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Author(s): Amaia Zabala-Letona; Amaia Arruabarrena-Aristorena; Natalia Martn-Martn; Sonia Fernandez-Ruiz; James D. Sutherland; Michelle Clasquin; Julen Tomas-Cortazar; Jose Jimenez; Ines Torres; Phong Quang; Pilar Ximenez-Embun; Ruzica Bago; Aitziber Ugalde-Olano; Ana Loizaga-Iriarte; [...]

Author(s): Amaia Zabala-Letona [1, 2]; Amaia Arruabarrena-Aristorena [1]; Natalia Martn-Martn [1, 2]; Sonia Fernandez-Ruiz [1, 2]; James D. Sutherland [1]; Michelle Clasquin [3]; Julen Tomas-Cortazar [1]; Jose Jimenez [4]; Ines [...]

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)Pbinding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4- phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K. [ABSTRACT FROM AUTHOR]

Nucleoside diphosphate kinases (NDPKs) are ubiquitous phosphotransfer enzymes responsible for producing most of the nucleoside triphosphates except for ATP. This role is important for the synthesis of nucleic acids and proteins and the metabolism of sugars and lipids. Apart from this housekeeping role NDPKs have been shown to have many regulatory functions in diverse cellular processes including proliferation and endocytosis. Although the protein has been shown to have a positive regulatory role in clathrin- and dynamin-mediated micropinocytosis, its roles in macropinocytosis and phagocytosis have not been studied. The additional non-housekeeping roles of NDPK are often independent of enzyme activity but dependent on the expression level of the protein. In this study we altered the expression level of NDPK in the model eukaryotic organism Dictyostelium discoideum through antisense inhibition and overexpression. We demonstrate that NDPK levels affect growth, endocytosis and exocytosis. In particular we find that Dictyostelium NDPK negatively regulates endocytosis in contrast to the positive regulatory role identified in higher eukaryotes. This can be explained by the differences in types of endocytosis that have been studied in the different systems - phagocytosis and macropinocytosis in Dictyostelium compared with micropinocytosis in mammalian cells. This is the first report of a role for NDPK in regulating macropinocytosis and phagocytosis, the former being the major fluid phase uptake mechanism for macrophages, dendritic cells and other (non dendritic) cells exposed to growth factors. [ABSTRACT FROM AUTHOR]

nm23-H1 was found to diminish metastatic potential of carcinoma cell lines and therefore was placed in the group of metastatic suppressor genes. Its protein product has a function of a nucleoside diphosphate kinase (NDPK) as well as protein kinase and nuclease. Though it was found that Nm23-H1 is involved in many cellular processes, it is still not known how it promotes metastatic suppressor activity. Since the process of metastasis is dependent on adhesion properties of cells, the goal of our work was to describe the adhesion properties of CAL 27 cells (oral squamous cell carcinoma of the tongue) overexpressing FLAG/nm23-H1. In our experiments, cells overexpressing nm23-H1 show reduced migratory and invasive potential. Additionally, cells overexpressing nm23-H1 adhere stronger on substrates (collagen IV and fibronectin) and show more spread morphology than the control cells. Results obtained by EGF induction of migration revealed that the adhesion strength predetermined cell response to chemoattractant and that Nm23-H1, in this cell type, does not interfere with, EGF induced, Ras signaling pathway. These data contribute to the overall knowledge about nm23-H1 and its role in cell adhesion, migration, and invasion, especially in oral squamous cell carcinoma.

The family of Nm23/NDPK (nucleoside diphosphate kinase) proteins regulates a vast variety of cellular processes and, therefore, participates in important physiological events like proliferation, differentiation, molecular transport, and apoptosis. The majority of experimental data concerning this gene family has been focused on their engagement in metastasis formation--a critical point in tumor progression. In spite of the growing amount of evidence suggesting the multifunctional role of nm23/NDPKs the specific functions of every particular family member is still elusive. This review focuses on recent evidence on specific subcellular localization of Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins and attempts to connect the findings to their diverse biological functions in the normal and/or malignantly transformed cell.

The human nm23-H1 was discovered as a tumor metastasis suppressor based on its reduced expression in melanoma cell lines with low versus high metastatic potential. It encodes for one of two subunits of the nucleoside-diphosphate kinase. Besides its role in the maintenance of the cells NTP pool, nm23 plays a key role in different cellular processes. The role of nm23-H1 in these processes still has to be elucidated. Our goal was to identify Nm23-H1 downstream targets by subjecting Nm23-H1 overexpressing CAL 27 cells oral squamous cell carcinoma (OSSC) to microarray analysis. The genes with changed expression patterns could be clustered into several groups: transforming growth factor beta (TGFbeta) signaling pathway, cell adhesion, invasion and motility, proteasome machinery, cell-cycle, epithelial structural and related molecules and others. Based on the expression patterns observed we presume that nm23-H1 might have a role in OSSCs, which should be confirmed by future experiments.

Combined cytogenetic, fluorescence in situ hybridization (FISH), and molecular analysis are useful in the diagnosis of sex chromosome aberrations. These methods were used in karyotype analysis of a 4-year-old girl with mild dysmorphism and growth retardation. Standard cytogenetic and FISH analysis was done on slides obtained from peripheral blood lymphocyte culture, and the molecular study was performed by using DNA polymorphism analysis. Both parents had normal karyotypes. Chromosome analysis of the proband identified the karyotype with 46 chromosomes and a late replicating dicentric X. Interphase FISH with an alpha satellite X centromere probe revealed two mosaic cell lines. Three signals were observed at 84.5% and one signal at 15.5% of the interphase cells. Molecular analysis showed that the dicentric X chromosome was of paternal origin. Based on this study, we concluded that the karyotype of the patient was 45,X/46,X, psu idic(X)(q22.3), with the trisomy Xpter-->q22.3 and monosomy Xqter-->q22.3. Dicentric X was the result of an isolocal break in both chromatids of the paternal X chromosome and subsequent rejoining of the broken ends, followed by the inactivation of one centromere. This study illustrates the usefulness of combined cytogenetic and molecular investigations for the detection of mosaicism, understanding the mechanism of the formation and parental origin of chromosomal rearrangement, and establishing the diagnosis.

We present a case of trisomy for the whole short arm of chromosome 6 in a 3-year-old girl with moderate mental retardation, mild facial dysmorphism, short stature, failure to thrive, and no abnormalities of the visceral organs. Cytogenetic and fluorescence in situ hybridization (FISH) analysis revealed a 46, X, der(X)t(X;6)(q22; p11.1) karyotype. The derived X was late replicating with variable spreading of X chromosome inactivation onto the translocated 6p. A normal karyotype was observed in the father, while the mother presented 46,XX/46,XX, der(10)t(6;10)(p11;p11). The mother is a mosaic with unbalanced t(6;10) in 4.7% of cells. To the best of our knowledge, this unusual mosaicism has not yet been reported. In this family the short arm of chromosome 6 was involved in an unbalanced rearrangement with chromosome X in the proband and with chromosome 10 in the mother. In order to study the mechanism of the formation of t(X;6) in the girl we performed DNA polymorphism analysis. These investigations revealed that chromosomes X and 6 involved in the rearrangement are of paternal origin. Our patient exhibits only discrete facial features characteristic of partial trisomy 6p. We suggest that mild phenotypic expression be probably due to X chromosome inactivation spreading onto the translocated 6p. This report show that combined cytogenetic, FISH, and molecular analysis of chromosomal aberrations are necessary for the understanding of the mechanism of formation, parental origin, and genetic counseling., (Copyright 2003 Wiley-Liss, Inc.)