Proximity-dependent proteomics reveals extensive interactions of Protocadherin-19 with regulators of Rho GTPases and the microtubule cytoskeleton

AbstractProtocadherin-19 belongs to the cadherin family of cell surface receptors and has been shown to play essential roles in the development of the vertebrate nervous system. Mutations in human Protocadherin-19 (PCDH19) lead to PCDH19 Female-limited epilepsy (PCDH19 FLE) in humans, characterized by the early onset of epileptic seizures in children and a range of cognitive and behavioral problems in adults. Despite being considered the second most prevalent gene in epilepsy, very little is known about the intercellular pathways in which it participates. In order to characterize the protein complexes within which Pcdh19 functions, we generated Pcdh19-BioID fusion proteins and utilized proximity-dependent biotinylation to identify neighboring proteins. Proteomic identification and analysis revealed that the Pcdh19 interactome is enriched in proteins that regulate Rho family GTPases, microtubule binding proteins and proteins that regulate cell divisions. We cloned the centrosomal protein Nedd1 and the RacGEF Dock7 and verified their interactions with Pcdh19 in vitro. Our findings provide the first comprehensive insights into the interactome of Pcdh19, and provide a platform for future investigations into the cellular and molecular biology of this protein critical to the proper development of the nervous system.

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Rho family GTPases

Biochemical Society Transactions ◽

10.1042/bst20120103 ◽

2012 ◽

Vol 40 (6) ◽

pp. 1378-1382 ◽

Cited By ~ 292

Author(s):

Alan Hall

Keyword(s):

Actin Cytoskeleton ◽

Rho Gtpases ◽

Cellular Response ◽

Molecular Switches ◽

Effector Proteins ◽

Eukaryotic Cells ◽

Cell Behaviour ◽

Wide Range ◽

Rho Family Gtpases ◽

Rho Family

Rho GTPases comprise a family of molecular switches that control signal transduction pathways in eukaryotic cells. A conformational change induced upon binding GTP promotes an interaction with target (effector) proteins to generate a cellular response. A highly conserved function of Rho GTPases from yeast to humans is to control the actin cytoskeleton, although, in addition, they promote a wide range of other cellular activities. Changes in the actin cytoskeleton drive many dynamic aspects of cell behaviour, including morphogenesis, migration, phagocytosis and cytokinesis, and the dysregulation of Rho GTPases is associated with numerous human diseases and disorders.

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Differential activation and function of Rho GTPases during Salmonella–host cell interactions

The Journal of Cell Biology ◽

10.1083/jcb.200605144 ◽

2006 ◽

Vol 175 (3) ◽

pp. 453-463 ◽

Cited By ~ 188

Author(s):

Jayesh C. Patel ◽

Jorge E. Galán

Keyword(s):

Rho Gtpases ◽

Food Poisoning ◽

Bacterial Pathogen ◽

Cellular Responses ◽

Host Cells ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Rho Family Gtpases ◽

Rho Family ◽

And Function

Salmonella enterica, the cause of food poisoning and typhoid fever, has evolved sophisticated mechanisms to modulate Rho family guanosine triphosphatases (GTPases) to mediate specific cellular responses such as actin remodeling, macropinocytosis, and nuclear responses. These responses are largely the result of the activity of a set of bacterial proteins (SopE, SopE2, and SopB) that, upon delivery into host cells via a type III secretion system, activate specific Rho family GTPases either directly (SopE and SopE2) or indirectly (SopB) through the stimulation of an endogenous exchange factor. We show that different Rho family GTPases play a distinct role in Salmonella-induced cellular responses. In addition, we report that SopB stimulates cellular responses by activating SH3-containing guanine nucleotide exchange factor (SGEF), an exchange factor for RhoG, which we found plays a central role in the actin cytoskeleton remodeling stimulated by Salmonella. These results reveal a remarkable level of complexity in the manipulation of Rho family GTPases by a bacterial pathogen.

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Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions

International Journal of Molecular Sciences ◽

10.3390/ijms222212493 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12493

Author(s):

Niloufar Mosaddeghzadeh ◽

Neda S. Kazemein Jasemi ◽

Jisca Majolée ◽

Si-Cai Zhang ◽

Peter L. Hordijk ◽

...

Keyword(s):

Rho Gtpases ◽

Rho Gtpase ◽

Kinetic Mechanism ◽

Membrane Extraction ◽

Rho Protein ◽

Switch Regions ◽

Charged Clusters ◽

Rho Family ◽

Hydrophobic Cleft

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

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Spire contains actin binding domains and is related to ascidian posterior end mark-5

Development ◽

10.1242/dev.126.23.5267 ◽

1999 ◽

Vol 126 (23) ◽

pp. 5267-5274 ◽

Cited By ~ 5

Author(s):

A. Wellington ◽

S. Emmons ◽

B. James ◽

J. Calley ◽

M. Grover ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Posterior Pole ◽

Actin Binding ◽

Binding Domains ◽

Trap System ◽

Rho Family Gtpases ◽

Rho Family ◽

Interaction Trap ◽

Anterior Posterior

Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence supports the model that SPIRE links rho family signaling to the actin cytoskeleton.

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Rho-family GTPases modulate Ca2+-dependent ATP release from astrocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00175.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. C231-C241 ◽

Cited By ~ 43

Author(s):

Andrew E. Blum ◽

Sheldon M. Joseph ◽

Ronald J. Przybylski ◽

George R. Dubyak

Keyword(s):

Rho Gtpases ◽

Atp Release ◽

Rho Kinase ◽

Astrocytoma Cells ◽

Gap Junction Channel ◽

Rho Family Gtpases ◽

Clostridium Difficile Toxin ◽

Protease Activated Receptor 1 ◽

Rho Family ◽

Clostridium Difficile Toxin B

Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a Gq/phophospholipase C (PLC)/Ca2+ mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca2+ mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain Gq-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca2+-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.

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Pxl1p, a Paxillin-like Protein inSaccharomyces cerevisiae, May Coordinate Cdc42p and Rho1p Functions during Polarized Growth

Molecular Biology of the Cell ◽

10.1091/mbc.e04-01-0079 ◽

2004 ◽

Vol 15 (9) ◽

pp. 3977-3985 ◽

Cited By ~ 25

Author(s):

Xiang-Dong Gao ◽

Juliane P. Caviston ◽

Serguei E. Tcheperegine ◽

Erfei Bi

Keyword(s):

Dependent Manner ◽

Polarized Growth ◽

Amino Acid Sequence Homology ◽

Yeast Saccharomyces Cerevisiae ◽

Lim Domains ◽

Rho Family Gtpases ◽

Primary Determinant ◽

Rho Family ◽

Share Amino Acid Sequence

Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the “ancient paxillin” in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.

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The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading

The Journal of Cell Biology ◽

10.1083/jcb.200108030 ◽

2002 ◽

Vol 156 (2) ◽

pp. 377-388 ◽

Cited By ~ 36

Author(s):

Simona Degani ◽

Fiorella Balzac ◽

Mara Brancaccio ◽

Simona Guazzone ◽

Saverio Francesco Retta ◽

...

Keyword(s):

Cell Spreading ◽

Cytoplasmic Domain ◽

Nih3t3 Cells ◽

Cell Matrix ◽

Rho Family Gtpases ◽

Rho Family ◽

First Time ◽

Cell Matrix Adhesion

Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the β1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor–induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.

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The Large GTPase, GBP-2, Regulates Rho Family GTPases to Inhibit Migration and Invadosome Formation in Breast Cancer Cells

Cancers ◽

10.3390/cancers13225632 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5632

Author(s):

Geoffrey O. Nyabuto ◽

John P. Wilson ◽

Samantha A. Heilman ◽

Ryan C. Kalb ◽

Jonathan P. Kopacz ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Rho Gtpases ◽

Breast Cancer Cells ◽

Primary Site ◽

Common Cancer ◽

Rho Family Gtpases ◽

Rho Family ◽

4T1 Cells ◽

Guanylate Binding Protein

Breast cancer is the most common cancer in women. Despite advances in early detection and treatment, it is predicted that over 43,000 women will die of breast cancer in 2021. To lower this number, more information about the molecular players in breast cancer are needed. Guanylate-Binding Protein-2 has been correlated with better prognosis in breast cancer. In this study, we asked if the expression of GBP-2 in breast cancer merely provided a biomarker for improved prognosis or whether it actually contributed to improving outcome. To answer this, the 4T1 model of murine breast cancer was used. 4T1 cells themselves are highly aggressive and highly metastatic, while 67NR cells, isolated from the same tumor, do not leave the primary site. The expression of GBP-2 was examined in the two cell lines and found to be inversely correlated with aggressiveness/metastasis. Proliferation, migration, and invadosome formation were analyzed after altering the expression levels of GBP-2. Our experiments show that GBP-2 does not alter the proliferation of these cells but inhibits migration and invadosome formation downstream of regulation of Rho GTPases. Together these data demonstrate that GBP-2 is responsible for cell autonomous activities that make breast cancer cells less aggressive.

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A conceptual view at microtubule plus end dynamics in neuronal axons

10.1101/062711 ◽

2016 ◽

Author(s):

André Voelzmann ◽

Ines Hahn ◽

Simon P. Pearce ◽

Natalia Sánchez-Soriano ◽

Andreas Prokop

Keyword(s):

Nervous System ◽

Conceptual Understanding ◽

Molecular Mechanisms ◽

Protein Complexes ◽

Building Blocks ◽

Cellular Level ◽

Cellular Functions ◽

Dynamic Changes ◽

Plastic Rearrangement

AbstractAxons are the cable-like protrusions of neurons which wire up the nervous system. Polar bundles of microtubules (MTs) constitute their structural backbones and are highways for life-sustaining transport between proximal cell bodies and distal synapses. Any morphogenetic changes of axons during development, plastic rearrangement, regeneration or degeneration depend on dynamic changes of these MT bundles. A key mechanism for implementing such changes is the coordinated polymerisation and depolymerisation at the plus ends of MTs within these bundles. To gain an understanding of how such regulation can be achieved at the cellular level, we provide here an integrated overview of the extensive knowledge we have about the molecular mechanisms regulating MT de/polymerisation. We first summarise insights gained from work in vitro, then describe the machinery which supplies the essential tubulin building blocks, the protein complexes associating with MT plus ends, and MT shaft-based mechanisms that influence plus end dynamics. We briefly summarise the contribution of MT plus end dynamics to important cellular functions in axons, and conclude by discussing the challenges and potential strategies of integrating the existing molecular knowledge into conceptual understanding at the level of axons.

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Dictyostelium LIS1 Is a Centrosomal Protein Required for Microtubule/Cell Cortex Interactions, Nucleus/Centrosome Linkage, and Actin Dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.e05-01-0069 ◽

2005 ◽

Vol 16 (6) ◽

pp. 2759-2771 ◽

Cited By ~ 66

Author(s):

Markus Rehberg ◽

Julia Kleylein-Sohn ◽

Jan Faix ◽

Thi-Hieu Ho ◽

Irene Schulz ◽

...

Keyword(s):

Golgi Apparatus ◽

Traveling Waves ◽

Neuronal Migration ◽

Rho Gtpases ◽

Actin Polymerization ◽

Actin Dynamics ◽

Cell Cortex ◽

Centrosomal Protein ◽

Associated Functions

The widespread LIS1-proteins were originally identified as the target for sporadic mutations causing lissencephaly in humans. Dictyostelium LIS1 (DdLIS1) is a microtubule-associated protein exhibiting 53% identity to human LIS1. It colocalizes with dynein at isolated, microtubule-free centrosomes, suggesting that both are integral centrosomal components. Replacement of the DdLIS1 gene by the hypomorphic D327H allele or overexpression of an MBP-DdLIS1 fusion disrupted various dynein-associated functions. Microtubules lost contact with the cell cortex and were dragged behind an unusually motile centrosome. Previously, this phenotype was observed in cells overexpressing fragments of dynein or the XMAP215-homologue DdCP224. DdLIS1 was coprecipitated with DdCP224, suggesting that both act together in dynein-mediated cortical attachment of microtubules. Furthermore, DdLIS1-D327H mutants showed Golgi dispersal and reduced centrosome/nucleus association. Defects in DdLIS1 function also altered actin dynamics characterized by traveling waves of actin polymerization correlated with a reduced F-actin content. DdLIS1 could be involved in actin dynamics through Rho-GTPases, because DdLIS1 interacted directly with Rac1A in vitro. Our results show that DdLIS1 is required for maintenance of the microtubule cytoskeleton, Golgi apparatus and nucleus/centrosome association, and they suggest that LIS1-dependent alterations of actin dynamics could also contribute to defects in neuronal migration in lissencephaly patients.

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