Type I Phosphatidylinositol-4-phosphate 5-Kinases

Abstract

Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5K␥) and the characterization of members of the type I PIP5K family. Type I PIP5K␥ has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the ␣ and ␤ isoforms. Northern blot analysis revealed that the ␥ isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the ␤ isoform has the greatest V max value for the PtdIns(4)P kinase activity and the ␥ isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the ␤ and ␥ isoforms were found to confer the greatest V max value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the ␣ isoform is essential for the PtdIns(4)P kinase activity. As was the case with the ␣ isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578 -7581), overexpression of either the ␤ or the ␥ isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.Recent advances in cell biology have revealed that phosphoinositide metabolism plays an essential role in various cellular processes. Synthesis and breakdown of certain phosphoinositides at appropriate times and intracellular sites appear to be required for complex regulation of these cellular processes. One of the phosphoinositides, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), 1 is located at an important branchpoint in phosphoinositide metabolism. PtdIns(4,5)P 2 serves as a substrate for phosphoinositide-specific phospholipase C (EC 3

Identification and Characterization of a Phosphoinositide Phosphate Kinase Homolog

“…Control of cellular functions involving membrane trafficking and adhesion requires rapid, reversible, and localized assembly of the actin cytoskeleton as well as of other functional complexes such as focal adhesions (1,(3)(4)(5)(6). These assemblages of structural and signaling proteins provide a delimited zone of infrastructure, mechanical support, or directionality for dynamic membrane processes.…”

“…It is now well established that this phosphoinositide is a signaling molecule in its own right, acting through several actin-binding proteins, members of the Wiscott-Aldrich Syndrome protein (WASP) family of proteins, and the Arp2/3 complex (7), as well as by controlling the localization of a subset of proteins containing pleckstrin homology (PH) domains. Dynamin, a large GTPase that is required for fission of nascent vesicles from Golgi and plasma membranes, binds to PI(4,5)P 2 by its PH domain and is found at actin-rich sites in peripheral ruffles of eukaryotic cells as well as in actin comets and tails generated by the intracellular pathogen Listeria or by overexpression of type I PIPKs (5,8,9). In addition, PI(4,5)P 2 is converted by phosphoinositide 3-kinases to PI(3,4,5)P 3 , which can activate members of the Rho/Rac/CDC42 family of GTP-binding proteins to initiate actin cytoskeletal remodeling (1).…”

“…These assemblages of structural and signaling proteins provide a delimited zone of infrastructure, mechanical support, or directionality for dynamic membrane processes. Rapid and reversible assembly of the cytoskeletal framework for membrane vesicle formation, endoand exocytosis, and cell migration is regulated by the generation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 1 by phosphatidylinositol phosphate kinases (PIPKs) (5). It is now well established that this phosphoinositide is a signaling molecule in its own right, acting through several actin-binding proteins, members of the Wiscott-Aldrich Syndrome protein (WASP) family of proteins, and the Arp2/3 complex (7), as well as by controlling the localization of a subset of proteins containing pleckstrin homology (PH) domains.…”

Detección de blancos moleculares en la ruta de señalización del fosfatidilinositol en Leishmania spp. a través de herramientas bioinformáticas y de modelado matemático

“…La ruta de señalización del fosfatidil-inositol es fundamental para múltiples procesos celulares en eucariotas, como la diferenciación, la supervivencia, el transporte intracelular y la activación de factores de transcripción, entre muchos otros (5,8,9,(32)(33)(34)(35)(36)(37). Este trabajo permitió la reconstrucción de dicha ruta en Leishmania, y constituye una primera aproximación para entender el funcionamiento y las interacciones que subyacen a las proteínas que la conforman y, por lo tanto, a la postulación de nuevos blancos para el desarrollo de medicamentos contra la leishmaniasis.…”

“…Uno de ellos es el PI4,5P2, sustrato que cataliza PLC para producir DAG e IP3, protagonistas de la activación de liberación del calcio y de la unión a múltiples factores de transcripción (32). También, se ha encontrado que modula la función de algunas proteínas, como las de unión de la actina, con un importante papel en el proceso de exocitosis (32).…”