Phosphatidic acid binds to and regulates guanine nucleotide exchange factor 8 (GEF8) activity in Arabidopsis

2017 ◽  
Vol 44 (10) ◽  
pp. 1029 ◽  
Author(s):  
Chunyan Cao ◽  
Peipei Wang ◽  
Hongdi Song ◽  
Wen Jing ◽  
Like Shen ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Small Gtpase ◽  
Bimolecular Fluorescence Complementation ◽  
Titration Calorimetry ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Synergistic Activation ◽  
Wide Range

Phosphatidic acid (PA) forms part of plant lipid metabolism and is a signalling molecule used in response to various external stresses. Guanine nucleotide exchange factors (GEFs) activate small GTPase ROPs, serving as molecular switches in a wide range of signalling pathways. However, the interaction between PA and GEFs in plants has not yet been reported. Here we show that PA bound specifically to GEF8 by using fat-Western blot and isothermal titration calorimetry assays. A C-terminal truncation of GEF8 exhibited strong PA binding, and mutation of lysines 13 and 18 in GEF8 PRONE domain caused a total loss of binding to PA. Two ROPs, ROP7 and ROP10, were identified as preferred substrates of GEF8 by pull-down and bimolecular fluorescence complementation assays. GEF8 activity towards ROP7, but not ROP10, was stimulated by PA both in vitro and in cells. Moreover, the PA- or ABA-induced activation of GEF8 was completely lost in the mutant GEF8, which did not bind to PA. Together, these findings identify a direct interconnection between PA-mediated GEFs activity and small GTPase signalling in plants and provide evidence for a synergistic activation of GEF8 by direct PA-binding to its PRONE domain.

Corrigendum to: Phosphatidic acid binds to and regulates guanine nucleotide exchange factor 8 (GEF8) activity in Arabidopsis

2021 ◽  
Vol 48 (3) ◽  
pp. 359
Author(s):  
Chunyan Cao ◽  
Peipei Wang ◽  
Hongdi Song ◽  
Wen Jing ◽  
Like Shen ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Small Gtpase ◽  
Bimolecular Fluorescence Complementation ◽  
Titration Calorimetry ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Synergistic Activation ◽  
Wide Range

Phosphatidic acid (PA) forms part of plant lipid metabolism and is a signalling molecule used in response to various external stresses. Guanine nucleotide exchange factors (GEFs) activate small GTPase ROPs, serving as molecular switches in a wide range of signalling pathways. However, the interaction between PA and GEFs in plants has not yet been reported. Here we show that PA bound specifically to GEF8 by using fat-Western blot and isothermal titration calorimetry assays. A C-terminal truncation of GEF8 exhibited strong PA binding, and mutation of lysines 13 and 18 in GEF8 PRONE domain caused a total loss of binding to PA. Two ROPs, ROP7 and ROP10, were identified as preferred substrates of GEF8 by pull-down and bimolecular fluorescence complementation assays. GEF8 activity towards ROP7, but not ROP10, was stimulated by PA both in vitro and in cells. Moreover, the PA- or ABA-induced activation of GEF8 was completely lost in the mutant GEF8, which did not bind to PA. Together, these findings identify a direct interconnection between PA-mediated GEFs activity and small GTPase signalling in plants and provide evidence for a synergistic activation of GEF8 by direct PA-binding to its PRONE domain.

scholarly journals The PDZ Domain of the Guanine Nucleotide Exchange Factor PDZGEF Directs Binding to Phosphatidic Acid during Brush Border Formation

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e98253 ◽  
Author(s):  
Sarah V. Consonni ◽  
Patricia M. Brouwer ◽  
Eleonora S. van Slobbe ◽  
Johannes L. Bos
Keyword(s):  
Phosphatidic Acid ◽  
Brush Border ◽  
Pdz Domain ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

scholarly journals Essential Role of Vav Family Guanine Nucleotide Exchange Factors in EphA Receptor-Mediated Angiogenesis

2006 ◽  
Vol 26 (13) ◽  
pp. 4830-4842 ◽  
Author(s):  
Sonja G. Hunter ◽  
Guanglei Zhuang ◽  
Dana Brantley-Sieders ◽  
Wojciech Swat ◽  
Christopher W. Cowan ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Rac1 Gtpase ◽  
Rho Family ◽  
Epha Receptor

ABSTRACT Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2−/− Vav3−/− mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.

A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions

2002 ◽  
Vol 115 (14) ◽  
pp. 2867-2879 ◽  
Author(s):  
Valérie Derrien ◽  
Carole Couillault ◽  
Michel Franco ◽  
Stéphanie Martineau ◽  
Philippe Montcourrier ◽  
...  
Keyword(s):  
Amino Acid ◽  
Coiled Coil ◽  
Terminal Region ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Sec7 Domain

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a ∼150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.

scholarly journals Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system

2002 ◽  
Vol 365 (3) ◽  
pp. 669-676 ◽  
Author(s):  
Francisco MANSILLA ◽  
Irene FRIIS ◽  
Mandana JADIDI ◽  
Karen M. NIELSEN ◽  
Brian F.C. CLARK ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Translation Elongation Factor ◽  
Yeast Two Hybrid ◽  
Translation Elongation ◽  
Guanine Nucleotide Exchange ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1Bα, eEF1Bβ and eEF1Bγ. The first two subunits possess the nucleotide-exchange activity, whereas the role of the last remains poorly defined. In mammals, two active tissue-specific isoforms of eEF1A have been identified. The reason for this pattern of differential expression is unknown. Several models on the basis of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1Bβ and eEF1Bγ subunits in vitro. Here, the human eEF1H complex is for the first time mapped using the yeast two-hybrid system, which is a powerful in vivo technique for analysing protein—protein interactions. The following complexes were observed: eEF1A1:eEF1Bα, eEF1A1:eEF1Bβ, eEF1Bβ:eEF1Bβ, eEF1Bα:eEF1Bγ, eEF1Bβ:eEF1Bγ and eEF1Bα:eEF1Bγ:eEF1Bβ, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit is a pentamer composed of two molecules of eEF1A, each interacting with either eEF1Bα or eEF1Bβ held together by eEF1Bγ. These units can dimerize via eEF1Bβ. Our model is compared with other models, and structural as well as functional aspects of the model are discussed.

Insights into K-Ras 4B regulation by post-translational lysine acetylation

2016 ◽  
Vol 397 (10) ◽  
pp. 1071-1085 ◽  
Author(s):  
Philipp Knyphausen ◽  
Franziska Lang ◽  
Linda Baldus ◽  
Antje Extra ◽  
Michael Lammers
Keyword(s):  
Genetic Code ◽  
Bound State ◽  
Molecular Switch ◽  
Mutant Protein ◽  
Lysine Acetylation ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Genetic Code Expansion

Abstract Ras is a molecular switch cycling between an active, GTP-bound and an inactive, GDP-bound state. Mutations in Ras, mostly affecting the off-switch, are found in many human tumours. Recently, it has been shown that K-Ras 4B is targeted by lysine acetylation at K104. Based on results obtained for an acetylation mimetic Ras mutant (K104Q), it was hypothesised that K104-acetylation might interfere with its oncogenicity by impairing SOS-catalysed guanine-nucleotide exchange. We prepared site-specifically K104-acetylated K-Ras 4B and the corresponding oncogenic mutant protein G12V using the genetic-code expansion concept. We found that SOS-catalysed nucleotide exchange, also of allosterically activated SOS, was neither affected by acetylation of K104 in wildtype K-Ras 4B nor in the G12V mutant, suggesting that glutamine is a poor mimetic for acetylation at this site. In vitro, the lysine-acetyltransferases CBP and p300 were able to acetylate both, wildtype and G12V K-Ras 4B. In addition to K104 we identified further acetylation sites in K-Ras 4B, including K147, within the important G5/SAK-motif. However, the intrinsic and the SOS-catalysed nucleotide exchange was not affected by K147-acetylation of K-Ras 4B. Finally, we show that Sirt2 and HDAC6 do neither deacetylate K-Ras 4B if acetylated at K104 nor if acetylated at K147 in vitro.

scholarly journals Gef1p, a New Guanine Nucleotide Exchange Factor for Cdc42p, Regulates Polarity inSchizosaccharomyces pombe

2003 ◽  
Vol 14 (1) ◽  
pp. 313-323 ◽  
Author(s):  
Pedro M. Coll ◽  
Yadira Trillo ◽  
Amagoia Ametzazurra ◽  
Pilar Perez
Keyword(s):  
Cell Morphology ◽  
Rho Gtpases ◽  
Genetic Interaction ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Schizosaccharomyces pombe cdc42+regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1+increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression ofgef1+causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1+deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1Δ scd1Δ is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1+orscd1+causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.

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