scholarly journals PAK and other Rho-associated kinases – effectors with surprisingly diverse mechanisms of regulation

2005 ◽  
Vol 386 (2) ◽  
pp. 201-214 ◽  
Author(s):  
Zhou-shen ZHAO ◽  
Ed MANSER
Keyword(s):  
Rho Gtpases ◽  
Cell Biology ◽  
Rho Gtpase ◽  
Rho Kinase ◽  
Molecular Switches ◽  
Eukaryotic Cell ◽  
Effector Proteins ◽  
Downstream Effector ◽  
P21 Activated Kinase ◽  
Do So

The Rho GTPases are a family of molecular switches that are critical regulators of signal transduction pathways in eukaryotic cells. They are known principally for their role in regulating the cytoskeleton, and do so by recruiting a variety of downstream effector proteins. Kinases form an important class of Rho effector, and part of the biological complexity brought about by switching on a single GTPase results from downstream phosphorylation cascades. Here we focus on our current understanding of the way in which different Rho-associated serine/threonine kinases, denoted PAK (p21-activated kinase), MLK (mixed-lineage kinase), ROK (Rho-kinase), MRCK (myotonin-related Cdc42-binding kinase), CRIK (citron kinase) and PKN (protein kinase novel), interact with and are regulated by their partner GTPases. All of these kinases have in common an ability to dimerize, and in most cases interact with a variety of other proteins that are important for their function. A diversity of known structures underpin the Rho GTPase–kinase interaction, but only in the case of PAK do we have a good molecular understanding of kinase regulation. The ability of Rho GTPases to co-ordinate spatial and temporal phosphorylation events explains in part their prominent role in eukaryotic cell biology.

scholarly journals Distinct Rho GTPase Activities Regulate Epithelial Cell Localization of the Adhesion Molecule CEACAM1: Involvement of the CEACAM1 Transmembrane Domain

2003 ◽  
Vol 23 (20) ◽  
pp. 7291-7304 ◽  
Author(s):  
Bénédicte Fournès ◽  
Jennifer Farrah ◽  
Melanie Olson ◽  
Nathalie Lamarche-Vane ◽  
Nicole Beauchemin
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Transmembrane Domain ◽  
Rho Kinase ◽  
Cell Contacts ◽  
Cell Localization ◽  
Downstream Effector ◽  
Cell Cell

ABSTRACT CEACAM1 is an intercellular adhesion glycoprotein. As CEACAM1 plays an important role in epithelial cell signaling and functions, we have examined its localization in epithelial cells. We have observed that distribution at cell contacts is not always seen in these cells, suggesting that CEACAM1 localization might be regulated. In Swiss 3T3 cells, the targeting of CEACAM1 at cell-cell boundaries is regulated by the Rho GTPases. In the present study, we have used the MDCK epithelial cells to characterize the effects of the Rho GTPases and their effectors on CEACAM1 intercellular targeting. Activated Cdc42 and Rac1 or their downstream effector PAK1 targeted CEACAM1 to sites of cell-cell contacts. On the other hand, neither activated RhoA nor activated Rho kinase directed CEACAM1 to cell boundaries, resulting in a condensed distribution of CEACAM1 at the cell surface. Interestingly, inhibition of this pathway resulted in CEACAM1 intercellular localization suggesting that a tightly regulated balance of Rho GTPase activities is necessary to target CEACAM1 at cell-cell boundaries. In addition, using CEACAM1 mutants and chimeric fusion constructs containing domains of the colony-stimulating factor receptor, we have shown that the transmembrane domain of CEACAM1 is responsible for the Cdc42-induced targeting at cell-cell contacts.

scholarly journals The GTP/GDP Cycling of Rho GTPase TCL Is an Essential Regulator of the Early Endocytic Pathway

2003 ◽  
Vol 14 (12) ◽  
pp. 4846-4856 ◽  
Author(s):  
Marion de Toledo ◽  
Francesca Senic-Matuglia ◽  
Jean Salamero ◽  
Gilles Uze ◽  
Franck Comunale ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Early Endosome ◽  
Effector Proteins ◽  
Endocytic Pathway ◽  
Actin Dynamics ◽  
Recycling Endosomes ◽  
Downstream Effector ◽  
C Terminus ◽  
Interfering Rna

Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1–positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1–positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.

Abstract 524: Identification of Roles for the Rho-Specific Guanine Nucleotide Dissociation Inhibitor (RhoGDI) Ly-Gdi in Platelet Function

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Anh T Ngo ◽  
Marisa L Thierheimer ◽  
Özgün Babur ◽  
Anne D Rocheleau ◽  
Xiaolin Nan ◽  
...  
Keyword(s):  
Platelet Function ◽  
Rho Gtpases ◽  
Cell Biology ◽  
Rho Gtpase ◽  
Thrombus Formation ◽  
Super Resolution ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Morphological Alterations ◽  
Guanine Nucleotide Dissociation Inhibitor

Introduction: Upon activation, platelets undergo specific morphological alterations critical to hemostatic plug and thrombus formation via actin cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in regulating platelet function. Methods and Hypothesis: Through an approach combining pharmacology, cell biology and systems biology methods we assessed the hypothesis that RhoGDI proteins regulate Rho GTPase-driven platelet functions downstream of platelet integrin and glycoprotein receptors. Results: We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular staining and super resolution microscopy assays find that Ly-GDI displays an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI in platelets, as Ly-GDI is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Notably, inhibition of PKC diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Conclusion: In conclusion, our results support roles for Ly-GDI as a localized regulator of Rho GTPases in platelets and link PKC and Rho GTPase signaling systems to platelet function.

Rho family GTPases

2012 ◽  
Vol 40 (6) ◽  
pp. 1378-1382 ◽  
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.

scholarly journals Targeting and activation of Rac1 are mediated by the exchange factor β-Pix

2006 ◽  
Vol 172 (5) ◽  
pp. 759-769 ◽  
Author(s):  
Jean Paul ten Klooster ◽  
Zahara M. Jaffer ◽  
Jonathan Chernoff ◽  
Peter L. Hordijk
Keyword(s):  
Rho Gtpases ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Exchange Factor ◽  
Downstream Signaling ◽  
Intracellular Targeting ◽  
Dynamics And Control ◽  
P21 Activated Kinase ◽  
Gtpase Activation

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator β-Pix (p21-activated kinase [Pak]–interacting exchange factor). The interaction with β-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1–β-Pix interaction is required for Rac1 activation by β-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the β-Pix–binding kinase Pak1, we show that Pak1 regulates the Rac1–β-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor β-Pix.

scholarly journals Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase Acts as a Cdc42 Effector in Promoting Cytoskeletal Reorganization

1998 ◽  
Vol 18 (1) ◽  
pp. 130-140 ◽  
Author(s):  
Thomas Leung ◽  
Xiang-Qun Chen ◽  
Ivan Tan ◽  
Edward Manser ◽  
Louis Lim
Keyword(s):  
Myotonic Dystrophy ◽  
Rho Gtpases ◽  
Pleckstrin Homology Domain ◽  
Cell Periphery ◽  
Cytoskeletal Reorganization ◽  
Binding Domains ◽  
Downstream Effector ◽  
Kinase C ◽  
Multidomain Structure ◽  
P21 Activated Kinase

ABSTRACT The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These ∼190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C andn-chimaerin and a putative pleckstrin homology domain. MRCKα and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCKα resulted in actin and myosin reorganization. Expression of kinase-dead MRCKα blocked Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCKα mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCKα plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCKα mutants. These results suggest that MRCKα may act as a downstream effector of Cdc42 in cytoskeletal reorganization.

scholarly journals Competitive Inhibitors of Ras Effector Binding

2019 ◽  
Author(s):  
Svenja Wiechmann ◽  
Pierre Maisonneuve ◽  
Britta M. Grebbin ◽  
Meike Hoffmeister ◽  
Manuel Kaulich ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Growth Arrest ◽  
Molecular Switches ◽  
Small Gtpases ◽  
Effector Proteins ◽  
Ras Signaling ◽  
Downstream Effector ◽  
Competitive Inhibitors ◽  
Tumorigenic Cells ◽  
Effector Binding

AbstractThe small GTPases H, K and NRas are molecular switches that are indispensable for the correct regulation of cellular proliferation and growth. Mutations in Ras are associated with cancer and result in unwanted activation of signaling processes caused by aberrant recruitment of downstream effector proteins. In this study, we have engineered variants of the Ras-binding domain (RBD) of CRAF kinase that bind with highly improved affinity the effector binding site of Ras. Structural characterization demonstrates how the engineered RBD variants outcompete effector binding and inhibit Ras signaling in cells leading to apoptosis, growth arrest and senescence. The optimized RBD variants provide new insights in Ras biology and enabled the functional stratification of Ras dependency in patient-derived colorectal cancer organoids.One sentence summaryInhibition of effector kinase binding to Ras induces senescence in non-tumorigenic cells and reveals Ras dependency in patient-derived cancer organoids.

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

2012 ◽  
Vol 441 (3) ◽  
pp. 869-880 ◽  
Author(s):  
Erik L. Anderson ◽  
Michael J. Hamann
Keyword(s):  
Rho Gtpases ◽  
Regulatory Protein ◽  
Effector Proteins ◽  
Luciferase Assay ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
P21 Activated Kinase ◽  
Rho Gef ◽  
Split Luciferase

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.

scholarly journals Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling

2019 ◽  
Author(s):  
Michelle S. Lu ◽  
David G. Drubin
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Rho Gtpase ◽  
Molecular Switches ◽  
Cell Types ◽  
Eukaryotic Cell ◽  
Small Gtpases ◽  
Cytoskeletal Remodeling ◽  
Rho Family

AbstractSmall GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none involved in the maintenance of nuclear envelope integrity or endoplasmic reticulum (ER) morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission, and by genetic analysis, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.SummaryThe small Rho GTPase Cdc42 is a well-known regulator of cytoskeletal rearrangement and polarity development in all eukaryotic cell types. Here, Lu and Drubin report the serendipitous discovery of a novel Cdc42-ESCRT-nuclear envelope/endoplasmic reticulum connection.

scholarly journals Ephrin-A5 Induces Collapse of Growth Cones by Activating Rho and Rho Kinase

2000 ◽  
Vol 149 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Siegfried Wahl ◽  
Holger Barth ◽  
Thomas Ciossek ◽  
Klaus Aktories ◽  
Bernhard K. Mueller
Keyword(s):  
Ganglion Cell ◽  
Tyrosine Kinases ◽  
Rho Gtpase ◽  
Rho Kinase ◽  
Signal Transduction Pathway ◽  
Specific Inhibitor ◽  
Growth Cones ◽  
Small Gtpase ◽  
Eph Receptor ◽  
Downstream Effector

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5–treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.

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