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1.
Biochem J ; 466(2): 359-67, 2015 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-25495341

RESUMEN

NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1 µM but did not inhibit the α and ß PI5P4K isoforms at concentrations up to 100 µM. A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme's ATP-binding site and direct exploration of binding using hydrogen-deuterium exchange (HDX)-MS (HDX-MS) revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and ß homologue to render PI5P4Kγ resistant NIH-12848 inhibition. NIH-12848 (10 µM) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4Kγ that increases when the cells grow to confluence and polarize. NIH-12848 inhibited the translocation of Na⁺/K⁺-ATPase to the plasma membrane that occurs when mpkCCD cells grow to confluence and also prevented reversibly their forming of 'domes' on the culture dish. Both these NIH-12848-induced effects were mimicked by specific RNAi knockdown of PI5P4Kγ, but not that of PI5P4Ks α or ß. Overall, the data reveal a probable contribution of PI5P4Kγ to the development and maintenance of epithelial cell functional polarity and show that NIH-12848 is a potentially powerful tool for exploring the cell physiology of PI5P4Ks.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Corteza Renal/enzimología , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Quinazolinas/farmacología , Tiofenos/farmacología , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Medición de Intercambio de Deuterio , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Corteza Renal/citología , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Ratones , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosfatos de Fosfatidilinositol/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Conformación Proteica , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
2.
Biochem Biophys Res Commun ; 411(2): 416-20, 2011 Jul 29.
Artículo en Inglés | MEDLINE | ID: mdl-21756881

RESUMEN

Phosphatidylinositol 4-phosphate 5-kinase Iγ (PIP5KIγ) is subject to extensive C-terminal splice variation, with four variants, PIP5KIγ_v1, 2, 4 and 5, described in humans Schill and Anderson (2009) [7]. Here firstly, we report a new rodent splice variant, which includes the exon that was previously unique to the rodent neuron-specific PIP5KIγ93 Giudici et al. (2006) [6], but which omits the C-terminal exon of PIP5KIγ93; this new variant shows a wide tissue expression pattern in mouse. Secondly, we show that in humans there is an alternative splicing site 78 nucleotides from the start of exon 16c, such that humans additionally express both PIP5KIγ93 (which we now call PIP5KIγ_v3) specifically in brain and, again expressed more widely, the new variant described here, which we now name PIP5KIγ_v6.


Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Humanos , Isoenzimas/química , Isoenzimas/genética , Ratones , Datos de Secuencia Molecular , Ratas
3.
FEBS Lett ; 580(30): 6933-7, 2006 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-17157843

RESUMEN

There are three known splice variants of Type Igamma phosphatidylinositol 4-phosphate 5-kinase (PIPkin Igamma): PIPkins Igamma87, Igamma90, and the most recently cloned (Giudici, M.L., Emson, P.C. and Irvine, R.F. (2004) A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma. Biochem. J. 379, 489-496) PIPkin IgammaC (here called PIPkin Igamma93). Here, we have explored the subcellular localisation and mobility of Type I PIPkins in transfected cells by confocal microscopy and flourescence recovery after photobleaching. The unique behaviour shown by PIPkin Igamma93 is consistent with its suggested distinct function. Moreover, the markedly different localisation and mobility of active versus inactive PIPkin Igamma93 provide insights into the factors that dictate cellular targeting of Type Igamma PIPkins.


Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Humanos , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Fluorescente , Peso Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/clasificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Transporte de Proteínas , Transfección
4.
Adv Biol Regul ; 61: 47-50, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26710750

RESUMEN

The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are an important family of enzymes, whose physiological roles are being teased out by a variety of means. Phosphatidylinositol-5-phosphate 4-kinase γ (PI5P4Kγ) is especially intriguing as its in vitro activity is very low. Here we review what is known about this enzyme and discuss some recent advances towards an understanding of its physiology. Additionally, the effects of the ATP-competitive inhibitor I-OMe Tyrphostin AG-538 on all three mammalian PI5P4Ks was explored, including two PI5P4Kγ mutants with altered ATP- or PI5P-binding sites. The results suggest a strategy for targeting non-ATP binding sites on inositol lipid kinases.


Asunto(s)
Catecoles/farmacología , Inhibidores Enzimáticos/farmacología , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Transducción de Señal , Tirfostinos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Expresión Génica , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica
5.
Biochem J ; 379(Pt 2): 489-96, 2004 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-14741049

RESUMEN

Type I PIPkins (phosphatidylinositol 4-phosphate 5-kinases) are the enzymes that catalyse the major cellular route of synthesis of PtdIns(4,5) P2, and three isoforms (alpha, beta and gamma) with several splice variants have been found to date. In the present paper, we describe the discovery of a novel splice variant of the gamma isoform, which we call PIPkin Igammac, and which is characterized by the inclusion of a 26-amino-acid insert near the C-terminus. Its transcript appears to be selectively expressed in brain, where it locates in the neurons of restricted regions, such as cerebellum, hippocampus, cortex and olfactory bulb, as indicated by in situ hybridization studies. Overexpression of two different catalytically inactive constructs of PIPkin Igammac in rat cerebellar granule cells causes a progressive loss of their neuronal processes, whereas equivalent kinase-dead versions of PIPkin Igammaa did not induce any such effect, suggesting the possible existence of a specific PtdIns(4,5) P2 pool synthesized by PIPkin Igammac, which is involved in the maintenance of some neuronal cellular processes.


Asunto(s)
Encéfalo/enzimología , Neuronas/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Encéfalo/citología , ADN Complementario/aislamiento & purificación , Hibridación in Situ , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Neuronas/citología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido
6.
Adv Biol Regul ; 57: 193-202, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25311266

RESUMEN

The family of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) is emerging from a comparative backwater in inositide signalling into the mainstream, as is their substrate, phosphatidylinositol 5-phosphate (PI5P). Here we review some of the key questions about the PI5P4Ks, their localisation, interaction, and regulation and also we summarise our current understanding of how PI5P is synthesised and what its cellular functions might be. Finally, some of the evidence for the involvement of PI5P4Ks in pathology is discussed.


Asunto(s)
Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal/fisiología , Animales , Humanos
7.
Biochem J ; 363(Pt 3): 563-70, 2002 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-11964157

RESUMEN

The phosphatidylinositol phosphate kinases (PIPkins) are a family of enzymes involved in regulating levels of several functionally important inositol phospholipids within cells. The PIPkin family is subdivided into three on the basis of substrate specificity, each subtype presumably regulating levels of different subsets of the inositol lipids. The physiological function of the type II isoforms, which exhibit a preference for phosphatidylinositol 5-phosphate, a lipid about which very little is known, is particularly poorly understood. In the present study, we demonstrate interaction between, and co-immunoprecipitation of, type IIalpha PIPkin with the related, but biochemically and immunologically distinct, type I PIPkin isoforms. Type IIalpha PIPkin interacts with all three known type I PIPkins (alpha, beta and gamma), and in each case co-expression of the type I isoform with type IIalpha results in recruitment of the latter from the cytosol to the plasma membrane of the cell. This change in subcellular localization could result in improved access of the type IIalpha PIPkin to its lipid substrates.


Asunto(s)
Membrana Celular/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Ácido Aspártico/metabolismo , Sitios de Unión , Western Blotting , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/enzimología , Células HeLa , Humanos , Lisina/metabolismo , Antígenos de Histocompatibilidad Menor , Mutagénesis Sitio-Dirigida , Porcinos
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