PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-17 (17)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  The RalB-RLIP76 (RalBP1) complex reveals a novel mode of Ral-effector interaction 
Summary
RLIP76 (RalBP1) is a multidomain protein that interacts with multiple small G protein families: Ral via a specific binding domain and Rho and R-Ras via a GTPase activating domain. RLIP76 interacts with endocytosis proteins and has also been shown to behave as a membrane ATPase that transports chemotherapeutic agents from the cell. We have determined the structure of the Ral binding domain of RLIP76 and show that it comprises a coiled-coil motif. The structure of the RLIP76-RalB complex reveals a novel mode of binding compared to the structures of RalA complexed with the exocyst components Sec5 and Exo84. RLIP76 interacts with both nucleotide-sensitive regions of RalB and key residues in the interface have been identified using affinity measurements of RalB mutants. Sec5, Exo84 and RLIP76 bind Ral proteins competitively and with similar affinities in vitro.
doi:10.1016/j.str.2010.05.013
PMCID: PMC4214634  PMID: 20696399
2.  SH3BP1, an exocyst-associated RhoGAP, inactivates Rac1 at the front to drive cell motility 
Molecular cell  2011;42(5):650-661.
Summary
The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex, and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss-of-function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 down-regulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.
doi:10.1016/j.molcel.2011.03.032
PMCID: PMC3488376  PMID: 21658605
3.  RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly 
Cell  2011;144(2):253-267.
The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G-protein, RalB, is localized to nascent autophagosomes and is activated upon nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.
doi:10.1016/j.cell.2010.12.018
PMCID: PMC3038590  PMID: 21241894
4.  Cell motility 
The Ras proto-oncogenic proteins, prototypes of the small GTPases, work as molecular switches: they are active when bound to GTP and inactive when bound to GDP. A variety of evidence suggested that the Ras paradigm is not fully valid for the Rho-family of small GTPases. Indeed, permanent activation is not sufficient but it is rather the continuous oscillation between the GDP-bound and GTP-bound conformations (namely the GDP/GTP cycling or GTPase flux), that is required for Rho-GTPases to perform their biological functions and properly coordinate actin cytoskeleton reorganization. In our recent study, we show that Rac1 needs to cycle between the GDP and GTP states in order to efficiently control cell motility. Similarly, it was previously reported that GDP/GTP cycling is required by RhoA for cytokinesis and by Cdc42 for cell polarization. The future challenge is to understand why the GTPase flux is so important for the biological actions of Rho GTPases.
PMCID: PMC3306356  PMID: 22446552
5.  Characterization of single chain antibody targets through yeast two hybrid 
BMC Biotechnology  2010;10:59.
Background
Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.
Results
We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.
Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed.
Conclusions
Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.
doi:10.1186/1472-6750-10-59
PMCID: PMC2936416  PMID: 20727208
6.  An aPKC-Exocyst Complex Controls Paxillin Phosphorylation and Migration through Localised JNK1 Activation 
PLoS Biology  2009;7(11):e1000235.
The exocyst/aPKC complex controls the spatiotemporal activation of the kinases JNK and ERK at the leading edge of migrating cells and thereby controls the dynamic behaviour of the adhesion protein paxillin during cell migration.
Atypical protein kinase C (aPKC) isoforms have been implicated in cell polarisation and migration through association with Cdc42 and Par6. In distinct migratory models, the Exocyst complex has been shown to be involved in secretory events and migration. By RNA interference (RNAi) we show that the polarised delivery of the Exocyst to the leading edge of migrating NRK cells is dependent upon aPKCs. Reciprocally we demonstrate that aPKC localisation at the leading edge is dependent upon the Exocyst. The basis of this inter-dependence derives from two-hybrid, mass spectrometry, and co-immunoprecipitation studies, which demonstrate the existence of an aPKC–Exocyst interaction mediated by Kibra. Using RNAi and small molecule inhibitors, the aPKCs, Kibra, and the Exocyst are shown to be required for NRK cell migration and it is further demonstrated that they are necessary for the localized activation of JNK at the leading edge. The migration associated control of JNK by aPKCs determines JNK phosphorylation of the plasma membrane substrate Paxillin, but not the phosphorylation of the nuclear JNK substrate, c-jun. This plasma membrane localized JNK cascade serves to control the stability of focal adhesion complexes, regulating migration. The study integrates the polarising behaviour of aPKCs with the pro-migratory properties of the Exocyst complex, defining a higher order complex associated with the localised activation of JNK at the leading edge of migrating cells that determines migration rate.
Author Summary
Cell migration is an essential process in multicellular organisms during such events as embryonic development, the immune response, and wound healing. Cell migration is also instrumental in the development of pathologies such as cancer cell invasion of healthy tissues. To make cells move, key molecules must be engaged in a coordinated manner; understanding which molecules, and how and when they work (for example, under physiological versus pathological conditions) will impact on new therapies designed to suppress abnormal migration. Migrating cells must coordinate two key processes: extension of the front or ‘leading’ edge of the cell and retraction of the back edge. Both processes require the turnover of protein assemblies known as focal adhesion complexes. In this paper we show that two different groups of regulators of migration – aPKC, a protein kinase, and exocyst, a complex of proteins also known to be required for exocytosis – interact physically via the scaffold protein kibra. All these components are required for efficient cell migration and all are enriched at the leading edge of moving cells, in a mutually dependent manner. At the leading edge, these components control the local activation of two additional protein kinases, ERK and JNK. The activation of ERK and JNK at the front of migrating cells in turn controls the phosphorylation of paxillin, a component of focal adhesions. Phosphorylation of paxillin is associated with the presence of more dynamic focal adhesions. Our data thus indicate that an aPKC-kibra-exocyst complex plays a crucial role in delivering local stimulatory signals to the leading edge of migrating cells.
doi:10.1371/journal.pbio.1000235
PMCID: PMC2762617  PMID: 19885391
7.  IKs response to protein kinase A-dependent KCNQ1 phosphorylation requires direct interaction with microtubules 
Cardiovascular Research  2008;79(3):427-435.
Aims
KCNQ1 (alias KvLQT1 or Kv7.1) and KCNE1 (alias IsK or minK) co-assemble to form the voltage-activated K+ channel responsible for IKs—a major repolarizing current in the human heart—and their dysfunction promotes cardiac arrhythmias. The channel is a component of larger macromolecular complexes containing known and undefined regulatory proteins. Thus, identification of proteins that modulate its biosynthesis, localization, activity, and/or degradation is of great interest from both a physiological and pathological point of view.
Methods and results
Using a yeast two-hybrid screening, we detected a direct interaction between β-tubulin and the KCNQ1 N-terminus. The interaction was confirmed by co-immunoprecipitation of β-tubulin and KCNQ1 in transfected COS-7 cells and in guinea pig cardiomyocytes. Using immunocytochemistry, we also found that they co-localized in cardiomyocytes. We tested the effects of microtubule-disrupting and -stabilizing agents (colchicine and taxol, respectively) on the KCNQ1–KCNE1 channel activity in COS-7 cells by means of the permeabilized-patch configuration of the patch-clamp technique. None of these agents altered IKs. In addition, colchicine did not modify the current response to osmotic challenge. On the other hand, the IKs response to protein kinase A (PKA)-mediated stimulation depended on microtubule polymerization in COS-7 cells and in cardiomyocytes. Strikingly, KCNQ1 channel and Yotiao phosphorylation by PKA—detected by phospho-specific antibodies—was maintained, as was the association of the two partners.
Conclusion
We propose that the KCNQ1–KCNE1 channel directly interacts with microtubules and that this interaction plays a major role in coupling PKA-dependent phosphorylation of KCNQ1 with IKs activation.
doi:10.1093/cvr/cvn085
PMCID: PMC2781743  PMID: 18390900
K+ channel; Myocytes; PKA; Signal transduction; KCNQ1; β-tubulin
8.  The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA 
The Journal of Cell Biology  2008;181(6):985-998.
Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.
doi:10.1083/jcb.200709076
PMCID: PMC2426946  PMID: 18541705
9.  The Ral/Exocyst Effector Complex Counters c-Jun N-Terminal Kinase-Dependent Apoptosis in Drosophila melanogaster▿ †  
Molecular and Cellular Biology  2006;26(23):8953-8963.
Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK.
doi:10.1128/MCB.00506-06
PMCID: PMC1636832  PMID: 17000765
10.  RalB Mobilizes the Exocyst To Drive Cell Migration†  
Molecular and Cellular Biology  2006;26(2):727-734.
The Ras family GTPases RalA and RalB have been defined as central components of the regulatory machinery supporting tumor initiation and progression. Although it is known that Ral proteins mediate oncogenic Ras signaling and physically and functionally interact with vesicle trafficking machinery, their mechanistic contribution to oncogenic transformation is unknown. Here, we have directly evaluated the relative contribution of Ral proteins and Ral effector pathways to cell motility and directional migration. Through loss-of-function analysis, we find that RalA is not limiting for cell migration in normal mammalian epithelial cells. In contrast, RalB and the Sec6/8 complex or exocyst, an immediate downstream Ral effector complex, are required for vectorial cell motility. RalB expression is required for promoting both exocyst assembly and localization to the leading edge of moving cells. We propose that RalB regulation of exocyst function is required for the coordinated delivery of secretory vesicles to the sites of dynamic plasma membrane expansion that specify directional movement.
doi:10.1128/MCB.26.2.727-734.2006
PMCID: PMC1346891  PMID: 16382162
11.  ARF6 controls post-endocytic recycling through its downstream exocyst complex effector 
The Journal of Cell Biology  2003;163(5):1111-1121.
The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.
doi:10.1083/jcb.200305029
PMCID: PMC2173613  PMID: 14662749
ARF6; exocyst complex; recycling; endocytosis; small GTP-binding protein
12.  A Ral Guanine Exchange Factor-Ral Pathway Is Conserved in Drosophila melanogaster and Sheds New Light on the Connectivity of the Ral, Ras, and Rap Pathways 
Molecular and Cellular Biology  2003;23(3):1112-1124.
Ras GTPases are central to many physiological and pathological signaling pathways and act via a combination of effectors. In mammals, at least three Ral exchange factors (RalGEFs) contain a Ras association domain and constitute a discrete subgroup of Ras effectors. Despite their ability to bind activated Rap as well as activated Ras, they seem to act downstream of Ras but not downstream of Rap. We have revisited the Ras/Rap-Ral connections in Drosophila melanogaster by using iterative two-hybrid screens with these three GTPases as primary baits and a subsequent genetic approach. We show that (i) the Ral-centered protein network appears to be extremely conserved in human and flies, (ii) in this network, RGL is a functional Drosophila orthologue of RalGEFs, and (iii) the RGL-Ral pathway functionally interacts with both the Ras and Rap pathways. Our data do not support the paradigmatic model where Ral is in the effector pathway of Ras. They reveal a signaling circuitry where Ral is functionally downstream of the Rap GTPase, at odds with the pathways described for mammalian cell lines. Thus, in vivo data show variations in the connectivity of pathways described for cell lines which might display only a subset of the biological possibilities.
doi:10.1128/MCB.23.3.1112-1124.2003
PMCID: PMC140692  PMID: 12529414
13.  Tyrosine Phosphorylation Regulates Alpha II Spectrin Cleavage by Calpain 
Molecular and Cellular Biology  2002;22(10):3527-3536.
Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of αII-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The αII-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the αI-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in αII-spectrin. Western blotting showed that αII-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of αII-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.
doi:10.1128/MCB.22.10.3527-3536.2002
PMCID: PMC133798  PMID: 11971983
14.  Ral GTPases Contribute to Regulation of Cyclin D1 through Activation of NF-κB 
Molecular and Cellular Biology  2000;20(21):8084-8092.
Ral GTPases have been implicated as mediators of Ras-induced signal transduction from observations that Ral-specific guanine nucleotide exchange factors associate with Ras and are activated by Ras. The cellular role of Ral family proteins is unclear, as is the contribution that Ral may make to Ras-dependent signaling. Here we show that expression of activated Ral in quiescent rodent fibroblasts is sufficient to induce activation of NF-κB-dependent gene expression and cyclin D1 transcription, two key convergence points for mitogenic and survival signaling. The regulation of cyclin D1 transcription by Ral is dependent on NF-κB activation and is mediated through an NF-κB binding site in the cyclin D1 promoter. Ral activation of these responses is likely through an as yet uncharacterized effector pathway, as we find activation of NF-κB and the cyclin D1 promoter by Ral is independent of association of Ral with active phospholipase D1 or Ral-binding protein 1, two proteins proposed to mediate Ral function in cells.
PMCID: PMC86418  PMID: 11027278
15.  Cloning and Characterization of SCHIP-1, a Novel Protein Interacting Specifically with Spliced Isoforms and Naturally Occurring Mutant NF2 Proteins 
Molecular and Cellular Biology  2000;20(5):1699-1712.
The neurofibromatosis type 2 (NF2) protein, known as schwannomin or merlin, is a tumor suppressor involved in NF2-associated and sporadic schwannomas and meningiomas. It is closely related to the ezrin-radixin-moesin family members, implicated in linking membrane proteins to the cytoskeleton. The molecular mechanism allowing schwannomin to function as a tumor suppressor is unknown. In attempt to shed light on schwannomin function, we have identified a novel coiled-coil protein, SCHIP-1, that specifically associates with schwannomin in vitro and in vivo. Within its coiled-coil region, this protein is homologous to human FEZ proteins and the related Caenorhabditis elegans gene product UNC-76. Immunofluorescent staining of transiently transfected cells shows a partial colocalization of SCHIP-1 and schwannomin, beneath the cytoplasmic membrane. Surprisingly, immunoprecipitation assays reveal that in a cellular context, association with SCHIP-1 can be observed only with some naturally occurring mutants of schwannomin, or a schwannomin spliced isoform lacking exons 2 and 3, but not with the schwannomin isoform exhibiting growth-suppressive activity. Our observations suggest that SCHIP-1 interaction with schwannomin is regulated by conformational changes in schwannomin, possibly induced by posttranslational modifications, alternative splicing, or mutations.
PMCID: PMC85353  PMID: 10669747
16.  hSiah2 Is a New Vav Binding Protein Which Inhibits Vav-Mediated Signaling Pathways 
Molecular and Cellular Biology  1999;19(5):3798-3807.
The hematopoietic proto-oncogene vav has been characterized as a Rac1-GDP/GTP exchanger protein which regulates cytoskeletal reorganization as well as signaling pathways leading to the activation of stress-activated protein kinases (SAPK/JNKs). Furthermore, vav overexpression enhances basal and T-cell receptor (TCR)-mediated stimulation of the nuclear factor of activated T cells (NFAT). We report here the interaction between Vav and hSiah2, a mammalian homolog of Drosophila Seven in absentia (Sina) that has been implicated in R7 photoreceptor cell formation during Drosophila eye development via the proteasome degradation pathway. Vav and hSiah2 interact in vitro and in vivo and colocalize in the cytoplasm of hematopoietic cells. The Src homology domain of Vav and the C-terminal region of hSiah2 are required for this interaction. We provide evidence for a negative regulation by hSiah2 of Vav-induced basal and TCR-mediated NFAT-dependent transcription. Overexpression of hSiah2 also inhibits the onco-Vav-induced JNK activation. Although the Vav-interacting domain is located in the C-terminal portion of hSiah2, the N-terminal region of hSiah2 is necessary for the inhibitory role that seems to be independent of the proteasome degradation.
PMCID: PMC84217  PMID: 10207103
17.  Activation of the Small GTPase Ral in Platelets 
Molecular and Cellular Biology  1998;18(5):2486-2491.
Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including α-thrombin. In contrast, the platelet antagonist prostaglandin I2 inhibited α-thrombin-induced Ral activation. Activation of Ral by α-thrombin could be inhibited by depletion of intracellular Ca2+, whereas the induction of intracellular Ca2+ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca2+ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.
PMCID: PMC110628  PMID: 9566869

Results 1-17 (17)