scholarly journals Interactions between Cdc42 and the scaffold protein Scd2: requirement of SH3 domains for GTPase binding

2005 ◽  
Vol 388 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Edward WHEATLEY ◽  
Katrin RITTINGER
Keyword(s):  
Binding Sites ◽  
Sh3 Domain ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Titration Calorimetry ◽  
Nucleotide Exchange ◽  
Sh3 Domains ◽  
Binding Surface ◽  
P21 Activated Kinase ◽  
Conventional Manner

The multi-domain protein Scd2 acts as a scaffold upon which the small GTPase Cdc42 (cell division cycle 42), its nucleotide-exchange factor Scd1 and the p21-activated kinase Shk1 assemble to regulate cell polarity and the mating response in fission yeast. In the present study, we show using isothermal titration calorimetry that Scd2 binds two molecules of active GTP-bound Cdc42 simultaneously, but independently of one another. The two binding sites have significantly different affinities, 21 nM and 3 μM, suggesting that they play distinct roles in the Shk1 signalling network. Each of the Cdc42-binding sites includes one of the SH3 (Src homology 3) domains of Scd2. Our data indicate that complex formation does not occur in a conventional manner via the conserved SH3 domain ligand-binding surface. Neither of the isolated SH3 domains is sufficient to interact with the GTPase, and they both require adjacent regions to either stabilize their conformations or contribute to the formation of the Cdc42-binding surface. Furthermore, we show that there is no evidence for an intramolecular PX–SH3 domain interaction, which could interfere with SH3 domain function. This work suggests that SH3 domains might contribute directly to signalling through small GTPases and thereby adds another aspect to the diverse nature of SH3 domains as protein–protein-interaction modules.

Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs

2017 ◽  
Vol 398 (5-6) ◽  
pp. 523-533 ◽  
Author(s):  
Klaus Gerwert ◽  
Daniel Mann ◽  
Carsten Kötting
Keyword(s):  
G Protein ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleophilic Attack ◽  
Protein Signaling ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Molecular Reaction ◽  
External Signals

Abstract GTPases are central switches in cells. Their dysfunctions are involved in severe diseases. The small GTPase Ras regulates cell growth, differentiation and apoptosis by transmitting external signals to the nucleus. In one group of oncogenic mutations, the ‘switch-off’ reaction is inhibited, leading to persistent activation of the signaling pathway. The switch reaction is regulated by GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis in Ras, and by guanine nucleotide exchange factors, which catalyze the exchange of GDP for GTP. Heterotrimeric G-proteins are activated by G-protein coupled receptors and are inactivated by GTP hydrolysis in the Gα subunit. Their GAPs are called regulators of G-protein signaling. In the same way that Ras serves as a prototype for small GTPases, Gαi1 is the most well-studied Gα subunit. By utilizing X-ray structural models, time-resolved infrared-difference spectroscopy, and biomolecular simulations, we elucidated the detailed molecular reaction mechanism of the GTP hydrolysis in Ras and Gαi1. In both proteins, the charge distribution of GTP is driven towards the transition state, and an arginine is precisely positioned to facilitate nucleophilic attack of water. In addition to these mechanistic details of GTP hydrolysis, Ras dimerization as an emerging factor in signal transduction is discussed in this review.

scholarly journals Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

2009 ◽  
Vol 30 (3) ◽  
pp. 829-844 ◽  
Author(s):  
Jeffrey M. Robens ◽  
Lee Yeow-Fong ◽  
Elsa Ng ◽  
Christine Hall ◽  
Ed Manser
Keyword(s):  
Binding Sites ◽  
Sh3 Domain ◽  
Domain Swapping ◽  
Regulatory Proteins ◽  
Effector Protein ◽  
Membrane Protrusion ◽  
Sh3 Domains ◽  
Bar Domain ◽  
Actin Regulatory Proteins ◽  
Dynamic Structures

ABSTRACT Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.

scholarly journals Essential Role of the NH2-Terminal Region of Cdc24 Guanine Nucleotide Exchange Factor in Its Initial Polarized Localization in Saccharomyces cerevisiae

2011 ◽  
Vol 11 (1) ◽  
pp. 2-15 ◽  
Author(s):  
Konomi Fujimura-Kamada ◽  
Tomoe Hirai ◽  
Kazuma Tanaka
Keyword(s):  
Fluorescent Protein ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Terminal Region ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Mutant Proteins ◽  
Pb1 Domain ◽  
P21 Activated Kinase ◽  
Green Fluorescent

ABSTRACTThe cortical recruitment and accumulation of the small GTPase Cdc42 are crucial steps in the establishment of polarity, but this process remains obscure. Cdc24 is an upstream regulator of budding yeast Cdc42 that accelerates the exchange of GDP for GTP in Cdc42 via its Dbl homology (DH) domain. Here, we isolated five novel temperature-sensitive (ts)cdc24mutants, the green fluorescent protein (GFP)-fused proteins of which lose their polarized localization at the nonpermissive temperature. All amino acid substitutions in the mutants were mapped to the NH2-terminal region of Cdc24, including the calponin homology (CH) domain. These Cdc24-ts mutant proteins did not interact with Bem1 at the COOH-terminal PB1 domain, suggesting a lack of exposure of the PB1 domain in the mutant proteins. Thecdc24-tsmutants were also defective in polarization in the absence of Bem1. It was previously reported that a fusion protein containing Cdc24 and the p21-activated kinase (PAK)-like kinase Cla4 could bypass the requirement for Bem1 in polarity cue-independent budding (i.e., symmetry breaking). Cdc24-ts–Cla4 fusion proteins also showed ts localization at the polarity site. We propose that the NH2-terminal region unmasks the DH and PB1 domains, leading to the activation of Cdc42 and interaction with Bem1, respectively, to initiate cell polarization.

scholarly journals Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.

1995 ◽  
Vol 15 (10) ◽  
pp. 5627-5634 ◽  
Author(s):  
Z Weng ◽  
R J Rickles ◽  
S Feng ◽  
S Richard ◽  
A S Shaw ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Binding Proteins ◽  
Hydrophobic Interactions ◽  
Function Analysis ◽  
Binding Specificity ◽  
Sh3 Domain ◽  
Single Amino Acid ◽  
Sh3 Domains ◽  
Cell Extracts

SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.

scholarly journals Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases in parallel

2019 ◽  
Author(s):  
Ryan C. Killoran ◽  
Matthew J. Smith
Keyword(s):  
Binding Pocket ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Selective Labelling ◽  
Activation Potential ◽  
Single Complex ◽  
Gtpase Activation ◽  
Ras Isoforms

AbstractSmall GTPase proteins alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly three RAS isoforms HRAS, KRAS and NRAS, and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is evidence of direct biochemical crosstalk between the functional G-domains of these proteins. The activation potential of a given GTPase is contingent on a co-dependent interaction with nucleotide and a Mg2+ ion, which bind to individual variants via distinct affinities coordinated by residues in the nucleotide binding pocket. Here, we utilize a selective-labelling strategy coupled with real-time nuclear magnetic resonance (NMR) spectroscopy to monitor nucleotide exchange, GTP hydrolysis and effector interactions of multiple small GTPases in a single complex system. We provide new insight on nucleotide preference and the role of Mg2+ in activating both wild-type and oncogenic mutant enzymes. Multiplexing reveals GEF, GAP and effector binding specificity in mixtures of GTPases and establishes the complete biochemical equivalence of the three related RAS isoforms. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately resolve GTPase activation potential, as GTPases such as RALA or the G12C mutant of KRAS demonstrate fast exchange kinetics but have a high affinity for GDP. Further, we propose that the G-domains of small GTPases behave autonomously in solution and nucleotide cycling proceeds independent of protein concentration but is highly impacted by Mg2+ abundance.

scholarly journals A non-linear system patterns Rab5 GTPase on the membrane

2019 ◽  
Author(s):  
A Cezanne ◽  
J Lauer ◽  
A Solomatina ◽  
IF Sbalzarini ◽  
M Zerial
Keyword(s):  
Lipid Bilayers ◽  
Lipid Membrane ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Membrane Composition ◽  
Dynamic Interactions ◽  
Universal System ◽  
Non Linear ◽  
Non Linear System

AbstractProteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effector binding, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers as a model system for membrane patterning. We show that there is a “handover” of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Surprisingly, a lipid membrane composition mimicking that of the early endosome was required for Rab5 patterning. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.

scholarly journals Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification

2021 ◽  
Vol 8 ◽  
Author(s):  
Natsuki Osaka ◽  
Yoshihisa Hirota ◽  
Doshun Ito ◽  
Yoshiki Ikeda ◽  
Ryo Kamata ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Guanine Nucleotide Exchange ◽  
Ras Superfamily ◽  
Switch Regions

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound “OFF” state and GTP-bound “ON” state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound “ON” state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

scholarly journals A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

1996 ◽  
Vol 16 (2) ◽  
pp. 548-556 ◽  
Author(s):  
N L Freeman ◽  
T Lila ◽  
K A Mintzer ◽  
Z Chen ◽  
A J Pahk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Adenylyl Cyclase ◽  
Sh3 Domain ◽  
Regulatory Function ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Rich Region ◽  
Sh3 Domains ◽  
Proline Rich Region

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

scholarly journals The Small GTPases in Fungal Signaling Conservation and Function

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1039
Author(s):  
Mitzuko Dautt-Castro ◽  
Montserrat Rosendo-Vargas ◽  
Sergio Casas-Flores
Keyword(s):  
General Rule ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Plant Interactions ◽  
New Family ◽  
Ras Gtpases ◽  
Guanine Nucleotide Exchange ◽  
Small Proteins ◽  
And Function

Monomeric GTPases, which belong to the Ras superfamily, are small proteins involved in many biological processes. They are fine-tuned regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several families have been identified in organisms from different kingdoms. Overall, the most studied families are Ras, Rho, Rab, Ran, Arf, and Miro. Recently, a new family named Big Ras GTPases was reported. As a general rule, the proteins of all families have five characteristic motifs (G1–G5), and some specific features for each family have been described. Here, we present an exhaustive analysis of these small GTPase families in fungi, using 56 different genomes belonging to different phyla. For this purpose, we used distinct approaches such as phylogenetics and sequences analysis. The main functions described for monomeric GTPases in fungi include morphogenesis, secondary metabolism, vesicle trafficking, and virulence, which are discussed here. Their participation during fungus–plant interactions is reviewed as well.

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