scholarly journals S-Palmitoylation-Dependent Regulation of Cardiomyocyte Rac1 Signaling Activity and Cardiac Hypertrophy

2021 ◽  
Author(s):  
Matthew J Brody ◽  
Tanya A. Baldwin ◽  
Arasakumar Subramani ◽  
Onur Kanisicak ◽  
Ronald J Vagnozzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transgenic Mice ◽  
Cardiac Disease ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Transferase Activity ◽  
Related Enzyme ◽  
Rho Family ◽  
Novel Therapeutic Strategies

S-palmitoylation is a reversible lipid modification that regulates trafficking, localization, activity, and/or stability of protein substrates by serving as a fatty acid anchor to cell membranes. However, S-palmitoylation-dependent control of signal transduction in cardiomyocytes and its effects on cardiac physiology are not well understood. We performed an in vivo gain-of-function screen of zinc finger Asp-His-His-Cys (zDHHC) family S-acyl transferases that catalyze S-palmitoylation and identified the Golgi-localized enzyme zDHHC3 as a critical regulator of cardiac maladaptation. The closely-related enzyme, zDHHC7, also induced severe cardiomyopathy but this effect was not observed with overexpression of plasma membrane enzyme zDHHC5, endoplasmic reticulum enzyme zDHHC6, or Golgi enzyme zDHHC13. To identify effectors that may underlie zDHHC3-induced cardiomyopathy we performed quantitative site-specific S-acyl proteomics in zDHHC3-overexpressing cells that revealed the small GTPase Rac1 as a novel substrate. We generated cardiomyocyte-specific transgenic mice overexpressing zDHHC3, which develop severe cardiac disease. Cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice are preceded by enhanced S-palmitoylation of Rac1 and induction of additional Rho family small GTPases including RhoA, Cdc42, and the Rho family-specific chaperone RhoGDI. In contrast, transgenic mice overexpressing an enzymatically-dead mutant of zDHHC3 do not exhibit this profound induction of RhoGTPase signaling or develop cardiac disease. Rac1 S-palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts. Taken together, these data suggest inhibition of zDHHC3/7 S-acyl transferase activity at the cardiomyocyte Golgi or disruption of Rac1 S-palmitoylation as novel therapeutic strategies to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.

scholarly journals PseudoGTPase domains in p190RhoGAP proteins: a mini-review

2018 ◽  
Vol 46 (6) ◽  
pp. 1713-1720 ◽  
Author(s):  
Amy L. Stiegler ◽  
Titus J. Boggon
Keyword(s):  
Family Members ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Signaling Proteins ◽  
New Family ◽  
Gtpase Activating Proteins ◽  
Rho Family ◽  
Biochemical Analyses ◽  
Catalytic Cleft

Pseudoenzymes generally lack detectable catalytic activity despite adopting the overall protein fold of their catalytically competent counterparts, indeed ‘pseudo’ family members seem to be incorporated in all enzyme classes. The small GTPase enzymes are important signaling proteins, and recent studies have identified many new family members with noncanonical residues within the catalytic cleft, termed pseudoGTPases. To illustrate recent discoveries in the field, we use the p190RhoGAP proteins as an example. p190RhoGAP proteins (ARHGAP5 and ARHGAP35) are the most abundant GTPase activating proteins for the Rho family of small GTPases. These are key regulators of Rho signaling in processes such as cell migration, adhesion and cytokinesis. Structural biology has complemented and guided biochemical analyses for these proteins and has allowed discovery of two cryptic pseudoGTPase domains, and the re-classification of a third, previously identified, GTPase-fold domain as a pseudoGTPase. The three domains within p190RhoGAP proteins illustrate the diversity of this rapidly expanding pseudoGTPase group.

scholarly journals Identification and characterization of a new isoform of small GTPase RhoE

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Yuan Dai ◽  
Weijia Luo ◽  
Xiaojing Yue ◽  
Wencai Ma ◽  
Jing Wang ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Biological Functions ◽  
Coding Region ◽  
Alternative Translation ◽  
Rho Family ◽  
Binding Capability ◽  
Identification And Characterization

Abstract The Rho family of GTPases consists of 20 members including RhoE. Here, we discover the existence of a short isoform of RhoE designated as RhoEα, the first Rho GTPase isoform generated from alternative translation. Translation of this new isoform is initiated from an alternative start site downstream of and in-frame with the coding region of the canonical RhoE. RhoEα exhibits a similar subcellular distribution while its protein stability is higher than RhoE. RhoEα contains binding capability to RhoE effectors ROCK1, p190RhoGAP and Syx. The distinct transcriptomes of cells with the expression of RhoE and RhoEα, respectively, are demonstrated. The data propose distinctive and overlapping biological functions of RhoEα compared to RhoE. In conclusion, this study reveals a new Rho GTPase isoform generated from alternative translation. The discovery provides a new scope of understanding the versatile functions of small GTPases and underlines the complexity and diverse roles of small GTPases.

scholarly journals Co-ordinated Ras and Rac activity shapes macropinocytic cups and enables phagocytosis of geometrically diverse bacteria

2019 ◽  
Author(s):  
Catherine M. Buckley ◽  
Henderikus Pots ◽  
Aurelie Gueho ◽  
Ben A. Phillips ◽  
Bernd Gilsbach ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Extracellular Material ◽  
Multidomain Protein ◽  
Complex Shapes ◽  
Rho Family ◽  
Phagocytic Cups ◽  
Interior Domain ◽  
Professional Phagocyte

AbstractEngulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialised cup shaped protrusions. To effectively capture and internalise their targets, these cups are organised into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac and phospholipid interactions to localise at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, RGBARG shapes the protrusion by driving Rac activation at the rim whilst suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of co-ordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.

Angiopoietin-related growth factor (AGF) promotes angiogenesis

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3760-3765 ◽  
Author(s):  
Yuichi Oike ◽  
Yasuhiro Ito ◽  
Hiromitsu Maekawa ◽  
Tohru Morisada ◽  
Yoshiaki Kubota ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transgenic Mice ◽  
Vascular Endothelial Cells ◽  
Direct Injection ◽  
Angiogenic Factor ◽  
Epidermal Keratinocytes ◽  
Vascular Endothelial ◽  
Novel Therapeutic Strategies

Abstract We report here the identification of angiopoietin-related growth factor (AGF) as a positive mediator for angiogenesis. To investigate the biologic function of AGF in angiogenesis, we analyzed the vasculature in the dermis of transgenic mice expressing AGF in mouse epidermal keratinocytes (K14-AGF). K14-AGF transgenic mice were grossly red, especially in the ears and snout, suggesting that hypervascularization had occurred in their skin. Histologic examination of ear skin from K14-AGF transgenic mice revealed increased numbers of microvessels in the dermis, whereas the expression of several angiogenic factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factors (VEGFs), and angiopoietin-1 (Ang-1), was decreased. We showed that AGF is a secreted protein and does not bind to tyrosine kinase with immunoglobulin and EGF-homology domain (Tie1) or Tie2 receptors. An in vitro chamber assay revealed that AGF directly promotes chemotactic activity of vascular endothelial cells. Both mouse corneal and matrigel plug assays showed that AGF induces neovascularization in vivo. Furthermore, we found that plasma leakage occurred after direct injection of AGF into the mouse dermis, suggesting that AGF directly induces a permeability change in the local vasculature. On the basis of these observations, we propose that AGF is a novel angiogenic factor and that handling of its biologic functions could lead to novel therapeutic strategies for control of angiogenesis.

scholarly journals Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

2011 ◽  
Vol 22 (2) ◽  
pp. 189-201 ◽  
Author(s):  
Roman Gorelik ◽  
Changsong Yang ◽  
Vasumathi Kameswaran ◽  
Roberto Dominguez ◽  
Tatyana Svitkina
Keyword(s):  
Plasma Membrane ◽  
Electrostatic Interactions ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Coincidence Detection ◽  
Membrane Targeting ◽  
Amino Terminal ◽  
N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

scholarly journals Regulation of the Rho Family Small GTPase Wrch-1/RhoU by C-Terminal Tyrosine Phosphorylation Requires Src

2010 ◽  
Vol 30 (17) ◽  
pp. 4324-4338 ◽  
Author(s):  
Jamie K. Alan ◽  
Anastacia C. Berzat ◽  
Brian J. Dewar ◽  
Lee M. Graves ◽  
Adrienne D. Cox
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Three Dimensional ◽  
Small Gtpase ◽  
Cell Morphogenesis ◽  
New Paradigm ◽  
Nonreceptor Tyrosine Kinase ◽  
Serum Stimulation ◽  
Rho Family

ABSTRACT Wrch-1 is an atypical Rho family small GTPase with roles in migration, epithelial cell morphogenesis, osteoclastogenesis, and oncogenic transformation. Here, we observed rapid relocalization of Wrch-1 from the plasma membrane upon serum stimulation. Studies revealed a requirement for serum-stimulated tyrosine phosphorylation of Wrch-1 at residue Y254 within its C-terminal membrane targeting domain, mediated by the nonreceptor tyrosine kinase Src. Genetic or pharmacological loss of Src kinase activity blocked both phosphorylation and relocalization of Wrch-1. Functionally, Y254 was required for proper Wrch-1 modulation of cystogenesis in three-dimensional culture, and the phospho-deficient mutant, Y254F, was enhanced in Wrch-1-mediated anchorage-independent growth. Mechanistically, C-terminal tyrosine phosphorylation and subsequent relocalization of Wrch-1 downregulated its ability to interact with and activate its effectors by decreasing active Wrch-1-GTP, perhaps by altering proximity to a GEF or GAP. Phospho-deficient Wrch-1(Y254F) remained at the plasma membrane and GTP bound and continued to recruit and activate its effector PAK, even upon serum stimulation. In contrast, a phospho-mimetic mutant, Y254E, was constitutively endosomally localized and GDP bound and failed to recruit PAK unless mutated to be constitutively active/GAP insensitive. C-terminal tyrosine phosphorylation thus represents a new paradigm in posttranslational control of small GTPase localization, activation, and biological function.

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst

2010 ◽  
Vol 38 (2) ◽  
pp. 723-728 ◽  
Author(s):  
Viktor Žárský ◽  
Martin Potocký
Keyword(s):  
Plasma Membrane ◽  
Plant Cell ◽  
Secretory Pathway ◽  
Root Hairs ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Cell Cortex ◽  
Membrane Phospholipids ◽  
Recycling Endosome ◽  
Regulatory Module

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a ‘recycling domain’ (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase–effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

scholarly journals The small GTPases ARL-13 and ARL-3 coordinate intraflagellar transport and ciliogenesis

2010 ◽  
Vol 189 (6) ◽  
pp. 1039-1051 ◽  
Author(s):  
Yujie Li ◽  
Qing Wei ◽  
Yuxia Zhang ◽  
Kun Ling ◽  
Jinghua Hu
Keyword(s):  
Intraflagellar Transport ◽  
Adenosine Diphosphate ◽  
Joubert Syndrome ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Causal Gene ◽  
Guanosine Triphosphatase ◽  
Bidirectional Movement ◽  
Dependent Pathway

Intraflagellar transport (IFT) machinery mediates the bidirectional movement of cargos that are required for the assembly and maintenance of cilia. However, little is known about how IFT is regulated in vivo. In this study, we show that the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor–like protein 13 (ARL-13) encoded by the Caenorhabditis elegans homologue of the human Joubert syndrome causal gene ARL13B, localizes exclusively to the doublet segment of the cilium. arl-13 mutants have shortened cilia with various ultrastructural deformities and a disrupted association between IFT subcomplexes A and B. Intriguingly, depletion of ARL-3, another ciliary small GTPase, partially suppresses ciliogenesis defects in arl-13 mutants by indirectly restoring binding between IFT subcomplexes A and B. Rescue of arl-13 mutants by ARL-3 depletion is mediated by an HDAC6 deacetylase-dependent pathway. Thus, we propose that two conserved small GTPases, ARL-13 and ARL-3, coordinate to regulate IFT and that perturbing this balance results in cilia deformation.

scholarly journals Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation.

1996 ◽  
Vol 16 (6) ◽  
pp. 2689-2699 ◽  
Author(s):  
R Foster ◽  
K Q Hu ◽  
Y Lu ◽  
K M Nolan ◽  
J Thissen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structural Features ◽  
Biochemical Properties ◽  
Small Gtpases ◽  
Evolutionary Divergence ◽  
Gtpase Activity ◽  
Activated State ◽  
Rho Protein ◽  
Rho Family

We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.

scholarly journals Defective Dendrite Elongation but Normal Fertility in Mice Lacking the Rho-Like GTPase Activator Dbl

2002 ◽  
Vol 22 (9) ◽  
pp. 3140-3148 ◽  
Author(s):  
Emilio Hirsch ◽  
Michela Pozzato ◽  
Alessandro Vercelli ◽  
Laura Barberis ◽  
Ornella Azzolino ◽  
...  
Keyword(s):  
Pyramidal Neurons ◽  
Embryonic Stem ◽  
Large Family ◽  
Small Gtpases ◽  
Normal Morphology ◽  
Targeted Deletion ◽  
Cortical Pyramidal Neurons ◽  
Rho Family

ABSTRACT Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation.

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