scholarly journals hVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells

2009 ◽  
Vol 417 (3) ◽  
pp. 747-755 ◽  
Author(s):  
Ying Yan ◽  
Rory J. Flinn ◽  
Haiyan Wu ◽  
Rachel S. Schnur ◽  
Jonathan M. Backer
Keyword(s):  
Mammalian Cells ◽  
Specific Activity ◽  
Gene Activation ◽  
Nutrient Sensing ◽  
Beclin 1 ◽  
Class Iii ◽  
Acetoxymethyl Ester ◽  
Phosphoinositide 3 Kinase ◽  
Vacuolar Protein

The mammalian Class III PI3K (phosphoinositide 3-kinase), hVps34 [mammalian Vps (vacuolar protein sorting) 34 homologue], is an important regulator of vesicular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with a putative serine/threonine protein kinase, Vps15, which is required for Vps34p activity. The mammalian homologue of Vps15p, hVps15 (formerly called p150), also binds to hVps34, but its role in hVps34 signalling has not been evaluated. In the present study we have therefore compared the activity and regulation of hVps34 expressed without or with hVps15. We find that hVps34 has low specific activity when expressed alone; co-expression with hVps15 leads to a marked increase in activity. Notably, beclin-1/UVRAG (UV radiation resistance-associated gene) activation of hVps34 requires co-expression with hVps15; this may be explained by the observation that beclin-1/UVRAG expression increases hVps34/hVps15 binding. Regulation of hVps34 activity by nutrients also requires co-expression with hVps15. Finally, given a recent report that hVps34 activity requires Ca2+/CaM (calmodulin), we considered whether hVps15 might be involved in this regulation. Although hVps34 does bind CaM, we find its activity is not affected by treatment of cells with BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] or W7. Removal of CaM by EDTA or EGTA washes has no effect on hVps34 activity, and hVps34 activity in vitro is unaffected by Ca2+ chelation. The results of the present study show that, in mammalian cells, hVps34 activity is regulated through its interactions with hVps15, but is independent of Ca2+/CaM.

Regulation of class III (Vps34) PI3Ks

2007 ◽  
Vol 35 (2) ◽  
pp. 239-241 ◽  
Author(s):  
Y. Yan ◽  
J.M. Backer
Keyword(s):  
Mammalian Cells ◽  
Mammalian Target Of Rapamycin ◽  
Nutrient Sensing ◽  
Class Iii ◽  
Lipid Kinase ◽  
Intracellular Targeting ◽  
Lipid Product ◽  
Important Regulator ◽  
Phosphoinositide 3 Kinase ◽  
Vacuolar Protein

The class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar protein sorting 34), was first identified as a regulator of vacuolar hydrolase sorting in yeast. Unlike other PI3Ks, the Vps34 lipid kinase specifically utilizes phosphatidylinositol as a substrate, producing the single lipid product PtdIns3P. While Vps34 has been studied for some time in the context of endocytosis and vesicular trafficking, it has more recently been implicated as an important regulator of autophagy, trimeric G-protein signalling, and the mTOR (mammalian target of rapamycin) nutrient-sensing pathway. The present paper will focus on studies that describe the regulation of hVps34 (human Vps34) intracellular targeting and enzymatic activity in yeast and mammalian cells.

scholarly journals Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

2008 ◽  
Vol 19 (12) ◽  
pp. 5360-5372 ◽  
Author(s):  
Eisuke Itakura ◽  
Chieko Kishi ◽  
Kinji Inoue ◽  
Noboru Mizushima
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Pi3 Kinase ◽  
Beclin 1 ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Interacting Protein ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.

The intricate regulation and complex functions of the Class III phosphoinositide 3-kinase Vps34

2016 ◽  
Vol 473 (15) ◽  
pp. 2251-2271 ◽  
Author(s):  
Jonathan M. Backer
Keyword(s):  
Protein Sorting ◽  
Nutrient Sensing ◽  
Endocytic Trafficking ◽  
Class Iii ◽  
Cellular Functions ◽  
Lipid Kinase ◽  
Complex Functions ◽  
Phosphoinositide 3 Kinase ◽  
Vacuolar Protein ◽  
Important Enzyme

The Class III phosphoinositide 3-kinase Vps34 (vacuolar protein sorting 34) plays important roles in endocytic trafficking, macroautophagy, phagocytosis, cytokinesis and nutrient sensing. Recent studies have provided exciting new insights into the structure and regulation of this lipid kinase, and new cellular functions for Vps34 have emerged. This review critically examines the wealth of new data on this important enzyme, and attempts to integrate these findings with current models of Vps34 signalling.

The regulation and function of Class III PI3Ks: novel roles for Vps34

2008 ◽  
Vol 410 (1) ◽  
pp. 1-17 ◽  
Author(s):  
Jonathan M. Backer
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Mammalian Target Of Rapamycin ◽  
Class Iii ◽  
Endocytic Sorting ◽  
Vacuolar Sorting ◽  
Phosphoinositide 3 Kinase ◽  
Sorting System ◽  
Vacuolar Protein ◽  
And Function

The Class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar protein sorting 34), was first described as a component of the vacuolar sorting system in Saccharomyces cerevisiae and is the sole PI3K in yeast. The homologue in mammalian cells, hVps34, has been studied extensively in the context of endocytic sorting. However, hVps34 also plays an important role in the ability of cells to respond to changes in nutrient conditions. Recent studies have shown that mammalian hVps34 is required for the activation of the mTOR (mammalian target of rapamycin)/S6K1 (S6 kinase 1) pathway, which regulates protein synthesis in response to nutrient availability. In both yeast and mammalian cells, Class III PI3Ks are also required for the induction of autophagy during nutrient deprivation. Finally, mammalian hVps34 is itself regulated by nutrients. Thus Class III PI3Ks are implicated in the regulation of both autophagy and, through the mTOR pathway, protein synthesis, and thus contribute to the integration of cellular responses to changing nutritional status.

scholarly journals Recruitment of the SWI-SNF Chromatin Remodeling Complex as a Mechanism of Gene Activation by the Glucocorticoid Receptor τ1 Activation Domain

2000 ◽  
Vol 20 (6) ◽  
pp. 2004-2013 ◽  
Author(s):  
Annika E. Wallberg ◽  
Kristen E. Neely ◽  
Ahmed H. Hassan ◽  
Jan-Åke Gustafsson ◽  
Jerry L. Workman ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Mammalian Cells ◽  
Gene Activation ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Chromatin Remodeling Complex ◽  
Nucleosomal Dna ◽  
Target Promoters

ABSTRACT The SWI-SNF complex has been shown to alter nucleosome conformation in an ATP-dependent manner, leading to increased accessibility of nucleosomal DNA to transcription factors. In this study, we show that the SWI-SNF complex can potentiate the activity of the glucocorticoid receptor (GR) through the N-terminal transactivation domain, τ1, in both yeast and mammalian cells. GR-τ1 can directly interact with purified SWI-SNF complex, and mutations in τ1 that affect the transactivation activity in vivo also directly affect τ1 interaction with SWI-SNF. Furthermore, the SWI-SNF complex can stimulate τ1-driven transcription from chromatin templates in vitro. Taken together, these results support a model in which the GR can directly recruit the SWI-SNF complex to target promoters during glucocorticoid-dependent gene activation. We also provide evidence that the SWI-SNF and SAGA complexes represent independent pathways of τ1-mediated activation but play overlapping roles that are able to compensate for one another under some conditions.

Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies

2007 ◽  
Vol 74 ◽  
pp. 47-57 ◽  
Author(s):  
David J. Gill ◽  
Hsiangling Teo ◽  
Ji Sun ◽  
Olga Perisic ◽  
Dmitry B. Veprintsev ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Multivesicular Body ◽  
Binding Pocket ◽  
Lipid Binding ◽  
Pleckstrin Homology Domain ◽  
Class Iii ◽  
Membrane Recruitment ◽  
Early Endosomes ◽  
Phosphoinositide 3 Kinase ◽  
Vacuolar Protein

Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.

scholarly journals A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast

2004 ◽  
Vol 166 (2) ◽  
pp. 205-211 ◽  
Author(s):  
Prasenjit Mitra ◽  
Yingjie Zhang ◽  
Lucia E. Rameh ◽  
Maria P. Ivshina ◽  
Dannel McCollum ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Mammalian Cells ◽  
Class I ◽  
Unexpected Finding ◽  
Class Iii ◽  
Growth And Survival ◽  
Higher Eukaryotes ◽  
Phosphoinositide 3 Kinase ◽  
Phosphatidylinositol 4 ◽  
Phosphatase And Tensin Homologue

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.

scholarly journals Histone H3 K4 Demethylation during Activation and Attenuation of GAL1 Transcription in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (22) ◽  
pp. 7856-7864 ◽  
Author(s):  
Kristin Ingvarsdottir ◽  
Chris Edwards ◽  
Min Gyu Lee ◽  
Jung Shin Lee ◽  
David C. Schultz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Gene Activation ◽  
Histone H3 ◽  
Histone Lysine ◽  
Insight Into ◽  
Demethylation Activity

ABSTRACT In mammalian cells, histone lysine demethylation is carried out by two classes of enzymes, the LSD1/BHC110 class and the jumonji class. The enzymes of the jumonji class in the yeast Saccharomyces cerevisiae have recently also been shown to have lysine demethylation activity. Here we report that the protein encoded by YJR119c (termed KDM5), coding for one of five predicted jumonji domain proteins in yeast, specifically demethylates trimethylated histone H3 lysine 4 (H3K4me3), H3K4me2, and H3K4me1 in vitro. We found that loss of KDM5 increased mono-, di-, and trimethylation of lysine 4 during activation of the GAL1 gene. Interestingly, cells deleted of KDM5 also displayed a delayed reduction of K4me3 upon reestablishment of GAL1 repression. These results indicate that K4 demethylation has two roles at GAL1, first to establish appropriate levels of K4 methylation during gene activation and second to remove K4 trimethylation during the attenuation phase of transcription. Thus, analysis of lysine demethylation in yeast provides new insight into the physiological roles of jumonji demethylase enzymes.

Nutrient sensing in the mTOR/S6K1 signalling pathway

2007 ◽  
Vol 35 (2) ◽  
pp. 236-238 ◽  
Author(s):  
P. Gulati ◽  
G. Thomas
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Ectopic Expression ◽  
Nutrient Sensing ◽  
Signalling Pathway ◽  
Small Interfering Rnas ◽  
Endosomal Membranes ◽  
Phosphoinositide 3 Kinase ◽  
Vacuolar Protein ◽  
Phox Homology ◽  
Prevailing View

Nutrient overload induces constitutive S6K1 (S6 kinase 1) activation, which leads to insulin resistance by suppressing insulin-induced class I PI3K (phosphoinositide 3-kinase) signalling [Um, Frigerio, Watanabe, Picard, Joaquin, Sticker, Fumagalli, Allegrini, Kozma, Auwerx and Thomas (2004) Nature 431, 200–205]. This finding gave rise to the question of the mechanism by which nutrients, such as AAs (amino acids), enter the mTOR (mammalian target of rapamycin)/S6K1 signalling pathway. Counter to the prevailing view, our recent studies have shown that the AA input into the mTOR/S6K1 signalling pathway is not mediated by the tumour suppressor TSC1 (tuberous sclerosis complex 1)/TSC2 or its target, the proto-oncogene Rheb (Ras homologue enriched in brain). Instead, we found that the AA input was mediated by class 3 PI3K, or hVps34 (human vacuolar protein sorting 34). In brief, ectopic expression of hVps34 drives S6K1 activation, but only in the presence of AAs, and this effect is blocked by small interfering RNAs directed against hVps34. Moreover, stimulation of cells with AAs increases hVps34 activity, as indicated by the production of PI3P (phosphatidylinositol 3-phosphate). PI3P mediates the recruitment of proteins containing FYVE (Fab1p, YOTB, Vac1p and EEA1) or PX (Phox homology) domains to endosomal membranes, with PI3P-rich micro-domains acting as signalling platforms. Additional evidence indicating hVps34 as the mediator of AA input to S6K1 came from experiments in which S6K1 activation was attenuated by ectopic expression of a cDNA containing two FYVE domains, which bind to PI3P, preventing binding of proteins containing either FYVE or PX domains [Nobukuni, Joaquin, Roccio, Dann, Kim, Gulati, Byfield, Backer, Natt, Bos, Zwartkruis and Thomas (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14238–14243].

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