scholarly journals Rabin8 regulates neurite outgrowth in both GEF activity–dependent and –independent manners

2016 ◽  
Vol 27 (13) ◽  
pp. 2107-2118 ◽  
Author(s):  
Yuta Homma ◽  
Mitsunori Fukuda
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Membrane Trafficking ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Recycling Endosomes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Activity Dependent

Many aspects of membrane-trafficking events are regulated by Rab-family small GTPases. Neurite outgrowth requires massive addition of proteins and lipids to the tips of growing neurites by membrane trafficking, and although several Rabs, including Rab8, Rab10, and Rab11, have been implicated in this process, their regulatory mechanisms during neurite outgrowth are poorly understood. Here, we show that Rabin8, a Rab8-guanine nucleotide exchange factor (GEF), regulates nerve growth factor (NGF)–induced neurite outgrowth of PC12 cells. Knockdown of Rabin8 results in inhibition of neurite outgrowth, whereas overexpression promotes it. We also find that Rab10 is a novel substrate of Rabin8 and that both Rab8 and Rab10 function during neurite outgrowth downstream of Rabin8. Surprisingly, however, a GEF activity–deficient isoform of Rabin8 also promotes neurite outgrowth, indicating the existence of a GEF activity–independent role of Rabin8. The Arf6/Rab8-positive recycling endosomes (Arf6/Rab8-REs) and Rab10/Rab11-positive REs (Rab10/Rab11-REs) in NGF-stimulated PC12 cells are differently distributed. Rabin8 localizes on both RE populations and appears to activate Rab8 and Rab10 there. These localizations and functions of Rabin8 are Rab11 dependent. Thus Rabin8 regulates neurite outgrowth both by coordinating with Rab8, Rab10, and Rab11 and by a GEF activity–independent mechanism.

scholarly journals ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment

2015 ◽  
Vol 26 (23) ◽  
pp. 4265-4279 ◽  
Author(s):  
Joseph C. Salem ◽  
Marta M. Reviriego-Mendoza ◽  
Lorraine C. Santy
Keyword(s):  
Adhesion Formation ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Recycling Endosomes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Migration ◽  
Regulated Trafficking ◽  
Migration Response

When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small the GTPase R-Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of α5-integrin and R-Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras–dependent effects on spreading and adhesion formation.

scholarly journals The role of Sec3p in secretory vesicle targeting and exocyst complex assembly

2014 ◽  
Vol 25 (23) ◽  
pp. 3813-3822 ◽  
Author(s):  
Guangzuo Luo ◽  
Jian Zhang ◽  
Wei Guo
Keyword(s):  
Membrane Trafficking ◽  
Secretory Vesicle ◽  
The Other ◽  
Rab Gtpase ◽  
Secretory Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Exocyst Complex ◽  
Nucleotide Exchange Factor

During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.

scholarly journals Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling

2014 ◽  
Vol 112 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Juanfei Wang ◽  
Jinqi Ren ◽  
Bin Wu ◽  
Shanshan Feng ◽  
Guoping Cai ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Egf Signaling

Exocytosis is tightly regulated in many cellular processes, from neurite expansion to tumor proliferation. Rab8, a member of the Rab family of small GTPases, plays an important role in membrane trafficking from the trans-Golgi network and recycling endosomes to the plasma membrane. Rabin8 is a guanine nucleotide exchange factor (GEF) and major activator of Rab8. Investigating how Rabin8 is activated in cells is thus pivotal to the understanding of the regulation of exocytosis. Here we show that phosphorylation serves as an important mechanism for Rabin8 activation. We identified Rabin8 as a direct phospho-substrate of ERK1/2 in response to EGF signaling. At the molecular level, ERK phosphorylation relieves the autoinhibition of Rabin8, thus promoting its GEF activity. We further demonstrate that blocking ERK1/2-mediated phosphorylation of Rabin8 inhibits transferrin recycling to the plasma membrane. Together, our results suggest that ERK1/2 activate Rabin8 to regulate vesicular trafficking to the plasma membrane in response to extracellular signaling.

scholarly journals Role of guanine-nucleotide exchange factor Epac in renal physiology and pathophysiology

2013 ◽  
Vol 304 (7) ◽  
pp. F831-F839 ◽  
Author(s):  
Shi-kun Yang ◽  
Li Xiao ◽  
Jun Li ◽  
Fuyou Liu ◽  
Lin Sun ◽  
...  
Keyword(s):  
Kidney Injury ◽  
Small Gtpases ◽  
Renal Physiology ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Nucleotide Exchange Factor ◽  
Renal Tubular

Exchange proteins directly activated by cAMP [Epac(s)] were discovered more than a decade ago as new sensors for the second messenger cAMP. The Epac family members, including Epac1 and Epac2, are guanine nucleotide exchange factors for the Ras-like small GTPases Rap1 and Rap2, and they function independently of protein kinase A. Given the importance of cAMP in kidney homeostasis, several molecular and cellular studies using specific Epac agonists have analyzed the role and regulation of Epac proteins in renal physiology and pathophysiology. The specificity of the functions of Epac proteins may depend upon their expression and localization in the kidney as well as their abundance in the microcellular environment. This review discusses recent literature data concerning the involvement of Epac in renal tubular transport physiology and renal glomerular cells where various signaling pathways are known to be operative. In addition, the potential role of Epac in kidney disorders, such as diabetic kidney disease and ischemic kidney injury, is discussed.

scholarly journals The DHR1 Domain of DOCK180 Binds to SNX5 and Regulates Cation-independent Mannose 6-phosphate Receptor Transport

2008 ◽  
Vol 19 (9) ◽  
pp. 3823-3835 ◽  
Author(s):  
Shigeo Hara ◽  
Etsuko Kiyokawa ◽  
Shun-ichiro Iemura ◽  
Tohru Natsume ◽  
Thomas Wassmer ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Retrograde Transport ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Sorting Nexins ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Mannose 6 Phosphate

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

scholarly journals The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

1997 ◽  
Vol 139 (3) ◽  
pp. 797-807 ◽  
Author(s):  
Frank N. van Leeuwen ◽  
Hendrie E.T. Kain ◽  
Rob A. van der Kammen ◽  
Frits Michiels ◽  
Onno W. Kranenburg ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Neuronal Morphology ◽  
Laminin Receptor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Neurite Formation

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.

Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere

2002 ◽  
Vol 115 (3) ◽  
pp. 629-640 ◽  
Author(s):  
Michel Souchet ◽  
Elodie Portales-Casamar ◽  
David Mazurais ◽  
Susanne Schmidt ◽  
Isabelle Léger ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Fiber Formation ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Intact Cells ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The Rho small GTPases are crucial proteins involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. It has been reported that these GTPases are directly associated with cardiovascular disorders. In this context, we have searched for novel modulators of Rho GTPases, and here we describe p63RhoGEF a new Db1-like guanine nucleotide exchange factor (GEF). P63RhoGEF encodes a 63 kDa protein containing a Db1 homology domain in tandem with a pleckstrin homology domain and is most closely related to the second Rho GEF domain of Trio. Northern blot and in situ analysis have shown that p63RhoGEF is mainly expressed in heart and brain. In vitro guanine nucleotide exchange assays have shown that p63RhoGEF specifically acts on RhoA. Accordingly, p63RhoGEF expression induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts. Moreover, we show that p63RhoGEF activation of RhoA in intact cells is dependent on the presence of the PH domain. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry in human heart and brain tissue sections. Confocal microscopy shows that p63RhoGEF is located in the sarcomeric I-band mainly constituted of cardiac sarcomeric actin. Together, these results show that p63RhoGEF is a RhoA-specific GEF that may play a key role in actin cytoskeleton reorganization in different tissues, especially in heart cellular morphology.

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