Guanine Nucleotide Exchange Factor Domain Research Articles

Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes

Rab GTPases and ubiquitination are critical regulators of transmembrane cargo sorting in endocytic and lysosomal targeting pathways. The endosomal protein Rabex-5 intersects these two layers of regulation by being both a guanine nucleotide exchange factor (GEF) for Rab5 and a substrate for ubiquitin (Ub) binding and conjugation. The ability of trafficking machinery components to bind ubiquitinated proteins is known to have a function in cargo sorting. Here, we demonstrate that Ub binding is essential for the recruitment of Rabex-5 from the cytosol to endosomes, independently of its GEF activity and of Rab5. We also show that monoubiquitinated Rabex-5 is enriched in the cytosol. These observations are consistent with a model whereby a cycle of Ub binding and monoubiquitination regulates the association of Rabex-5 with endosomes.

Crystal structure of the Sec4p:Sec2p complex in the nucleotide exchanging intermediate state

Vesicular transport during exocytosis is regulated by Rab GTPase (Sec4p in yeast), which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. Here, we report the crystal structure of the Sec2p GEF domain in a complex with the nucleotide-free Sec4p at 2.7 Å resolution. Upon complex formation, the Sec2p helices approach each other, flipping the side chain of Phe-109 toward Leu-104 and Leu-108 of Sec2p. These three residues provide a hydrophobic platform to attract the side chains of Phe-49, Ile-53, and Ile-55 in the switch I region as well as Phe-57 and Trp-74 in the interswitch region of Sec4p. Consequently, the switch I and II regions are largely deformed, to create a flat hydrophobic interface that snugly fits the surface of the Sec2p coiled coil. These drastic conformational changes disrupt the interactions between switch I and the bound guanine nucleotide, which facilitates the GDP release. Unlike the recently reported 3.3 Å structure of the Sec4p·Sec2p complex, our structure contains a phosphate ion bound to the P-loop, which may represent an intermediate state of the nucleotide exchange reaction.

Regulation of Kalirin by Cdk5

Kalirin, one of the few Rho guanine nucleotide exchange factors (GEFs) that contains spectrin-like repeats, plays a critical role in axon extension and maintenance of dendritic spines. PC12 cells were used to determine whether Cdk5, a critical participant in both processes, regulates the action of Kalirin. Expression of Kalirin-7 in nondifferentiated PC12 cells caused GEF-activity-dependent extension of broad cytoplasmic protrusions; coexpression of dominant-negative Cdk5 largely eliminated this response. The spectrin-like repeat region of Kalirin plays an essential role in this response, which is not mimicked by the GEF domain alone. Thr1590, which follows the first GEF domain of Kalirin, is the only Cdk5 phosphorylation site in Kalirin-7. Although mutant Kalirin-7 with Ala1590 retains GEF activity, it is unable to cause extension of protrusions. Kalirin-7 with an Asp1590 mutation has slightly increased GEF activity and dominant-negative Cdk5 fails to block its ability to cause extension of protrusions. Phosphorylation of Thr1590 causes a slight increase in GEF activity and Kalirin-7 solubility. Dendritic spines formed by cortical neurons in response to the expression of Kalirin-7 with Ala1590 differ in shape from those formed in response to wild-type Kalirin-7 or Kalirin-7 containing Asp1590. The presence of Thr1590 in each major Kalirin isoform would allow Cdk5 to regulate Kalirin function throughout development.

Linking Ras to myosin function: RasGEF Q, aDictyosteliumexchange factor for RasB, affects myosin II functions

Ras guanine nucleotide exchange factor (GEF) Q, a nucleotide exchange factor from Dictyostelium discoideum, is a 143-kD protein containing RasGEF domains and a DEP domain. We show that RasGEF Q can bind to F-actin, has the potential to form complexes with myosin heavy chain kinase (MHCK) A that contain active RasB, and is the predominant exchange factor for RasB. Overexpression of the RasGEF Q GEF domain activates RasB, causes enhanced recruitment of MHCK A to the cortex, and leads to cytokinesis defects in suspension, phenocopying cells expressing constitutively active RasB, and myosin-null mutants. RasGEF Q− mutants have defects in cell sorting and slug migration during later stages of development, in addition to cell polarity defects. Furthermore, RasGEF Q− mutants have increased levels of unphosphorylated myosin II, resulting in myosin II overassembly. Collectively, our results suggest that starvation signals through RasGEF Q to activate RasB, which then regulates processes requiring myosin II.

RanBP10 Is a Cytoplasmic Guanine Nucleotide Exchange Factor That Modulates Noncentrosomal Microtubules

Microtubule spindle assembly in mitosis is stimulated by Ran.GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including megakaryocytes. In these cells, platelet biogenesis requires massive mobilization of microtubules in the cell periphery, where they form proplatelets, the immediate precursors of platelets, in the apparent absence of centrioles. Here we identify a cytoplasmic Ran-binding protein, RanBP10, as a factor that binds beta-tubulin and associates with megakaryocyte microtubules. Unexpectedly, RanBP10 harbors GEF activity toward Ran. A point mutation in the candidate GEF domain abolishes exchange activity, and our results implicate RanBP10 as a localized cytoplasmic Ran-GEF. RNA interference-mediated loss of RanBP10 in cultured megakaryocytes disrupts microtubule organization. These results lead us to propose that spatiotemporally restricted generation of cytoplasmic Ran.GTP may influence organization of the specialized microtubules required in thrombopoiesis and that RanBP10 might serve as a molecular link between Ran and noncentrosomal microtubules.

Function of the N-terminus of zizimin1: autoinhibition and membrane targeting

Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.

EPAC regulation of cardiac EC coupling

EPAC regulation of cardiac EC coupling

Trio’s Rho-specific GEF domain is the missing Gαq effector in C. elegans

The Galpha(q) pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cbeta [PLCbeta]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Galpha(q) pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCbeta). Strains containing null mutations in both EGL-8 (PLCbeta) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Galpha(q)-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Galpha(q) synergizes with Trio RhoGEF to activate RhoA. Activated Galpha(q) and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio's Rho-specific GEF domain is a major Galpha(q) effector that, together with PLCbeta, mediates the Galpha(q) signaling that drives the locomotion, egg laying, and growth of the animal.

The RhoGEF domain of p210 Bcr-Abl activates RhoA and is required for transformation

The BCR-ABL oncogene encodes an in-frame fusion protein containing N-terminal sequences derived from Bcr and C-terminal sequences derived from Abl. Bcr contains a centrally located Rho-specific guanine nucleotide exchange factor (RhoGEF) domain that is retained within p210 Bcr-Abl. Although this domain is subject to autoinhibition in the context of Bcr, here we show that it is constitutively activated in p210 Bcr-Abl. p210 Bcr-Abl can stimulate RhoA activation independently of its tyrosine kinase activity, and mutations within the RhoGEF domain that are predicted to eliminate RhoGEF activity inhibit RhoA activation. The RhoGEF mutant of p210 Bcr-Abl does not affect the tyrosine kinase activity of the molecule, nor the ability of p210 Bcr-Abl to interact with XPB through the RhoGEF domain. Despite retaining normal levels of tyrosine kinase activity, the RhoGEF mutant of p210 Bcr-Abl is impaired in transforming activity as measured by anchorage-independent growth. However, the mutant is still able to confer the phenotype of growth factor independence in myeloid cells, suggesting that some, but not all parameters of p210 Bcr-Abl transformation, are dependent upon a catalytically active RhoGEF domain. Collectively, these observations identify a gain-of-function activity attributable to the RhoGEF domain of p210 Bcr-Abl that is required to support the transformed phenotype.

Kalirin/Trio Rho Guanine Nucleotide Exchange Factors Regulate a Novel Step in Secretory Granule Maturation

The molecular mechanisms involved in the maturation of secretory granules, organelles that store hormones and neuropeptides, are poorly understood. As granule content proteins are processed, the composition of granule membranes changes, yielding constitutive-like secretion of immature content proteins and producing secretagogue-responsive mature granules. Constitutive-like secretion was not previously recognized as a process subject to regulation. We show that Kalirin and Trio, homologous Rho guanine nucleotide exchange factors (GEFs), which interact with a secretory granule resident protein, modulate cargo secretion from immature granules. Some of the Kalirin and Trio isoforms expressed in neuroendocrine cells colocalize with immature granules. Overexpression of their N-terminal GEF domain (GEF1) enhances secretion from immature granules, depleting cells of secretory cargo in the absence of secretagogue. This response requires GEF1 activity and is mimicked by Kalirin/Trio substrates Rac1 and RhoG. Accordingly, selective pharmacological inhibition of endogenous GEF1 activity decreases secretagogue-independent release of hormone precursors, accumulating product peptide in mature secretory granules. Kalirin/Trio modulation of cargo secretion from immature granules provides secretory cells with an extra layer of control over the sets of peptides released. Control of this step enhances the range of physiological responses that can be elicited, whereas lack of control could have pathological consequences.

Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.

Critical Contacts between the Eukaryotic Initiation Factor 2B (eIF2B) Catalytic Domain and both eIF2β and -2γ Mediate Guanine Nucleotide Exchange

Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bepsilon C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15 degrees C. The mutation of W699, found on a separate surface approximately 40 A from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2beta, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2gamma. These data show that multiple contacts between eIF2gamma and eIF2Bepsilon mediate nucleotide exchange.

Crystal structure of the Sec4p.Sec2p complex in the nucleotide exchanging intermediate state.

Vesicular transport during exocytosis is regulated by Rab GTPase (Sec4p in yeast), which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. Here, we report the crystal structure of the Sec2p GEF domain in a complex with the nucleotide-free Sec4p at 2.7 A resolution. Upon complex formation, the Sec2p helices approach each other, flipping the side chain of Phe-109 toward Leu-104 and Leu-108 of Sec2p. These three residues provide a hydrophobic platform to attract the side chains of Phe-49, Ile-53, and Ile-55 in the switch I region as well as Phe-57 and Trp-74 in the interswitch region of Sec4p. Consequently, the switch I and II regions are largely deformed, to create a flat hydrophobic interface that snugly fits the surface of the Sec2p coiled coil. These drastic conformational changes disrupt the interactions between switch I and the bound guanine nucleotide, which facilitates the GDP release. Unlike the recently reported 3.3 A structure of the Sec4p.Sec2p complex, our structure contains a phosphate ion bound to the P-loop, which may represent an intermediate state of the nucleotide exchange reaction.

Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors

Eukaryotic cells mobilize the actin cytoskeleton to generate a remarkable diversity of morphological behaviours, including motility, phagocytosis and cytokinesis. Much of this diversity is mediated by guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases-the master regulators of the actin cytoskeleton. There are over 80 Rho GEFs in the human genome (compared to only 22 genes for the Rho GTPases themselves), and the evolution of new and diverse GEFs is thought to provide a mechanism for linking the core cytoskeletal machinery to a wide range of new control inputs. Here we test this hypothesis and ask if we can systematically reprogramme cellular morphology by engineering synthetic GEF proteins. We focused on Dbl family Rho GEFs, which have a highly modular structure common to many signalling proteins: they contain a catalytic Dbl homology (DH) domain linked to diverse regulatory domains, many of which autoinhibit GEF activity. Here we show that by recombining catalytic GEF domains with new regulatory modules, we can generate synthetic GEFs that are activated by non-native inputs. We have used these synthetic GEFs to reprogramme cellular behaviour in diverse ways. The GEFs can be used to link specific cytoskeletal responses to normally unrelated upstream signalling pathways. In addition, multiple synthetic GEFs can be linked as components in series to form an artificial cascade with improved signal processing behaviour. These results show the high degree of evolutionary plasticity of this important family of modular signalling proteins, and indicate that it may be possible to use synthetic biology approaches to manipulate the complex spatio-temporal control of cell morphology.

An Activating Mutation in sos-1 Identifies Its Dbl Domain as a Critical Inhibitor of the Epidermal Growth Factor Receptor Pathway during Caenorhabditis elegans Vulval Development

Proper regulation of receptor tyrosine kinase (RTK)-Ras-mitogen-activated protein kinase (MAPK) signaling pathways is critical for normal development and the prevention of cancer. SOS is a dual-function guanine nucleotide exchange factor (GEF) that catalyzes exchange on Ras and Rac. Although the physiologic role of SOS and its CDC25 domain in RTK-mediated Ras activation is well established, the in vivo function of its Dbl Rac GEF domain is less clear. We have identified a novel gain-of-function missense mutation in the Dbl domain of Caenorhabditis elegans SOS-1 that promotes epidermal growth factor receptor (EGFR) signaling in vivo. Our data indicate that a major developmental function of the Dbl domain is to inhibit EGF-dependent MAPK activation. The amount of inhibition conferred by the Dbl domain is equal to that of established trans-acting inhibitors of the EGFR pathway, including c-Cbl and RasGAP, and more than that of MAPK phosphatase. In conjunction with molecular modeling, our data suggest that the C. elegans mutation, as well as an equivalent mutation in human SOS1, activates the MAPK pathway by disrupting an autoinhibitory function of the Dbl domain on Ras activation. Our work suggests that functionally similar point mutations in humans could directly contribute to disease.

A Catalytic Coiled Coil: Structural Insights into the Activation of the Rab GTPase Sec4p by Sec2p

Rab GTPases, the largest subgroup in the superfamily of Ras-like GTPases, play regulatory roles in multiple steps of intracellular vesicle trafficking. They are activated by guanine nucleotide exchange factors (GEFs), which catalyze the interconversion of the GDP-bound, or inactive, form of Rab to the GTP-bound, or active, form. Relatively little is known of the mechanisms by which GEFs activate Rabs. Here, we present the crystal structure of the GEF domain of Sec2p in complex with its Rab partner Sec4p. The Sec2p GEF domain is a 220 Angstroms long coiled coil, striking in its simplicity and in the use of the coiled-coil motif for catalysis. The structure suggests a mechanism whereby Sec2p induces extensive structural rearrangements in the Sec4p switch regions and phosphate-binding loop that are incompatible with nucleotide binding. We show that Sec2p is specific for Sec4p and that specificity determinants reside in the two switch regions of Sec4p.

Asymmetric Coiled-Coil Structure with Guanine Nucleotide Exchange Activity

Vesicular traffic during exocytosis is regulated by Rab GTPase, Sec4p in yeast, which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. The GEF activity is localized in the N-terminal 160 residues of Sec2p, which lacks sequence similarity with any other GEFs with known structures, and thereby the guanine nucleotide exchange mechanism by Sec2p remains unknown. Here, we report the crystal structure of the Sec2p GEF domain at 3.0 A resolution. The structure unexpectedly consists of a homodimeric, parallel coiled coil that extends over 180 A. Pull-down and guanine nucleotide exchange analyses on a series of deletion and point mutants of Sec2p unveiled the catalytic residues for its GEF activity as well as the Sec4p binding site, thus presenting a nucleotide exchange mechanism by a simple coiled coil. The present functional analyses allow us to build the Sec2p:Sec4p complex model, which explains the specificity for Rab GTPases by their respective GEF proteins.

Morphological analysis of axons of newly generated granule cells in adult mouse hippocampus

Trio is a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Trio regulates cytoskeleton dynamics and actin remodeling and is involved in cell migration and axonal guidance in neuronal development. The null allele of the Trio gene led to embryonic lethality, and Trio null embryos displayed aberrant organization in several regions of the brain at E18.5, including hippocampus. Nestin-Trio−/− mice, in which the Trio gene was deleted specifically in the neuronal system by the Nestin-Cre system, displayed severe phenotypes, including low survival rate, ataxia and multiple developmental defects of the cerebellum. All Nestin-Trio−/− mice died before reaching adulthood, which hinders research on Trio gene function in adult mice. Thus, we generated EMX1-Trio−/− mice by crossing Trio-floxed mice with EMX1-Cre mice in which Cre is expressed in the brain cortex and hippocampus. EMX1-Trio−/− mice can survive to adulthood. Trio gene deletion results in smaller brains, an abnormal hippocampus and disordered granule cells in the dentate gyrus (DG) and cornu ammonis (CA). Behavior tests showed that Trio deletion interfered with the hippocampal-dependent spatial learning in the mice, suggesting that Trio plays critical roles in the learning ability of adult mice. We conclude that the Trio gene regulates the neuronal development of the hippocampus and that it affects the intelligence of adult mice.

The Hematopoietic Isoform of Cas-Hef1-Associated Signal Transducer Regulates Chemokine-Induced Inside-Out Signaling and T Cell Trafficking

Leukocyte migration and trafficking is dynamically regulated by various chemokine and adhesion molecules and is vital to the proper function of the immune system. We describe a role for the Cas and Hef-1-associated signal transducer in hematopoietic cells (Chat-H) as a critical regulator of T lymphocyte migration, by using lentivirus-mediated RNA interference (RNAi). Impaired migration of Chat-H-depleted cells coincided with defective inside-out signaling shown by diminished chemokine-induced activation of the Rap-1 GTPase and integrin-mediated adhesion. Localization of Chat-H to the plasma membrane, association with its binding partner Crk-associated substrate in lymphocytes (CasL), and Chat-H-mediated CasL serine-threonine phosphorylation were required for T cell migration. These results identify Chat-H as a critical signaling intermediate acting upstream of Rap1 to regulate chemokine-induced adhesion and migration.

Regulation of RhoGEF Activity by Intramolecular and Intermolecular SH3 Domain Interactions

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members.