scholarly journals Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling

2012 ◽  
Vol 23 (14) ◽  
pp. 2723-2740 ◽  
Author(s):  
Steve Jean ◽  
Sarah Cox ◽  
Eric J. Schmidt ◽  
Fred L. Robinson ◽  
Amy Kiger
Keyword(s):  
Endosomal Trafficking ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Membrane Compartment ◽  
Endosomal Pathway ◽  
Gtpase Activation ◽  
Phosphatidylinositol 3

Cells rely on the coordinated regulation of lipid phosphoinositides and Rab GTPases to define membrane compartment fates along distinct trafficking routes. The family of disease-related myotubularin (MTM) phosphoinositide phosphatases includes catalytically inactive members, or pseudophosphatases, with poorly understood functions. We found that Drosophila MTM pseudophosphatase Sbf coordinates both phosphatidylinositol 3-phosphate (PI(3)P) turnover and Rab21 GTPase activation in an endosomal pathway that controls macrophage remodeling. Sbf dynamically interacts with class II phosphatidylinositol 3-kinase and stably recruits Mtm to promote turnover of a PI(3)P subpool essential for endosomal trafficking. Sbf also functions as a guanine nucleotide exchange factor that promotes Rab21 GTPase activation associated with PI(3)P endosomes. Of importance, Sbf, Mtm, and Rab21 function together, along with Rab11-mediated endosomal trafficking, to control macrophage protrusion formation. This identifies Sbf as a critical coordinator of PI(3)P and Rab21 regulation, which specifies an endosomal pathway and cortical control.

scholarly journals Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity

2017 ◽  
Vol 28 (2) ◽  
pp. 252-260 ◽  
Author(s):  
Travis R. Ruch ◽  
David M. Bryant ◽  
Keith E. Mostov ◽  
Joanne N. Engel
Keyword(s):  
Apical Membrane ◽  
Protein Localization ◽  
Apical Surface ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Signaling Mechanism ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Chemically Induced Dimerization ◽  
Phosphatidylinositol 3

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens.

scholarly journals Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3

2014 ◽  
Vol 25 (10) ◽  
pp. 1608-1619 ◽  
Author(s):  
Gus Lawrence ◽  
Christopher C. Brown ◽  
Blake A. Flood ◽  
Surya Karunakaran ◽  
Margarita Cabrera ◽  
...  
Keyword(s):  
Fusion Reaction ◽  
Casein Kinase ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphatidic Acid Phosphatase ◽  
Phosphatidylinositol 3

Maturation of organelles in the endolysosomal pathway requires exchange of the early endosomal GTPase Rab5/Vps21 for the late endosomal Rab7/Ypt7. The Rab exchange depends on the guanine nucleotide exchange factor activity of the Mon1-Ccz1 heterodimer for Ypt7. Here we investigate vacuole binding and recycling of Mon1-Ccz1. We find that Mon1-Ccz1 is absent on vacuoles lacking the phosphatidic acid phosphatase Pah1, which also lack Ypt7, the phosphatidylinositol 3-kinase Vps34, and the lipid phosphatidylinositol 3-phosphate (PI3P). Interaction of Mon1-Ccz1 with wild-type vacuoles requires PI3P, as shown in competition experiments. We also find that Mon1 is released from vacuoles during the fusion reaction and its release requires its phosphorylation by the type 1 casein kinase Yck3. In contrast, Mon1 is retained on vacuoles lacking Yck3 or when Mon1 phosphorylation sites are mutated. Phosphorylation and release of Mon1 is restored with addition of recombinant Yck3. Together the results show that Mon1 is recruited to endosomes and vacuoles by PI3P and, likely after activating Ypt7, is phosphorylated and released from vacuoles for recycling.

scholarly journals αPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase

Oncogene ◽  
1999 ◽  
Vol 18 (41) ◽  
pp. 5680-5690 ◽  
Author(s):  
Shigeto Yoshii ◽  
Masamitsu Tanaka ◽  
Yoshirou Otsuki ◽  
Dong-Yu Wang ◽  
Rong-Jun Guo ◽  
...  
Keyword(s):  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Phosphatidylinositol 3

scholarly journals The substrate specificity of the human TRAPPII complex’s Rab-guanine nucleotide exchange factor activity

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Meredith L. Jenkins ◽  
Noah J. Harris ◽  
Udit Dalwadi ◽  
Kaelin D. Fleming ◽  
Daniel S. Ziemianowicz ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Binding Interface ◽  
Nucleotide Exchange Factor ◽  
Biochemical Characterisation ◽  
Hydrogen Deuterium ◽  
Insight Into

AbstractThe TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

scholarly journals Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

2015 ◽  
Vol 26 (6) ◽  
pp. 1119-1128 ◽  
Author(s):  
Bjorn D. M. Bean ◽  
Michael Davey ◽  
Jamie Snider ◽  
Matthew Jessulat ◽  
Viktor Deineko ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
New Yeast ◽  
Phosphatidylinositol 3

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.

scholarly journals An Arf/Rab cascade controls the growth and invasiveness of glioblastoma

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Gopinath Kulasekaran ◽  
Mathilde Chaineau ◽  
Valerio Emilio Crescenzo Piscopo ◽  
Federica Verginelli ◽  
Maryam Fotouhi ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Growth Factor Receptor ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Tumor Initiating Cells ◽  
Human Brain Tumor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Brain Tumor Initiating Cells

Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.

scholarly journals A Vps21 endocytic module regulates autophagy

2014 ◽  
Vol 25 (20) ◽  
pp. 3166-3177 ◽  
Author(s):  
Yong Chen ◽  
Fan Zhou ◽  
Shenshen Zou ◽  
Sidney Yu ◽  
Shaoshan Li ◽  
...  
Keyword(s):  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Downstream Effectors ◽  
Fluorescence And Electron Microscopy ◽  
Late Endosomes ◽  
Cellular Components ◽  
Mutant Cells ◽  
Additional Role

In autophagy, the double-membrane autophagosome delivers cellular components for their degradation in the lysosome. The conserved Ypt/Rab GTPases regulate all cellular trafficking pathways, including autophagy. These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors. Rab7 and its yeast homologue, Ypt7, in the context of such a module, regulate the fusion of both late endosomes and autophagosomes with the lysosome. In yeast, the Rab5-related Vps21 is known for its role in early- to late-endosome transport. Here we show an additional role for Vps21 in autophagy. First, vps21∆ mutant cells are defective in selective and nonselective autophagy. Second, fluorescence and electron microscopy analyses show that vps21∆ mutant cells accumulate clusters of autophagosomal structures outside the vacuole. Third, cells with mutations in other members of the endocytic Vps21 module, including the GEF Vps9 and factors that function downstream of Vps21, Vac1, CORVET, Pep12, and Vps45, are also defective in autophagy and accumulate clusters of autophagosomes. Finally, Vps21 localizes to PAS. We propose that the endocytic Vps21 module also regulates autophagy. These findings support the idea that the two pathways leading to the lysosome—endocytosis and autophagy—converge through the Vps21 and Ypt7 GTPase modules.

scholarly journals PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

2019 ◽  
Author(s):  
Sophie Vieweg ◽  
Katie Mulholland ◽  
Bastian Bräuning ◽  
Nitin Kachariya ◽  
Yu-Chiang Lai ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Therapeutic Potential ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Lrrk2 Kinase ◽  
Genetic Code Expansion

AbstractLoss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

scholarly journals An Arf/Rab cascade controls the growth and invasiveness of glioblastoma

2020 ◽  
Author(s):  
Gopinath Kulasekaran ◽  
Mathilde Chaineau ◽  
Valerio E. Piscopo ◽  
Federica Verginelli ◽  
Maryam Fotouhi ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Growth Factor Receptor ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Tumor Initiating Cells ◽  
Human Brain Tumor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Brain Tumor Initiating Cells

AbstractGlioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brain. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn and allosterically enhances its GEF activity towards Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals upregulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.

Identification and characterisation of novel Mss4-binding Rab GTPases

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Viktor Wixler ◽  
Ludmilla Wixler ◽  
Anika Altenfeld ◽  
Stephan Ludwig ◽  
Roger S. Goody ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mutational Analysis ◽  
Binding Capacity ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Binding Studies ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.

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