scholarly journals Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

2012 ◽  
Vol 23 (20) ◽  
pp. 4008-4019 ◽  
Author(s):  
Ryan Ard ◽  
Kirk Mulatz ◽  
Hanan Abramovici ◽  
Jean-Christian Maillet ◽  
Alexandra Fottinger ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Critical Factor ◽  
Diacylglycerol Kinase ◽  
Rhoa Activity ◽  
Signaling Complex ◽  
Rhoa Activation ◽  
C Terminus ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Protein Kinase Cα ◽  
Underlying Mechanisms

Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

scholarly journals MARCKS domain phosphorylation regulates the differential interaction of Diacylglycerol Kinase ζ with Rac1, RhoA and Syntrophin

2020 ◽  
Author(s):  
Ryan Ard ◽  
Jean-Christian Maillet ◽  
Elias Daher ◽  
Michael Phan ◽  
Radoslav Zinoviev ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Pdz Domain ◽  
Diacylglycerol Kinase ◽  
Binding Motif ◽  
Membrane Blebbing ◽  
Rhoa Activity ◽  
C Terminus ◽  
Mechanistic Basis

AbstractCells can switch between Rac1, lamellipodia-based and RhoA, blebbing-based migration modes but the molecular mechanisms regulating this choice are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation. Instead, DGKζ functions as a scaffold that stimulates RhoA release by enhancing RhoGDI phosphorylation by protein kinase Cα (PKCα). Here, PKCα-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification increased binding of the DGKζ C-terminus to the α1-syntrophin PDZ domain. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the Rho-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism involving DGKζ phosphorylation by PKCα that favours RhoA-driven blebbing over Rac1-driven lamellipodia formation and macropinocytosis. These findings provide a mechanistic basis for the effect of PKCα signaling on Rho GTPase activity and suggest PKCα activity plays a role in the interconversion between Rac1 and RhoA signaling that underlies different migration modes.

scholarly journals GDI-1 Phosphorylation Switch at Serine 96 Induces RhoA Activation and Increased Endothelial Permeability

2007 ◽  
Vol 27 (18) ◽  
pp. 6323-6333 ◽  
Author(s):  
Nebojsa Knezevic ◽  
Arun Roy ◽  
Barbara Timblin ◽  
Maria Konstantoulaki ◽  
Tiffany Sharma ◽  
...  
Keyword(s):  
Endothelial Permeability ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Mutant Form ◽  
Actin Stress Fiber ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Stress Fiber Formation ◽  
C Terminus ◽  
Actin Stress Fiber Formation

ABSTRACT We identified the GDI-1-regulated mechanism of RhoA activation from the Rho-GDI-1 complex and its role in mediating increased endothelial permeability. Thrombin stimulation failed to induce RhoA activation and actin stress fiber formation in human pulmonary arterial endothelial cells transduced with full-length GDI-1. Expression of a GDI-1 mutant form (C-GDI) containing the C terminus (aa 69 to 204) also prevented RhoA activation, whereas further deletions failed to alter RhoA activation. We observed that protein kinase Cα-mediated phosphorylation of the C terminus of GDI-1 at Ser96 reduced the affinity of GDI-1 for RhoA and thereby enabled RhoA activation. Rendering GDI-1 phosphodefective with a Ser96 → Ala substitution rescued the inhibitory activity of GDI-1 toward RhoA but did not alter the thrombin-induced activation of other Rho GTPases, i.e., Rac1 and Cdc42. Phosphodefective mutant GDI-1 also suppressed myosin light chain phosphorylation, actin stress fiber formation, and the increased endothelial permeability induced by thrombin. In contrast, expressing the phospho-mimicking mutant S96D-GDI-1 protein induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of the phosphorylation of the C terminus of GDI-1 at S96 in selectively activating RhoA. Inhibiting GDI-1 phosphorylation at S96 is a potential therapeutic target for modulating RhoA activity and thus preventing the increase in endothelial permeability associated with vascular inflammation.

scholarly journals Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

2017 ◽  
Vol 43 (5) ◽  
pp. 1777-1789 ◽  
Author(s):  
Lei Zhang ◽  
Tianrong Ji ◽  
Qin Wang ◽  
Kexin Meng ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Protective Action ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Dependent Manner ◽  
Calcium Sensing Receptor ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Calcium Sensing

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

scholarly journals Postprenylation CAAX Processing Is Required for Proper Localization of Ras but Not Rho GTPases

2005 ◽  
Vol 16 (4) ◽  
pp. 1606-1616 ◽  
Author(s):  
David Michaelson ◽  
Wasif Ali ◽  
Vi K. Chiu ◽  
Martin Bergo ◽  
Joseph Silletti ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Protein Trafficking ◽  
Rho Gtpases ◽  
Ras Proteins ◽  
Rho Proteins ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Individual Contributions ◽  
The Individual ◽  
And Function

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal– AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the –AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

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.

Suppression of Synaptotagmin II restrains phorbolester-induced downregulation of protein kinase Cα by diverting the kinase from a degradative pathway to the recycling endocytic compartment

2002 ◽  
Vol 115 (15) ◽  
pp. 3083-3092 ◽  
Author(s):  
Ze Peng ◽  
Elena Grimberg ◽  
Ronit Sagi-Eisenberg
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Secretory Granules ◽  
Critical Factor ◽  
Cellular Level ◽  
Endocytic Compartment ◽  
Early Endosomes ◽  
Protein Kinase Cα ◽  
Rbl Cells

Downregulation of protein kinase Cα (PKCα) following long-term exposure to phorbol esters such as TPA is traffic dependent and involves delivery of the active, membrane-associated PKCα to endosomes. In this study, we show that synaptotagmin II (Syt II), a member of the Syt family of proteins, is required for TPA-induced degradation of PKCα. Thus, whereas the kinase half-life in TPA-treated cultured mast cells (the mast cell line rat basophilic leukemia RBL-2H3) is 2 hours, it is doubled in RBL-Syt II- cells, in which the cellular level of Syt II is reduced by>95% by transfection with Syt II antisense cDNA. We demonstrate that in TPA-treated RBL cells, PKCα travels from the cytosol to the plasma membrane, where it is delivered to early endosomes on its route to degradation. By contrast, in TPA-treated RBL-Syt II- cells,PKCα is diverted to recycling endosomes and remains distributed between the plasma membrane and the perinuclear recycling endocytic compartment. Notably, in both RBL and RBL-Syt II- cells, a fraction of PKCα is delivered and maintained in the secretory granules (SG). These results implicate Syt II as a critical factor for the delivery of internalized cargo for degradation. As shown here, one consequence of Syt II suppression is a delay in PKCα downregulation, resulting in its prolonged signaling.

scholarly journals Differential Localization of Rho Gtpases in Live Cells

2001 ◽  
Vol 152 (1) ◽  
pp. 111-126 ◽  
Author(s):  
David Michaelson ◽  
Joseph Silletti ◽  
Gretchen Murphy ◽  
Peter D'Eustachio ◽  
Mark Rush ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Essential Feature ◽  
Hypervariable Region ◽  
Dominant Negative ◽  
Live Cells ◽  
Rho Proteins ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Membrane Compartment ◽  
Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

scholarly journals Disrupted filamin A/αIIbβ3 interaction induces macrothrombocytopenia by increasing RhoA activity

Blood ◽  
2019 ◽  
Vol 133 (16) ◽  
pp. 1778-1788 ◽  
Author(s):  
Alessandro Donada ◽  
Nathalie Balayn ◽  
Dominika Sliwa ◽  
Larissa Lordier ◽  
Valentina Ceglia ◽  
...  
Keyword(s):  
Cell Model ◽  
Filamin A ◽  
Rhoa Activity ◽  
Cellular Processes ◽  
Rhoa Activation ◽  
Actomyosin Contractility ◽  
Stem Cell Model ◽  
Proplatelet Formation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Filamin A (FLNa) links the cell membrane with the cytoskeleton and is central in several cellular processes. Heterozygous mutations in the X-linked FLNA gene are associated with a large spectrum of conditions, including macrothrombocytopenia, called filaminopathies. Using an isogenic pluripotent stem cell model derived from patients, we show that the absence of the FLNa protein in megakaryocytes (MKs) leads to their incomplete maturation, particularly the inability to produce proplatelets. Reduction in proplatelet formation potential is associated with a defect in actomyosin contractility, which results from inappropriate RhoA activation. This dysregulated RhoA activation was observed when MKs were plated on fibrinogen but not on other matrices (fibronectin, vitronectin, collagen 1, and von Willebrand factor), strongly suggesting a role for FLNa/αIIbβ3 interaction in the downregulation of RhoA activity. This was confirmed by experiments based on the overexpression of FLNa mutants deleted in the αIIbβ3-binding domain and the RhoA-interacting domain, respectively. Finally, pharmacological inhibition of the RhoA-associated kinase ROCK1/2 restored a normal phenotype and proplatelet formation. Overall, this work suggests a new etiology for macrothrombocytopenia, in which increased RhoA activity is associated with disrupted FLNa/αIIbβ3 interaction.

scholarly journals Molecular Mechanism of the Negative Regulation of Smad1/5 Protein by Carboxyl Terminus of Hsc70-interacting Protein (CHIP)

2011 ◽  
Vol 286 (18) ◽  
pp. 15883-15894 ◽  
Author(s):  
Le Wang ◽  
Yi-Tong Liu ◽  
Rui Hao ◽  
Lei Chen ◽  
Zhijie Chang ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Protein Chip ◽  
Independent Manner ◽  
Interacting Protein ◽  
Signaling Complex ◽  
Bone Morphogenetic Protein Pathway ◽  
C Terminus ◽  
Shock Proteins

The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner.

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