Determination of in Vivo Dissociation Constant, KD, of Cdc42-Effector Complexes in Live Mammalian Cells Using Single Wavelength Fluorescence Cross-correlation Spectroscopy

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane-bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (K_D) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine K_D of Cdc42-effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured K_D for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nm, respectively. The determination of K_D for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.Over the last 2 decades, we have been successful in describing a myriad of cell signaling pathways that regulate the biology of cells. These pathways are made of elements incorporating protein-protein, protein-lipid and protein-ligand interactions. With the advent of GFP² (1, 2) and its variants (3), it is now possible to genetically encode fluorescent probes into any protein of interest. GFP fusion proteins can be used in live cells giving spatial and temporal resolution to cell signaling pathways (4). To gain mechanistic insights into cellular processes, it is crucial that we measure quantitative parameters to describe cell signaling. In this study, we present an approach based on fluorescence cross-correlation spectroscopy (FCCS) (5, 6) and Förster resonance energy transfer (FRET) to determine quantitative parameters of cell signaling pathways, including the determination of the K_D for Cdc42-effector interactions in live CHO-K-1 (hereafter referred to as CHO) mammalian cells.The RhoGTPase Cdc42 (7, 8) regulates pathways that coordinate cell cycle, morphogenesis, and polarity. Cdc42 is a molecular switch that cycles between an inactive (GDP-bound) and active (GTP-bound) state. The V12 Cdc42 point mutation freezes the protein in an activated GTP-bound form, which binds effectors strongly. In contrast, Cdc42N17 is a dominant negative protein that is GDP-bound and interacts with effectors weakly if at all (9). A major Cdc42 binding site/domain in effector proteins is known as Cdc42- and Rac-interacting binding region (CRIB)³ and was originally found in activated Cdc42 kinase, p21 activated kinase (PAK), and neural Wiskott-Aldrich syndrome protein (N-WASP) (10). The inverse Bin-amphiphysins-Rvs domain adaptor protein IRSp53 is also an effector but binds Cdc42 through a partial CRIB domain (11, 12). Cdc42 interaction with its effectors has two main consequences, which are not mutually exclusive: (i) unfolding of effector to expose the active site and (ii) relocalization of effector to membrane compartments. Thus Cdc42-effector interactions serve as a good model for cell signaling as a whole.Fluorescence correlation spectroscopy and FCCS measure fluctuations in fluorescence of a small number of molecules as they pass through a defined confocal volume, respectively (13, 14, 15). Since the number of molecules in the confocal volume and the confocal volume itself can be determined, concentrations of protein can be measured by fluorescence correlation spectroscopy. Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) is an FCCS variant in which excitation of two or more probes is achieved by single wavelength one-photon excitation. To date SW-FCCS has been used successfully to follow receptors and receptor-ligand interactions in vitro and in vivo (6, 16, 17).In the present analysis, we take a two-step approach to determining the K_D of Cdc42 binding to CRIB (domain of PAK), N-WASP, and IRSp53. First, we show that the proteins under investigation are indeed interacting with each other directly in vivo by FRET analysis. Here we use acceptor photobleaching (AP)-FRET as well as changes in lifetime (through fluorescence lifetime imaging microscopy (FLIM)) as indicators of FRET. Second, we use SW-FCCS to determine the K_D of Cdc42 interacting with its effectors by measuring the concentration of free protein versus complexed protein. Thus, the combined use of FRET and FCCS allows quantitative analysis of cell signaling pathways in vivo.     

Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (K_ds) of biomolecules. The determination of a K_d depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ～0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Förster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the K_d.     

The Pseudomonas aeruginosa N-Acylhomoserine Lactone Quorum Sensing Molecules Target IQGAP1 and Modulate Epithelial Cell Migration

Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxo-dodecanoyl-L-homoserine lactone 3O-C₁₂-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C₁₂-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C₁₂-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C₁₂-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C₁₂-HSL with a sensor bioassay. Moreover, 3O-C₁₂-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P.aeruginosa 3O-C₁₂-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C₁₂-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial – human cell communication.     

Interactions among IQGAP1, Cdc42, and the cadherin/catenin protein complex regulate Sertoli-germ cell adherens junction dynamics in the testis

The movement of developing germ cells across the seminiferous epithelium during spermatogenesis involves extensive adherens junction (AJ) restructuring between Sertoli cells, as well as between Sertoli and germ cells. In this report, we show that the intricate interactions between Cdc42 (a Rho family protein of Mr approximately 23 kDa originally identified in membranes of human platelets and placenta, and is the homolog of CDC42Sc, which is known to regulate of bud-site assembly in Saccharomyces cerevisiae) and its effector, IQ motif containing GTPase activating protein (IQGAP1, Mr approximately 189 kDa, it is also an actin-binding protein known to interact with Cdc42 and Rac1 GTPases), regulate Sertoli-germ cell, but not Sertoli-Sertoli cell, AJ dynamics. Using testis lysates for immunoprecipitation (IP), IQGAP1 was shown to associate with E-cadherin, N-cadherin, and beta-catenin (but not beta1-integrin and nectin-2), as well as with actin and vimentin (but not alpha-tubulin). Moreover, IQGAP1 was found to localize to the periphery of both Sertoli and germ cells in the seminiferous epithelium, at sites of cell-cell contacts. Using fluorescent microscopy with dual fluorescent probes, IQGAP1 was found to co-localize, at least in part, with N-cadherin in the seminiferous epithelium consistent with their localization at the basal and apical ES. Using Sertoli-germ cell cocultures, it was demonstrated that AJ assembly associated with a transient induction of Cdc42 and IQGAP1, which was not found when Sertoli cells were cultured alone. Lastly, a shift in the interactions of Cdc42, IQGAP1, beta-catenin, and N-cadherin was detected in Sertoli-germ cell cocultures using an Ca2+-induced AJ disruption model, which was used to examine AJ disassembly and its reassembly. In the presence of Ca2+, IQGAP1 bound preferentially to Cdc42 rather than to beta-catenin. However, when Ca2+ was depleted from cocultures using EGTA, a Ca2+ chelating agent, IQGAP1 lost its affinity for Cdc42 and became tightly associated with beta-catenin, destabilizing cadherin-mediated AJs between Sertoli and germ cells. Yet this shift of protein-protein interaction was not detected in Sertoli cells cultured alone. These results illustrate that the interactions among IQGAP1, Cdc42, and beta-catenin are crucial to the regulation of Sertoli-germ cell, but not Sertoli-Sertoli cell, AJ dynamics in the seminiferous epithelium.     

Crystal Structure of the GTPase-activating Protein-related Domain from IQGAP1

IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic “arginine finger” seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099–1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42·GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.The small GTPase Ras functions as a binary switch in cell signaling processes. When bound to GTP, Ras is able to interact with effector proteins, including Raf kinase, and alter their activities. Ras signaling is terminated when bound GTP is hydrolyzed to GDP and inorganic phosphate. The basal rate of GTP hydrolysis on Ras is quite slow (∼1.2 × 10^–4 s^–1), but this rate of hydrolysis can be enhanced ∼10⁵-fold by interaction with a GTPase-activating protein (GAP)² (1). Several RasGAPs have been identified to date including p120 RasGAP and neurofibromin (NF1). The Rho family of Ras-related small GTPases also function as binary switches in cell signaling processes. Whereas the intrinsic rate of GTP hydrolysis on Rho proteins is faster than Ras, this rate can also be stimulated by interaction with a RhoGAP. Examination of the structures of the GAP domains of p120RasGAP (2), neurofibromin (3), SynGAP (4), and the GAP domains from the RhoGAPs p50 RhoGAP and the Bcr homology domain of phosphatidylinositol 3-kinase (5, 6) indicates that although ostensibly different, these all-helical domains are structurally related (7).IQGAP1 was discovered by chance during an attempt to isolate novel matrix metalloproteinases (8). Analysis reveals that the protein contains several discrete domains and motifs including a region containing four isoleucine- and glutamine-rich motifs (IQ repeats) and a region with sequence homology to the Ras-specific GAP domains of p120RasGAP, NF1, and SynGAP (2–4, 8). Subsequently, two homologs, IQGAP2 and IQGAP3, have been discovered. The IQ repeats have been shown to mediate binding to calmodulin and calmodulin-like proteins (e.g. S100, myosin essential light chain), whereas the GAP-related domain (GRD) does not appear to bind to Ras but instead is necessary for binding to the Rho family GTPases Cdc42 and Rac1, primarily in their active forms (9–11). However, instead of accelerating hydrolysis of GTP, IQGAP1 preserves the activated states of Cdc42 and Rac1 to the extent that overexpression of IQGAP1 in cells increases the levels of active GTPase (12). Because IQGAP1 expression increases the level of activated Cdc42, initially there was some confusion as to whether the protein might not represent a novel guanine nucleotide exchange factor. However it now appears that IQGAP1 is an effector of Cdc42 and Rac1 and preserves their activated states by tightly binding to the GTPases and stabilizing them in a conformation not conducive to GTP hydrolysis. IQGAP1 appears to be such an important effector for Cdc42 that abrogation of binding to IQGAP1 not only reduces the levels of active Cdc42, it also reduces membrane-localized Cdc42 and the cellular response to bradykinin (12).A growing body of evidence implicates IQGAP1 in carcinogenesis. Expression of IQGAP1 increases during the transition from a minimally to a highly metastastic form of melanoma, and IQGAP1 has been found to be overexpressed in ovarian, breast, lung, and colorectal cancers (13–17). In vitro, overexpressed IQGAP1 enhances cell motility and invasiveness in a process that requires Cdc42 and Rac (18). β-Catenin is one of the many binding partners of IQGAP1 identified to date. IQGAP1 has been shown to bind to β-catenin and interfere with β-catenin binding to α-catenin, an interaction necessary for stable cell-cell adhesion (19). Another study found that IQGAP2 knock-out mice overexpress IQGAP1 and developage-dependent liver cancer and apoptosis (20).To better understand how a protein domain homologous to others that accelerate GTP hydrolysis can function as an effector and preserve the GTP-bound state, we have determined the x-ray structure of the IQGAP1 GRD. Despite low sequence identity, the GRD structure is quite similar to the GAP domains of p120, neurofibromin, and SynGAP; however, unlike those domains, the GRD possesses a conserved threonine in place of the catalytic arginine finger and has a 31-residue insertion that projects from one end of the molecule. Using the coordinates of Ras·GDP·AlF₃ in complex with the GAP domain of p120, we built a model of Cdc42·GTP bound to the GRD. The model indicates that a steric clash between the conserved Thr¹⁰⁴⁶ and the phosphate-binding loop of Cdc42 and other subtle changes within the active site would likely preclude nucleotide hydrolysis. Sequence conservation mapped to the surface of the GRD indicates that the surface with the highest degree of conservation overlaps with the surface that makes contacts to Cdc42 in the model.     

Honokiol inhibits HepG2 migration via down‐regulation of IQGAP1 expression discovered by a quantitative pharmaceutical proteomic analysis

Honokiol (HNK), a natural small molecular product, inhibited proliferation of HepG2 cells and exhibited anti‐tumor activity in nude mice. In this article, we applied a novel sensitive stable isotope labeling with amino acids in cell culture‐based quantitative proteomic method and a model of nude mice to investigate the correlation between HNK and the hotspot migration molecule Ras GTPase‐activating‐like protein (IQGAP1). The quantitative proteomic analysis showed that IQGAP1 was 0.53‐fold down‐regulated under 10 μg/mL HNK exposure for 24 h on HepG2 cells. Migration ability of HepG2 cells under HNK treatment was correlated with its expression level of IQGAP1. In addition, the biochemical validation on HepG2 cells and the tumor xenograft model further demonstrated that HNK decreased the expression level of IQGAP1 and its upstream proteins Cdc42/Rac1. These data supported that HNK can modulate cell adhesion and cell migration by acting on Cdc42/Rac1 signaling via IQGAP1 interactions with its upstream Cdc42/Rac1 proteins, which is a new molecular mechanism of HNK to exert its anti‐tumor activity.     

Involvement of IQGAP3, a regulator of Ras/ERK‐related cascade,in hepatocyte proliferation in mouse liver regeneration and development

The spatio‐temporal regulation of hepatocyte proliferation is a critical issue in liver regeneration. Here, in normal and regenerating liver as well as in developing liver, we examined its expression/localization of IQGAP3, which was most recently reported as a Ras/Rac/Cdc42‐binding proliferation factor associated with cell–cell contacts in epithelial‐type cells. In parallel, the expression/localization of Rac/Cdc42‐binding IQGAP1/2 was examined. IQGAP3 showed a specific expression in proliferating hepatocytes positive for the proliferating marker Ki‐67, the levels of expressions of mRNAs and proteins were significantly increased in hepatocytes in liver regeneration and development. In immunofluorescence, IQGAP3 was highly enriched at cell–cell contacts of hepatocytes. IQGAP1 and IQGAP2 were exclusively expressed in Kupffer and sinusoidal endothelial cells, respectively, in normal, regenerating, and developing liver. The expression of IQGAP1, but not of IQGAP2, was increased in CCl₄‐induced (but not in partial hepatectomy‐induced) liver regeneration. Exclusive expression/localization of IQGAP3 to hepatocytes in the liver likely reflects the specific involvement of the IQGAP3/Ras/ERK signaling cascade in hepatocyte proliferation in addition to the previously identified signaling pathways, possibly by integrating cell–cell contact‐related proliferating signaling events. On the other hand, the Rac/Cdc42‐binding properties of IQGAP1/2/3 may be related to the distinct modes of remodeling due to the different strategies which induced proliferation of liver cells; partial hepatectomy, CCl₄ injury, or embryonic development. Thus, the functional orchestration of Ras and the Ras homologous (Rho) family proteins Rac/Cdc42 likely plays a critical role in liver regeneration and development. J. Cell. Physiol. 220: 621–631, 2009. © 2009 Wiley‐Liss, Inc.     

Identification and characterization of the Cdc42-binding site of IQGAP1

IQGAP1 is a multi-domained protein that integrates signaling of the Rho family GTPase Cdc42 with regulation of the cytoskeleton. Using SPOT analysis and in vitro peptide competition assays we have identified a 24 amino acid region of IQGAP1 that is necessary for Cdc42 binding. Both in vitro and in vivo analyses reveal that deletion of this sequence abolishes binding of IQGAP1 to Cdc42. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. An IQGAP1 mutant lacking the Cdc42 binding site mislocalizes to the cell periphery. These observations specifically define a short sequence of IQGAP1 that is required for its interaction with Cdc42 and demonstrate that Cdc42 binding is necessary for the normal subcellular distribution of IQGAP1.     

Fluorescence assays of Cdc42 interactions with target/effector proteins

The goal of these studies was to examine the interactions between the GTP-binding protein Cdc42 and its target/effectors by fluorescence spectroscopy. We have inserted fluorescent reporter groups at two distinct sites on Cdc42: N-methylanthraniloyl- (Mant-) derivatized nucleotides were complexed to the nucleotide-binding site of Cdc42, while a fluorescent succinimidyl ester was covalently attached to lysine 150. These two sites are separated by about 30 A on the Cdc42 molecule. Thus, the attachment of reporter groups to these sites enables the effects of target/effector binding to be viewed over a significant portion of the GTP-binding protein surface. We have taken advantage of fluorescence changes occurring at both sites to compare the interactions of activated Cdc42 with the limit binding domains from the following target/effectors: the serine/threonine kinase PAK, the tyrosine kinase ACK-2, and the RasGAP-related protein IQGAP. In addition, a unique lysine residue on the Cdc42-binding domain of ACK-2 (GBD-ACK) was covalently modified with a fluorescent succinimidyl ester. The distances separating this reactive lysine from the nucleotide binding site and lysine 150 of Cdc42 were determined by fluorescence resonance energy transfer and yielded a picture for Cdc42/GBD-ACK interactions that is consistent with recent NMR structural determinations for Cdc42/effector complexes. The changes in reporter group fluorescence at the reactive lysine of GBD-ACK, which were induced by the binding of activated Cdc42, were also examined. Overall, the results of these studies suggest not only that Cdc42 can induce conformational changes within an effector but also that in a reciprocal fashion the target/effectors induce conformational changes that span a significant distance on the GTP-binding protein.     

Phosphorylation of IQGAP1 modulates its binding to Cdc42, revealing a new type of rho-GTPase regulator

The Rho-GTPase Cdc42 is important for the establishment and maintenance of epithelial polarity. Signaling from Cdc42 is propagated via its effector molecules that specifically bind to Cdc42 in the GTP-bound form. The cell-cell contact regulator and actin-binding protein IQGAP1 is described as effector of Cdc42 and Rac. Unexpectedly, we show in this study that IQGAP1 bound also directly nucleotide-depleted Cdc42 (Cdc42-ND). This interaction was enhanced in the presence of phosphatase inhibitors and in epithelial cells without cell-cell contacts. Tandem mass spectrometry analysis and immunoprecipitation experiments revealed that IQGAP1 was Ser1443-phosphorylated in vivo, potentially by protein kinase Cepsilon and upon loss of cell-cell contacts. In addition, we identified two independent domains of the IQGAP1 C terminus that bound exclusively Cdc42-ND. These domains interacted with each other, favoring the binding to Cdc42-GTP. Moreover, phosphorylation on Ser1443 strongly inhibited this intramolecular interaction. Thus, we unraveled a molecular mechanism that reveals a novel type of Rho-GTPase regulator. We propose that, depending on its phosphorylation state, IQGAP1 might serve as an effector or sequester nucleotide-free Cdc42 to prevent signaling.     

Self-association of IQGAP1: characterization and functional sequelae

The scaffolding protein IQGAP1 participates in numerous cellular functions by binding to target proteins such as actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. IQGAP1 regulates the cytoskeleton, promotes cell motility, and modulates E-cadherin-mediated cell-cell adhesion. However, how IQGAP1 exerts its functions in vivo is still unclear. In this study we investigate the self-association of IQGAP1 and its role in IQGAP1 function. Endogenous IQGAP1 co-immunoprecipitated from MCF-7 cells with IQGAP1 tagged with enhanced green fluorescent protein, indicating that IQGAP1 self-associates in cells. In vitro assays confirmed that IQGAP1 can self-associate and that this effect is mediated by the N-terminal half of the protein. Gel filtration analysis suggested that full-length IQGAP1 exists as a combination of monomers, dimers, and larger oligomers. Analysis performed with multiple fragments of IQGAP1 narrowed the self-association region to amino acids 763-863. In support of this observation, a peptide comprising residues 763-863 disrupted self-association of full-length IQGAP1 in a dose-dependent manner. Similarly, deleting this sequence from IQGAP1 abolished binding to full-length IQGAP1. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. Consistent with these findings, transfection into cells of a peptide containing the self-association domain significantly reduced the amount of active Cdc42 in cell lysates. These observations define a sequence of IQGAP1 that is necessary for its oligomerization and demonstrate that self-association is required for the normal cellular function of IQGAP1.     

6-Phosphogluconate Dehydrogenase Mechanism: EVIDENCE FOR ALLOSTERIC MODULATION BY SUBSTRATE

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG ^D(V/K) and ^D(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases ^D(V/K) to 2.84 and decreases ^D(V) to 1.38. A similar increase of ^T(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K_m of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.     

A Phosphatidylinositol-Transfer Protein and Phosphatidylinositol-4-phosphate 5-Kinase Control Cdc42 to Regulate the Actin Cytoskeleton and Secretory Pathway in Yeast

The actin cytoskeleton rapidly depolarizes in yeast secretory (sec) mutants at restrictive temperatures. Thus, an unknown signal conferred upon secretion is necessary for actin polarity and exocytosis. Here, we show that a phosphatidylinositol (PI) transfer protein, Sfh5, and a phosphatidylinositol-4-phosphate 5-kinase, Mss4, facilitate Cdc42 activation to concomitantly regulate both actin and protein trafficking. Defects in Mss4 function led to actin depolarization, an inhibition of secretion, reduced levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] in membranes, mislocalization of a pleckstrin homology domain fused to green fluorescent protein, and the mislocalization of Cdc42. Similar defects were observed in sec, myo2-66, and cdc42-6 mutants at elevated temperatures and were rescued by the overexpression of MSS4. Likewise, the overexpression of SFH5 or CDC42 could ameliorate these defects in many sec mutants, most notably in sec3Δ cells, indicating that Cdc42-mediated effects upon actin and secretion do not necessitate Sec3 function. Moreover, mutation of the residues involved in PI binding in Sfh5 led to the mislocalization and loss of function of both Sfh5 and Cdc42. Based upon these findings, we propose that the exocytic signal involves PI delivery to the PI kinases (i.e., Mss4) by Sfh5, generation of PI(4,5)P₂, and PI(4,5)P₂-dependent regulation of Cdc42 and the actin cytoskeleton.     

多态性蛋白Mad2与其配体Cdc20121-138的相互作用研究

多态性蛋白Mad2是有丝分裂纺锤体检测点(SAC)的关键蛋白,也是多态性蛋白质家族中研究最广泛的成员之一.Mad2有两种不同的天然构象:O-Mad2和C-Mad2.Mad2构象间的转变及其与配体Cdc20间的相互作用对SAC发挥其生物学功能至关重要.本文利用荧光各向异性技术对O-Mad2和C-Mad2与配体TAMRA-Cdc20~(121-138)间相互作用的热力学及动力学过程进行了系统表征.结果表明:在无盐和低盐溶液(100 mmol/L NaCl)中,Mad2两种构象与Cdc20~(121-138)的平衡解离常数(K_D)均在10~(-6) mol/L,但C-Mad2与Cdc20~(121-138)结合的K_D值约为O-Mad2的1/5;在高盐(300 mmol/L NaCl)溶液中,Mad2两种构象与TAMRA-Cdc20~(121-138)结合的K_D值无明显差别.动力学实验结果显示,在同一种缓冲液中Mad2两种构象与Cdc20~(121-138)相互作用的解离速率常数k_d没有显著差别,而C-Mad2与Cdc20~(121-138)的结合速率常数k_a却比O-Mad2高一个数量级,这表明C-Mad2与Cdc20~(121-138)不仅结合力更强,且结合速率要快很多.Mad2与Cdc20~(121-138)突变体间的相互作用以及离子强度对二者相互作用的影响结果提示,Mad2和Cdc20间的相互作用不是通过静电相互作用,而可能是通过疏水相互作用来实现的.本研究为揭示多态性蛋白Mad2的构象转变机理及其在有丝分裂过程中的作用机制提供了重要的实验基础.     

IQGAP1 promotes cell motility and invasion

The dynamic processes of cell migration and invasion are largely coordinated by Rho family GTPases. The scaffolding protein IQGAP1 binds to Cdc42, increasing the amount of active Cdc42 both in vitro and in cells. Here we show that overexpression of IQGAP1 in mammalian cells enhances cell migration in a Cdc42- and Rac1-dependent manner. Importantly, cell motility was significantly decreased both by knock down of endogenous IQGAP1 using small interfering RNA and by transfection of a dominant negative IQGAP1 construct, IQGAP1DeltaGRD. Cell invasion was similarly altered by manipulating intracellular IQGAP1 concentrations. Moreover, invasion mediated by constitutively active Cdc42 was attenuated by IQGAP1DeltaGRD. Thus, IQGAP1 has a fundamental role in cell motility and invasion.     

Identification and functional characterization of zebrafish K2P10.1 (TREK2) two-pore-domain K+ channels

Two-pore-domain potassium (K_2P) channels mediate K⁺ background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K_2P currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K_2P channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K_2P10.1 channel. K_2P10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK_2P10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K_2P10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K_2P10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K⁺ selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK_2P10.1, induced robust activation of zK_2P10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK_2P10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K_2P10.1 two-pore-domain K⁺ channels that exhibit structural and functional properties largely similar to human K_2P10.1. We conclude that the zebrafish represents a valid model to study K_2P10.1 function in vivo.     

IQGAP1 scaffolding links phosphoinositide kinases to cytoskeletal reorganization

IQGAP1 is a multidomain scaffold protein that coordinates the direction and impact of multiple signaling pathways by scaffolding its various binding partners. However, the spatial and temporal resolution of IQGAP1 scaffolding remains unclear. Here, we use fluorescence imaging and correlation methods that allow for real-time live-cell changes in IQGAP1 localization and complex formation during signaling. We find that IQGAP1 and PIPKIγ interact on both the plasma membrane and in cytosol. Epidermal growth factor (EGF) stimulation, which can initiate cytoskeletal changes, drives the movement of the cytosolic pool toward the plasma membrane to promote cytoskeletal changes. We also observe that a significant population of cytosolic IQGAP1-PIPKIγ complexes localize to early endosomes, and in some instances form aggregated clusters which become highly mobile upon EGF stimulation. Our imaging studies show that PIPKIγ and PI3K bind simultaneously to IQGAP1, which may accelerate conversion of PI4P to PI(3,4,5)P₃ that is required for cytoskeletal changes. Additionally, we find that IQGAP1 is responsible for PIPKIγ association with two proteins associated with cytoskeletal changes, talin and Cdc42, during EGF stimulation. These results directly show that IQGAP1 provides a physical link between phosphoinositides (through PIPKIγ), focal adhesion formation (through talin), and cytoskeletal reorganization (through Cdc42) upon EGF stimulation. Taken together, our results support the importance of IQGAP1 in regulating cell migration by linking phosphoinositide lipid signaling with cytoskeletal reorganization.     

The effect of IQGAP1 on Xenopus embryonic ectoderm requires Cdc42

IQGAP1 contains a number of protein recognition motifs through which it binds to targets. Several in vitro studies have documented that IQGAP1 interacts directly with calmodulin, actin, E-cadherin, beta-catenin, and the small GTPases Cdc42 and Rac. Nevertheless, direct demonstration of in vivo function of mammalian IQGAP1 is limited. Using a novel assay to evaluate in vivo function of IQGAP1, we document here that microinjection of IQGAP1 into early Xenopus embryos generates superficial ectoderm lesions at late blastula stages. This activity was retained by the mutated variants of IQGAP1 in which the calponin homology domain or the WW domain was deleted. By contrast, deletion of the IQ (IQGAP1-DeltaIQ), Ras-GAP-related (IQGAP1-DeltaGRD), or C-terminal (IQGAP1-DeltaC) domains abrogated the effect of IQGAP1 on the embryos. None of the latter mutants bound Cdc42, suggesting that the binding of Cdc42 by IQGAP1 is critical for its function. Moreover, overexpression of IQGAP1, but not IQGAP1-DeltaGRD, significantly increased the amount of active Cdc42 in embryonic cells. Co-injection of wild type IQGAP1 with dominant negative Cdc42, but not the dominant negative forms of Rac or Rho, blocked the effect of IQGAP1 on embryonic ectoderm. Together these data indicate that the activity of IQGAP1 in embryonic ectoderm requires Cdc42 function.     

IQGAP1 is a component of Cdc42 signaling to the cytoskeleton

The Ras-GAP related protein IQGAP1 binds several proteins, including actin, calmodulin, E-cadherin and the Rho family GTPase Cdc42. To gain insight into its in vivo function, IQGAP1 was overexpressed in mammalian cells. Transfection of IQGAP1 significantly increased the levels of active, GTP-bound Cdc42, resulting in the formation of peripheral actin microspikes. By contrast, transfection of an IQGAP1 mutant lacking part of the GAP-related domain (IQGAP1deltaGRD) substantially decreased the amount of GTP-bound Cdc42 in cell lysates. Consistent with these findings, IQGAP1DeltaGRD blocked Cdc42 function in cells that stably overexpress constitutively active Cdc42 and abrogated the effect of bradykinin on Cdc42. In cells transfected with IQGAP1deltaGRD, bradykinin was unable to activate Cdc42, translocate Cdc42 to the membrane fraction, or induce filopodia production. IQGAP1deltaGRD transfection altered cellular morphology, producing small, round cells that closely resemble Cdc42-/- cells. Some insight into the mechanism was provided by in vitro analysis, which revealed that IQGAP1deltaGRD increased the intrinsic GTPase activity of Cdc42, thereby increasing the amount of inactive, GDP-bound Cdc42. These data imply that IQGAP1 has a crucial role in transducing Cdc42 signaling to the cytoskeleton.