosphatase (PIPP) dephosphorylate the 5-position to produce PI(3,4)P2 (Park et al., 2001). The latter is in turn subjected to dephosphorylation by inositol polyphosphate 4-phosphatase type I (INPP4A) and type II (INPP4B) at the 4-position to generate PI(3)P, thus terminating PI3K signaling. During the past decades, INPP4B has been well established to be a tumor suppressor in a number of cancers. However, we have found that it is frequently upregulated in human colon cancer cells. Silencing of INPP4B blocks activation of Akt and serum- and glucocorticoid-regulated kinase 3 (SGK3), inhibits colon cancer cell proliferation and retards colon cancer xenograft growth. Conversely, overexpression of INPP4B increases proliferation and triggers anchorage-independent growth of normal colon epithelial cells. Moreover, we demonstrate that the effect of INPP4B on Akt and SGK3 is associated with inactivation of phosphate and tensin homolog through its protein phosphatase activity and that the increase in INPP4B is due to Ets-1-mediated transcriptional upregulation in colon cancer cells. To further consolidate the result, two genetically modified mouse model were generated by CRISP/CAS9 technique. The INPP4B^+/-; VillinCre^+/- mouse strain in which INPP4B expression limited to colon epithelium display increased proliferation potential than wild-type mice. Moreover, these mice appeared to be more sensitive to Colitis induced by Dextran Sulfate Sodium (DSS). Nevertheless, when these mice were crossed with adenomatous polyposis coli (APC) mutant mice, the resulting INPP4B^+/-; VillinCre^+/-; APC^+/- mice exhibited increased tumorigenesis in colon. Although results with these transgenic mouse strains are still under way, the available results strongly support the oncogenic role of INPP4B in colon cancer. In this study, we also discovered and cloned a novel small transcript variant of INPP4B generated by alternative splicing in cells of human colon and breast epithelium origins, which we have named INPP4B-S( INPP4B-short). Moreover, we found that INPP4B-S is translated into a protein product that is located to the cytoplasm and promotes proliferation in colon and breast cancer cells. To the best of our knowledge, this is the first time that INPP4B-S has been cloned and described in detail. It seems that INPP4B-S has an additive effect to INPP4B-FL (INPP4B-full length).

Research Doctorate - Doctor of Philosophy (PhD)
Aberrant activation of survival-signaling pathways causes uncontrolled cell proliferation and resistance to apoptosis, and plays an important role in cancer development, progression, and resistance to treatment (Courtney et al., 2010; Ferte et al., 2010). In colorectal cancer (CRC), activation of the phosphatidylinositol 3-kinase (PI3K) pathway is of particular importance, in that many common genetic and epigenetic anomalies in the disease, such as amplification of epidermal growth factor (EGF) receptor, activating mutations in KRAS, and loss of phosphate and tensin homolog deleted on chromosome 10 (PTEN), converge on activation of PI3K signalling (Colakoglu et al., 2008). Moreover, activating mutations of PIK3CA, the gene encoding the catalytic subunit of PI3K, is found in up to 40% of colon cancers (Colakoglu et al., 2008; De Roock et al., 2011). In melanoma, identification of activating mutations in BRAF as the major cause of constitutive activation of the mitogen activated protein kinase (MAPK) pathway has led to successful development of mutant BRAF-specific inhibitors in the treatment of the disease (Chapman et al., 2011; Davies et al., 2002; Houslay, 2011; Ribas and Flaherty, 2011). However, primary and acquired resistance, which is commonly associated with activation of other survival pathways, in particular, the phosphatidylinositol 3-kinase (PI3K) signaling pathway, remains a major obstacle in the quest for curative treatment (Jiang et al., 2011; Karreth et al., 2011; Paraiso et al., 2011; Poulikakos and Rosen, 2011). Indeed, activation of PI3K signaling has been shown to cooperate with mutant BRAF in melanomagenesis using in vivo models (Cheung et al., 2008; Dankort et al., 2009). Activation of PI3K signaling is negatively regulated by three classes of inositol polyphosphate phosphatases (Fedele et al., 2010; Gewinner et al., 2009; Kisseleva et al., 2002). The inositol polyphosphate 3-phosphatase (3-phosphatase) PTEN dephosphorylates the 3-position of PI(3,4,5)P₃ to generate PI(4,5)P₂ (Carracedo et al., 2011; Ma et al., 2008), whereas 5-phosphatases, such as Src homology 2-containing inositol 5- phosphatase (SHIP) and phosphatidylinositol 4,5-Bisphosphate 5- Phosphatase (PIB5PA)/proline-rich inositol polyphosphate phosphatase (PIPP) dephosphorylate the 5-position to produce PI(3,4)P₂ (Ooms et al., 2006; Ye et al., 2013a). PI(3,4)P₂ is in turn subjected to dephosphorylation by inositol polyphosphate 4-phosphatase type I (INPP4A) and type II (INPP4B) at the 4-position to generate PI(3)P, thus terminating PI3K signaling (Fedele et al., 2010; Gewinner et al., 2009; Hodgson et al., 2011). Interestingly, despite INPP4B tumor suppressive role in some other tissues, in this study we found that inositol polyphosphate 4-phosphatase type II (INPP4B) functions as an oncogenic regulator in human colon cancer and melanoma. While INPP4B is upregulated in two cancers and its high expression is associated with poor patient survival, INPP4B knockdown blocks activation of PI3K downstream signaling, inhibits proliferation, undermines survival of colon cancer and melanoma cells, and retards cancer growth in a xenograft model. Conversely, overexpression of INPP4B causes increased proliferation and anchorage-independent growth in normal colon epithelial cells and melanocytes. However, INPP4B regulates PI3K signalling pathway in two cancers by different mechanisms. It plays an important role for maintaining cellular PI(3,4,5)P₃ and PI(3,4)P₂ levels in colon cancer whereas PI(3)P levels in melanoma cells. Also, the increase in INPP4B is primarily due to Ets-1-mediated transcriptional upregulation in colon cancer cells, whereas a posttranscriptional increase via reduction of miRNA-494 and miRNA-599 in melanoma.