scholarly journals PIP2 Influences the Conformational Dynamics of Membrane bound KRAS4b

2019 ◽  
Author(s):  
Mark A. McLean ◽  
Andrew G. Stephen ◽  
Stephen G. Sligar
Keyword(s):  
Tyrosine Kinases ◽  
Membrane Surface ◽  
Conformational Dynamics ◽  
Small Gtpase ◽  
Phospholipid Bilayer ◽  
Activation Mechanism ◽  
Nucleotide Exchange ◽  
Dynamic Simulations ◽  
Fluorescence Anisotropy Decay

ABSTRACTKRAS4b is a small GTPase involved in cellular signaling through receptor tyrosine kinases. Activation of KRAS4b is achieved through the interaction with nucleotide exchange factors while inactivation is regulated by through interaction with GTPase activating proteins. The activation of KRAS4b only occurs after recruitment of the regulatory proteins to the plasma membrane thus making the role of the phospholipid bilayer an integral part of the activation mechanism. The phospholipids, primarily with anionic head groups, interact with both the membrane anchoring hypervariable region and the G-domain, thus influencing the orientation of KRAS at the membrane surface. The orientation of the G-domain at the membrane surface is believed to play a role in the regulation of KRAS activation. Much of the research has focused on the role of phosphatidyl serine but little has been done regarding the important signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We report here the use of fluorescence anisotropy decay, atomic force microscopy, and molecular dynamic simulations to show that the presence of PIP2 in the bilayer promotes the interaction of the G-domain with the bilayer surface. The stability of these interactions significantly alters the dynamics of KRAS4b bound to the membrane indicating a potential role for PIP2 in the regulation of KRAS4b activity.

Integrin-Independent Role of CalDAG-GEFI in Neutrophil Chemotaxis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1266-1266 ◽  
Author(s):  
Carla Carbo ◽  
Tobias Goerge ◽  
Hidenori Hattori ◽  
Daniel Duerschmied ◽  
Stephen M. Cifuni ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Small Gtpase ◽  
Calcium Flux ◽  
Migration Speed ◽  
Neutrophil Chemotaxis ◽  
Nucleotide Exchange ◽  
Transwell Assay ◽  
Nucleotide Exchange Factor

Abstract Neutrophil chemotaxis and transmigration towards a source of inflammation are two crucial processes for host defense against infection that rely on integrin function. Recently, integrin-independent migration of dendritic cells to the lymph node has been brought to light, although neutrophil migration in the presence of EDTA was reported many years ago. Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), is a small signaling protein that plays a key role in the activation of beta-1, beta-2, and beta-3 integrins in platelets and neutrophils by activating the small GTPase Rap1. We explored the role of CalDAG-GEFI in integrin-independent chemotaxis in neutrophils. Here we report that CalDAG-GEFI−/− neutrophils have impaired chemotaxis that is independent of integrin function. In a chemotaxis transwell assay towards LTB4 and in the presence of 10mM EDTA, CalDAG-GEFI−/− neutrophils had a 50% reduction in transmigration over 60 minutes compared to wild-type (WT) neutrophils (p<0.05). In separate experiments we confirmed that the transwell assay is independent of integrins using either CD18−/− neutrophils or WT neutrophils plus a blocking anti-CD18 monoclonal antibody. We previously showed that LTB4 signaling upstream of CalDAG-GEFI was not affected in CalDAG-GEFI−/− neutrophils, as assessed by intracellular calcium flux measurements. Using videomicroscopy to visualize the live migrating neutrophils in a horizontal plate in the presence of 10mM EDTA, we found that the reason CalDAG-GEFI−/− neutrophils fail to reach the chemotactic stimulus (10 pg/mL LTB4) is because they have a significantly reduced migration speed compared to WT neutrophils (16 um/sec vs. 23 um/sec, p<0.05), and also because they have an abnormal chemotactic directionality, with a directionality index (the distance between the start and finish points of a migrating neutrophil/total distance covered by the migrating neutrophil) of 0.84 vs 0.94 in WT neutrophils, p<0.05. We investigated whether the observed differences in chemotaxis between CalDAG-GEFI−/− and WT neutrophils could be explained by differences in F-actin polymerization. Using fluorescence microscopy, we found that the percentage of CalDAG-GEFI−/− neutrophils with F-actin pseudopodia after LTB4 stimulation was significantly lower compared to WT neutrophils (22% vs. 56.7%, p<0.05), suggesting that CalDAG-GEFI−/− neutrophils have a defect in F-actin polymerization. Overall, our studies suggest that CalDAG-GEFI plays a role in the mechanisms that regulate both the migration speed and direction of neutrophils during chemotaxis, independent of its established role in integrin activation.

Preliminary Studies on the Role of a Small GTPase, RhoA and Its Regulation in Imatinib Resistance

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4243-4243
Author(s):  
Song Zhang ◽  
Qingfeng Du ◽  
Hongqian Zhu ◽  
Rong Li ◽  
Zhi Liu ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Fusion Gene ◽  
Human Cancer ◽  
Chronic Phase ◽  
Small Gtpase ◽  
Signal Pathways ◽  
G1 Arrest ◽  
Imatinib Resistance ◽  
Point Mutant

Abstract Imatinib has impressive response rates and good tolerability quickly led to its adoption as frontline therapy for all patients with chronic-phase CML (chronic myeloid leukemia), but the therapeutic effect of imatinib is poor in the blast crisis, and imatinib resistance has become a major problem in CML. The possible mechanisms of imatinib resistance include the amplification of BCR-ABL fusion gene and its expression increase, the point mutant of BCR-ABL kinase domain and the effects of other tyrosine kinases such as Src, Hck and Lyn and so on. However, the second-generation tyrosine kinase inhibitors (such as nilotinib and dasatinib), which were developed to overcome imatinib resistance resulting from the point mutant or the activation of other tyrosine kinases, even can not prevent all patients with CML progression to drug resistance. So there would be the other potential factor in imatinib resistance. Our previous studies generated a new imatinib-resistant BCR/ABL-positive cell line, K562-R. The 50% inhibitory concentration of imatinib was 15-fold higher in K562-R than in the wild-type K562. The expression of RhoA gene is up-regulated in K562-R by microarray analyses. RhoA, a small GTPase (24KD), has been found overexpression in breast, colon, head and neck squamous cell carcinoma, bladderand testicular cancer, lung and gastric cancer. It plays an important role in the initiation as well as the progression of human cancer, but the potential role of RhoA related to imatinib resistance has yet been unknown. In this study, we firstly detect the biologic characteristic of K562-R cells with RhoA down-expression by RNA interference. When K562-R cells were transfected with 150nM siRNA-RhoA for 48 hours, the percentage of apoptotic K562-R cells is respectively 12.82% by AnnexinV-PI assay and 9.0% by Hoechst 33258 staining and both have significant increase, cell cycle analysis found significant G0/G1 arrest, the expression of CD29 increase and that of CD71 and GPA have no difference. Secondly, The K562-R cells were treated with three selective inhibitors, including PD98059 (Ras/MAPK inhibitor), LY294002 (PI3K/AKT inhibitor) and AG490(JAK/STAT inhibitor) for 2,4 and 8 hours and the expression of RhoA were analyzed by Western-Blotting. The expression of RhoA is arrested in the K562-R cells treated with PD98059 and AG490 and no different with LY294002. These results indicate that RhoA would be an important target in the down-stream of multi-signal pathways related to imatinib resistance and the potential function of RhoA in imatinib resistance involve in increasing of cell proliferation, resistance to cell apoptosis and changes of cell adhesion.

scholarly journals Specific Phosphorylation of p120-Catenin Regulatory Domain Differently Modulates Its Binding to RhoA

2006 ◽  
Vol 27 (5) ◽  
pp. 1745-1757 ◽  
Author(s):  
Julio Castaño ◽  
Guiomar Solanas ◽  
David Casagolda ◽  
Imma Raurell ◽  
Patricia Villagrasa ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Regulatory Mechanism ◽  
Adherens Junction ◽  
Small Gtpase ◽  
Regulatory Domain ◽  
P120 Catenin ◽  
Tyrosine Residues ◽  
Rhoa Activity ◽  
Intracellular Proteins

ABSTRACT p120-catenin is an adherens junction-associated protein that controls E-cadherin function and stability. p120-catenin also binds intracellular proteins, such as the small GTPase RhoA. In this paper, we identify the p120-catenin N-terminal regulatory domain as the docking site for RhoA. Moreover, we demonstrate that the binding of RhoA to p120-catenin is tightly controlled by the Src family-dependent phosphorylation of p120-catenin on tyrosine residues. The phosphorylation induced by Src and Fyn tyrosine kinases on p120-catenin induces opposite effects on RhoA binding. Fyn, by phosphorylating a residue located in the regulatory domain of p120-catenin (Tyr112), inhibits the interaction of this protein with RhoA. By contrast, the phosphorylation of Tyr217 and Tyr228 by Src promotes a better affinity of p120-catenin towards RhoA. In agreement with these biochemical data, results obtained in cell lines support the important role of these phosphorylation sites in the regulation of RhoA activity by p120-catenin. Taken together, these observations uncover a new regulatory mechanism acting on p120-catenin that contributes to the fine-tuned regulation of the RhoA pathways during specific signaling events.

scholarly journals A GAP that Divides

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1788 ◽  
Author(s):  
Angika Basant ◽  
Michael Glotzer
Keyword(s):  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
C Elegans ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Parallel Pathway ◽  
Mutant Phenotypes

Cytokinesis in metazoan cells is mediated by an actomyosin-based contractile ring that assembles in response to activation of the small GTPase RhoA. The guanine nucleotide exchange factor that activates RhoA during cytokinesis, ECT-2, is highly regulated. In most metazoan cells, with the notable exception of the early Caenorhabditis elegans embryo, RhoA activation and furrow ingression require the centralspindlin complex. This exception is due to the existence of a parallel pathway for RhoA activation in C. elegans. Centralspindlin contains CYK-4 which contains a predicted Rho family GTPase-activating protein (GAP) domain. The function of this domain has been the subject of considerable debate. Some publications suggest that the GAP domain promotes RhoA activation (for example, Zhang and Glotzer, 2015; Loria, Longhini and Glotzer, 2012), whereas others suggest that it functions to inactivate the GTPase Rac1 (for example, Zhuravlev et al., 2017). Here, we review the mechanisms underlying RhoA activation during cytokinesis, primarily focusing on data in C. elegans. We highlight the importance of considering the parallel pathway for RhoA activation and detailed analyses of cyk-4 mutant phenotypes when evaluating the role of the GAP domain of CYK-4.

scholarly journals R-Ras Regulates Exocytosis by Rgl2/Rlf-mediated Activation of RalA on Endosomes

2007 ◽  
Vol 18 (5) ◽  
pp. 1850-1860 ◽  
Author(s):  
Akiyuki Takaya ◽  
Takahiro Kamio ◽  
Michitaka Masuda ◽  
Naoki Mochizuki ◽  
Hirofumi Sawa ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Cellular Functions ◽  
Recycling Endosomes ◽  
Nucleotide Exchange Factor ◽  
Wide Range ◽  
Ras Family

R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.

scholarly journals Rab7a and Mitophagosome Formation

Cells ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 224 ◽  
Author(s):  
Esther Tan ◽  
Bor Tang
Keyword(s):  
Retrograde Transport ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Autophagosome Formation ◽  
Guanine Nucleotide Exchange ◽  
Gtpase Activating Proteins ◽  
Retromer Complex ◽  
Nucleotide Exchange Factor

The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to regulate endosomal retrograde transport and late endosome-lysosome fusion, earlier work has indicated a role for Rab7a in autophagosome-lysosome fusion as well as autolysosome maturation. However, as suggested by recent findings on PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy, Rab7a and its regulators are critical for the correct targeting of Atg9a-bearing vesicles to effect autophagosome formation around damaged mitochondria. This mitophagosome formation role for Rab7a is dependent on an intact Rab cycling process mediated by the Rab7a-specific guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs). Rab7a activity in this regard is also dependent on the retromer complex, as well as phosphorylation by the TRAF family-associated NF-κB activator binding kinase 1 (TBK1). Here, we discuss these recent findings and broadened perspectives on the role of the Rab7a network in PINK1-Parkin mediated mitophagy.

scholarly journals Biological and Regulatory Properties of Vav-3, a New Member of the Vav Family of Oncoproteins

1999 ◽  
Vol 19 (11) ◽  
pp. 7870-7885 ◽  
Author(s):  
Nieves Movilla ◽  
Xosé R. Bustelo
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinases ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Wild Type ◽  
Gtp Binding ◽  
Regulatory Properties ◽  
Identification And Characterization

ABSTRACT We report here the identification and characterization of a novel Vav family member, Vav-3. Signaling experiments demonstrate that Vav-3 participates in pathways activated by protein tyrosine kinases. Vav-3 promotes the exchange of nucleotides on RhoA, on RhoG and, to a lesser extent, on Rac-1. During this reaction, Vav-3 binds physically to the nucleotide-free states of those GTPases. These functions are stimulated by tyrosine phosphorylation in wild-type Vav-3 and become constitutively activated upon deletion of the entire calponin-homology region. Expression of truncated versions of Vav-3 leads to drastic actin relocalization and to the induction of stress fibers, lamellipodia, and membrane ruffles. Moreover, expression of Vav-3 alters cytokinesis, resulting in the formation of binucleated cells. All of these responses need only the expression of the central region of Vav-3 encompassing the Dbl homology (DH), pleckstrin homology (PH), and zinc finger (ZF) domains but do not require the presence of the C-terminal SH3-SH2-SH3 regions. Studies conducted with Vav-3 proteins containing loss-of-function mutations in the DH, PH, and ZF regions indicate that only the DH and ZF regions are essential for Vav-3 biological activity. Finally, we show that one of the functions of the Vav-3 ZF region is to work coordinately with the catalytic DH region to promote both the binding to GTP-hydrolases and their GDP-GTP nucleotide exchange. These results highlight the role of Vav-3 in signaling and cytoskeletal pathways and identify a novel functional cross-talk between the DH and ZF domains of Vav proteins that is imperative for the binding to, and activation of, Rho GTP-binding proteins.

scholarly journals ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

2015 ◽  
Vol 112 (15) ◽  
pp. 4642-4647 ◽  
Author(s):  
Henrik M. Hammarén ◽  
Daniela Ungureanu ◽  
Jean Grisouard ◽  
Radek C. Skoda ◽  
Stevan R. Hubbard ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Critical Role ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Nucleotide Binding ◽  
Inhibitory Effect ◽  
Atp Binding ◽  
Type I ◽  
Dynamic Simulations

Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase–kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.

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