scholarly journals Multiple interactions between an Arf/GEF complex and charged lipids determine activation kinetics on the membrane

2017 ◽  
Vol 114 (43) ◽  
pp. 11416-11421 ◽  
Author(s):  
Deepti Karandur ◽  
Agata Nawrotek ◽  
John Kuriyan ◽  
Jacqueline Cherfils
Keyword(s):  
Lipid Bilayer ◽  
Membrane Trafficking ◽  
Structural Model ◽  
High Efficiency ◽  
Structural Data ◽  
Lipid Binding ◽  
Small Gtpases ◽  
Coarse Grained ◽  
Nucleotide Exchange ◽  
Ph Domain

Lipidated small GTPases and their regulators need to bind to membranes to propagate actions in the cell, but an integrated understanding of how the lipid bilayer exerts its effect has remained elusive. Here we focused on ADP ribosylation factor (Arf) GTPases, which orchestrate a variety of regulatory functions in lipid and membrane trafficking, and their activation by the guanine-nucleotide exchange factor (GEF) Brag2, which controls integrin endocytosis and cell adhesion and is impaired in cancer and developmental diseases. Biochemical and structural data are available that showed the exceptional efficiency of Arf activation by Brag2 on membranes. We determined the high-resolution crystal structure of unbound Brag2 containing the GEF (Sec7) and membrane-binding (pleckstrin homology) domains, revealing that it has a constitutively active conformation. We used this structure to analyze the interaction of uncomplexed Brag2 and of the myristoylated Arf1/Brag2 complex with a phosphatidylinositol bisphosphate (PIP2) -containing lipid bilayer, using coarse-grained molecular dynamics. These simulations revealed that the system forms a close-packed, oriented interaction with the membrane, in which multiple PIP2 lipids bind the canonical lipid-binding site and unique peripheral sites of the PH domain, the Arf GTPase and, unexpectedly, the Sec7 domain. We cross-validated these predictions by reconstituting the binding and kinetics of Arf and Brag2 in artificial membranes. Our coarse-grained structural model thus suggests that the high efficiency of Brag2 requires interaction with multiple lipids and a well-defined orientation on the membrane, resulting in a local PIP2 enrichment, which has the potential to signal toward the Arf pathway.

scholarly journals NRas slows the rate at which a model lipid bilayer phase separates

2014 ◽  
Vol 169 ◽  
pp. 209-223 ◽  
Author(s):  
Elizabeth Jefferys ◽  
Mark S. P. Sansom ◽  
Philip W. Fowler
Keyword(s):  
Lipid Bilayer ◽  
Cell Signalling ◽  
Line Tension ◽  
Structural Data ◽  
Coarse Grained ◽  
Ras Proteins ◽  
C Terminus ◽  
Ras Family ◽  
Multiple Copies ◽  
Dynamics Simulations

The Ras family of small membrane-associated GTP-ases are important components in many different cell signalling cascades. They are thought to cluster on the cell membrane through association with cholesterol-rich nanodomains. This process remains poorly understood. Here we test the effect of adding multiple copies of NRas, one of the canonical Ras proteins, to a three-component lipid bilayer that rapidly undergoes spinodal decomposition (i.e.unmixing), thereby creating ordered and disordered phases. Coarse-grained molecular dynamics simulations of a large bilayer containing 6000 lipids, with and without protein, are compared. NRas preferentially localises to the interface between the domains and slows the rate at which the domains grow. We infer that this doubly-lipidated cell signalling protein is reducing the line tension between the ordered and disordered regions. This analysis is facilitated by our use of techniques borrowed from image-processing. The conclusions above are contingent upon several assumptions, including the use of a model lipid with doubly unsaturated tails and the limited structural data available for the C-terminus of NRas, which is where the lipid anchors are found.

scholarly journals Dependence of Dbl and Dbs Transformation on MEK and NF-κB Activation

1999 ◽  
Vol 19 (11) ◽  
pp. 7759-7770 ◽  
Author(s):  
Ian P. Whitehead ◽  
Que T. Lambert ◽  
Judith A. Glaven ◽  
Karon Abe ◽  
Kent L. Rossman ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Structural Integrity ◽  
Small Gtpases ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Transforming Activity ◽  
Rho Family ◽  
Altered Gene

ABSTRACT Dbs was identified initially as a transforming protein and is a member of the Dbl family of proteins (>20 mammalian members). Here we show that Dbs, like its rat homolog Ost and the closely related Dbl, exhibited guanine nucleotide exchange activity for the Rho family members RhoA and Cdc42, but not Rac1, in vitro. Dbs transforming activity was blocked by specific inhibitors of RhoA and Cdc42 function, demonstrating the importance of these small GTPases in Dbs-mediated growth deregulation. Although Dbs transformation was dependent upon the structural integrity of its pleckstrin homology (PH) domain, replacement of the PH domain with a membrane localization signal restored transforming activity. Thus, the PH domain of Dbs (but not Dbl) may be important in modulating association with the plasma membrane, where its GTPase substrates reside. Both Dbs and Dbl activate multiple signaling pathways that include activation of the Elk-1, Jun, and NF-κB transcription factors and stimulation of transcription from the cyclin D1 promoter. We found that Elk-1 and NF-κB, but not Jun, activation was necessary for Dbl and Dbs transformation. Finally, we have observed that Dbl and Dbs regulated transcription from the cyclin D1 promoter in a NF-κB-dependent manner. Previous studies have dissociated actin cytoskeletal activity from the transforming potential of RhoA and Cdc42. These observations, when taken together with those of the present study, suggest that altered gene expression, and not actin reorganization, is the critical mediator of Dbl and Rho family protein transformation.

Centaurin-α1 and KIF13B kinesin motor protein interaction in ARF6 signalling

2005 ◽  
Vol 33 (6) ◽  
pp. 1279-1281 ◽  
Author(s):  
V. Kanamarlapudi
Keyword(s):  
Membrane Trafficking ◽  
Motor Protein ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Kinesin Motor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Gtp Binding ◽  
Cytoskeletal Dynamics

The ARF (ADP-ribosylation factor) family of small GTPases regulate intracellular membrane trafficking by cycling between an inactive GDP- and an active GTP-bound form. Among the six known mammalian ARFs (ARF1–ARF6), ARF6 is the least conserved and plays critical roles in membrane trafficking and cytoskeletal dynamics near the cell surface. Since ARFs have undetectable levels of intrinsic GTP binding and hydrolysis, they are totally dependent on extrinsic GEFs (guanine nucleotide-exchange factors) for GTP binding and GAPs (GTPase-activating proteins) for GTP hydrolysis. We have recently isolated a novel KIF (kinesin) motor protein (KIF13B) that binds to centaurin-α1, an ARF6GAP that binds to the second messenger PIP3 [PtdIns(3,4,5)P3]. KIFs transport intracellular vesicles and recognize their cargo by binding to proteins (receptors) localized on the surface of the cargo vesicles. Identification of centaurin-α1 as a KIF13B interactor suggests that KIF13B may transport ARF6 and/or PIP3 using centaurin-α1 as its receptor. This paper reviews the studies carried out to assess the interaction and regulation of centaurin-α1 by KIF13B.

FYVE zinc-finger proteins in the plant model Arabidopsis thaliana: identification of PtdIns3P-binding residues by comparison of classic and variant FYVE domains

2001 ◽  
Vol 359 (1) ◽  
pp. 165-173 ◽  
Author(s):  
Rikke B. JENSEN ◽  
Tanja LACOUR ◽  
Jakob ALBRETHSEN ◽  
Michael NIELSEN ◽  
Karen SKRIVER
Keyword(s):  
Arabidopsis Thaliana ◽  
Zinc Finger ◽  
Consensus Sequence ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Dot Blot ◽  
Fyve Domain ◽  
Guanine Nucleotide Exchange

Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)1(R/K)HHCR6 (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins, named PRAF, consist of a pleckstrin homology (PH) domain, a regulator of chromosome condensation (RCC1) guanine nucleotide exchange factor repeat domain, and a variant FYVE domain containing an Asn residue and a Tyr residue at positions corresponding to the PtdIns3P-interacting His4 and Arg6 of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P2, the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast, specificity for PtdIns3P was obtained by mutagenic conversion of the variant into a classic FYVE domain (Asn4,Tyr6 → His4,Arg6). Separate substitutions of the variant residues were not sufficient to impose preferential binding of PtdIns3P, suggesting a co-operative effect of these residues in binding. A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides.

scholarly journals Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma

2021 ◽  
Author(s):  
Ryan J Boyd ◽  
Tien Olson ◽  
James Zook ◽  
Manuel Aceves ◽  
Derek Stein ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Computational Simulation ◽  
B Cell Lymphoma ◽  
Cd Spectroscopy ◽  
Small Gtpases ◽  
Size Exclusion ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Expression And Purification ◽  
Protein Expression And Purification

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein-protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co-elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic - Diffuse B-cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

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 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 Ubiquitination-Dependent Regulation of Small GTPases in Membrane Trafficking: From Cell Biology to Human Diseases

2021 ◽  
Vol 9 ◽  
Author(s):  
Zehui Lei ◽  
Jing Wang ◽  
Lingqiang Zhang ◽  
Cui Hua Liu
Keyword(s):  
Membrane Trafficking ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Neurological Diseases ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Human Diseases ◽  
Nucleotide Exchange ◽  
Temporal Regulation ◽  
Spatio Temporal

Membrane trafficking is critical for cellular homeostasis, which is mainly carried out by small GTPases, a class of proteins functioning in vesicle budding, transport, tethering and fusion processes. The accurate and organized membrane trafficking relies on the proper regulation of small GTPases, which involves the conversion between GTP- and GDP-bound small GTPases mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Emerging evidence indicates that post-translational modifications (PTMs) of small GTPases, especially ubiquitination, play an important role in the spatio-temporal regulation of small GTPases, and the dysregulation of small GTPase ubiquitination can result in multiple human diseases. In this review, we introduce small GTPases-mediated membrane trafficking pathways and the biological processes of ubiquitination-dependent regulation of small GTPases, including the regulation of small GTPase stability, activity and localization. We then discuss the dysregulation of small GTPase ubiquitination and the associated human membrane trafficking-related diseases, focusing on the neurological diseases and infections. An in-depth understanding of the molecular mechanisms by which ubiquitination regulates small GTPases can provide novel insights into the membrane trafficking process, which knowledge is valuable for the development of more effective and specific therapeutics for membrane trafficking-related human diseases.

scholarly journals Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides

2001 ◽  
Vol 154 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Jun Xu ◽  
Dan Liu ◽  
Gordon Gill ◽  
Zhou Songyang
Keyword(s):  
Membrane Trafficking ◽  
Direct Interaction ◽  
Lipid Binding ◽  
Ph Domain ◽  
Growth And Survival ◽  
Binding Domains ◽  
Px Domain ◽  
And Function ◽  
Phox Homology

PKB/Akt and serum and glucocorticoid–regulated kinase (SGK) family kinases are important downstream targets of phosphatidylinositol 3 (PI-3) kinase and have been shown to mediate a variety of cellular processes, including cell growth and survival. Although regulation of Akt can be achieved through several mechanisms, including its phosphoinositide-binding Pleckstrin homology (PH) domain, how SGK kinases are targeted and regulated remains to be elucidated. Unlike Akt, cytokine-independent survival kinase (CISK)/SGK3 contains a Phox homology (PX) domain. PX domains have been implicated in several cellular events involving membrane trafficking. However, their precise function remains unknown. We demonstrate here that the PX domain of CISK interacts with phosphatidylinositol (PtdIns)(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2. The CISK PX domain is required for targeting CISK to the endosomal compartment. Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo. These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides. Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

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