The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, βPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets.

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Grit, a GTPase-Activating Protein for the Rho Family, Regulates Neurite Extension through Association with the TrkA Receptor and N-Shc and CrkL/Crk Adapter Molecules

Molecular and Cellular Biology ◽

10.1128/mcb.22.24.8721-8734.2002 ◽

2002 ◽

Vol 22 (24) ◽

pp. 8721-8734 ◽

Cited By ~ 70

Author(s):

Takeshi Nakamura ◽

Misako Komiya ◽

Kiyoaki Sone ◽

Eiji Hirose ◽

Noriko Gotoh ◽

...

Keyword(s):

Tyrosine Kinases ◽

Rho Gtpases ◽

Rho Gtpase ◽

Neuronal Cells ◽

Small Gtpases ◽

Actin Dynamics ◽

Gtpase Activating Protein ◽

Interacting Protein ◽

High Affinity Receptor ◽

Rho Family

ABSTRACT Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.

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Prepatterning by RhoGEFs governs Rho GTPase spatiotemporal dynamics during wound repair

The Journal of Cell Biology ◽

10.1083/jcb.201704145 ◽

2017 ◽

Vol 216 (12) ◽

pp. 3959-3969 ◽

Cited By ~ 22

Author(s):

Mitsutoshi Nakamura ◽

Jeffrey M. Verboon ◽

Susan M. Parkhurst

Keyword(s):

Wound Repair ◽

Rho Gtpase ◽

Single Cells ◽

Small Gtpases ◽

Nucleotide Exchange ◽

Spatial And Temporal Patterns ◽

Cytoskeletal Remodeling ◽

Guanine Nucleotide Exchange ◽

Rho Family ◽

Membrane Resealing

Like tissues, single cells are subjected to continual stresses and damage. As such, cells have a robust wound repair mechanism comprised of dynamic membrane resealing and cortical cytoskeletal remodeling. One group of proteins, the Rho family of small guanosine triphosphatases (GTPases), is critical for this actin and myosin cytoskeletal response in which they form distinct dynamic spatial and temporal patterns/arrays surrounding the wound. A key mechanistic question, then, is how these GTPase arrays are formed. Here, we show that in the Drosophila melanogaster cell wound repair model Rho GTPase arrays form in response to prepatterning by Rho guanine nucleotide exchange factors (RhoGEFs), a family of proteins involved in the activation of small GTPases. Furthermore, we show that Annexin B9, a member of a class of proteins associated with the membrane resealing, is involved in an early, Rho family–independent, actin stabilization that is integral to the formation of one RhoGEF array. Thus, Annexin proteins may link membrane resealing to cytoskeletal remodeling processes in single cell wound repair.

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Interaction between a Ras and a Rho GTPase Couples Selection of a Growth Site to the Development of Cell Polarity in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0426 ◽

2003 ◽

Vol 14 (12) ◽

pp. 4958-4970 ◽

Cited By ~ 67

Author(s):

Keith G. Kozminski ◽

Laure Beven ◽

Elizabeth Angerman ◽

Amy Hin Yan Tong ◽

Charles Boone ◽

...

Keyword(s):

Cell Polarity ◽

Rho Gtpase ◽

Cell Cortex ◽

Nucleotide Exchange ◽

Yeast Saccharomyces Cerevisiae ◽

Growth Site ◽

Ras Family ◽

Rho Family ◽

Polarity Establishment ◽

Selection Of

Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment.

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Bacterial Guanine Nucleotide Exchange Factors SopE-Like and WxxxE Effectors

Infection and Immunity ◽

10.1128/iai.01250-09 ◽

2010 ◽

Vol 78 (4) ◽

pp. 1417-1425 ◽

Cited By ~ 58

Author(s):

Richard Bulgin ◽

Benoit Raymond ◽

James A. Garnett ◽

Gad Frankel ◽

Valerie F. Crepin ◽

...

Keyword(s):

Escherichia Coli ◽

Rho Gtpases ◽

Shigella Flexneri ◽

Small Gtpases ◽

Effector Proteins ◽

Actin Dynamics ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

T3ss Effector

ABSTRACT Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce “the trigger mechanism of cell entry.” Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

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Identification and characterization of a new isoform of small GTPase RhoE

Communications Biology ◽

10.1038/s42003-020-01295-4 ◽

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.

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Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation in primary human endothelial cells

10.1101/541219 ◽

2019 ◽

Author(s):

Nathalie R. Reinhard ◽

Sanne van der Niet ◽

Anna Chertkova ◽

Marten Postma ◽

Peter L. Hordijk ◽

...

Keyword(s):

Endothelial Cells ◽

Single Cell ◽

Rho Gtpases ◽

Rho Gtpase ◽

Actin Dynamics ◽

Pleckstrin Homology Domain ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Dh Domain

AbstractThe Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signaling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the full-length GEFs. Our data reveal a specific GEF dependent activation profile, with most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and pRex1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may activate Cdc42, which will be of great value for the field of vascular biology.Abstract FigureGraphical Abstract

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Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling

Cells ◽

10.3390/cells9051132 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1132

Author(s):

Xiaolong Wang ◽

Meiqian Weng ◽

Yuting Ke ◽

Ellen Sapp ◽

Marian DiFiglia ◽

...

Keyword(s):

Low Frequency ◽

Cell Movement ◽

Mass Spectrometry Analysis ◽

Nucleotide Exchange ◽

Vesicle Budding ◽

Recycling Endosomes ◽

Spectrometry Analysis ◽

Cellular Processes ◽

Nucleotide Exchange Factor ◽

Elevated Expression

Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex. The small RNA-mediated depletion of kalirin diminished activities in cellular membranes for activating Rab11 and resulted in a shift in size of Rab11 positive structures from small to larger ones and tubulation of recycling endosomes. Our study suggests that kalirin and TRAPP form a dual GEF complex to choreograph actions of Rab11 and Rac1 at recycling endosomes.

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MicroRNA Regulation of the Small Rho GTPase Regulators—Complexities and Opportunities in Targeting Cancer Metastasis

Cancers ◽

10.3390/cancers12051092 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1092 ◽

Cited By ~ 2

Author(s):

Brock A. Humphries ◽

Zhishan Wang ◽

Chengfeng Yang

Keyword(s):

Transcriptional Activation ◽

Rho Gtpases ◽

Cancer Metastasis ◽

Rho Gtpase ◽

Critical Role ◽

Large Family ◽

Actin Dynamics ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange

The small Rho GTPases regulate important cellular processes that affect cancer metastasis, such as cell survival and proliferation, actin dynamics, adhesion, migration, invasion and transcriptional activation. The Rho GTPases function as molecular switches cycling between an active GTP-bound and inactive guanosine diphosphate (GDP)-bound conformation. It is known that Rho GTPase activities are mainly regulated by guanine nucleotide exchange factors (RhoGEFs), GTPase-activating proteins (RhoGAPs), GDP dissociation inhibitors (RhoGDIs) and guanine nucleotide exchange modifiers (GEMs). These Rho GTPase regulators are often dysregulated in cancer; however, the underlying mechanisms are not well understood. MicroRNAs (miRNAs), a large family of small non-coding RNAs that negatively regulate protein-coding gene expression, have been shown to play important roles in cancer metastasis. Recent studies showed that miRNAs are capable of directly targeting RhoGAPs, RhoGEFs, and RhoGDIs, and regulate the activities of Rho GTPases. This not only provides new evidence for the critical role of miRNA dysregulation in cancer metastasis, it also reveals novel mechanisms for Rho GTPase regulation. This review summarizes recent exciting findings showing that miRNAs play important roles in regulating Rho GTPase regulators (RhoGEFs, RhoGAPs, RhoGDIs), thus affecting Rho GTPase activities and cancer metastasis. The potential opportunities and challenges for targeting miRNAs and Rho GTPase regulators in treating cancer metastasis are also discussed. A comprehensive list of the currently validated miRNA-targeting of small Rho GTPase regulators is presented as a reference resource.

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The Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (LARG) /ARHGEF12 Is Important For Erythroid Differentiation

Blood ◽

10.1182/blood.v122.21.3682.3682 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3682-3682

Author(s):

Li Gao ◽

Yong Zhou ◽

JingYan Tang ◽

Shuhong Shen

Keyword(s):

Rho Gtpase ◽

Conflicts Of Interest ◽

Regulatory Protein ◽

Erythroid Differentiation ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Antibody Array ◽

Fusion Partner ◽

Nucleotide Exchange ◽

Rho A

Abstract ARHGEF12,which was initially identified as a fusion partner of MLL in acute myeloid leukemia, is a regulatory protein involved in the GDP/GTP exchange reaction of the Rho A and activates a Rho-GTPase-dependent signaling pathway. However, the role of ARHGEF12 in hematopoiesis remains unknown. Here, we first report that ARHGEF12 takes part in erythroid differentiation not only in erythroleukemia cell line K562 but also in zebra fish based on knockdown technology. Although primitive hematopoiesis including erythroid differentiation progressed normally, double silencing of both orthologues arhgef12a and arhgef12b in Danio rerio with morpholino caused erythropenia during definitive hematopoiesis. Meanwhile, cytology assay reveal less mature red blood cells with large and loose nuclear in morpholino injected embryos. The phenotype of blocked erythroid differentiation can be rescured by mRNA overexpression of RhoA active mutant. On the other hand, injection of mRNA transcripted from the dominant negative mutant of RhoA could replicate the phenotype of arhgef12 silencing. Antibody array screen of kinase phosphorylation suggested STAT1 and MAPK pathways downstream of Rho A may be important for erythroid differentiation in our modles. Disclosures: No relevant conflicts of interest to declare.

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Hsp110 is required for spindle length control

The Journal of Cell Biology ◽

10.1083/jcb.201111105 ◽

2012 ◽

Vol 198 (4) ◽

pp. 623-636 ◽

Cited By ~ 17

Author(s):

Taras Makhnevych ◽

Philip Wong ◽

Oxana Pogoutse ◽

Franco J. Vizeacoumar ◽

Jack F. Greenblatt ◽

...

Keyword(s):

Affinity Purification ◽

Spindle Assembly ◽

S Phase ◽

Mass Spectrometry Analysis ◽

Nucleotide Exchange ◽

Spectrometry Analysis ◽

Nucleotide Exchange Factor ◽

Spindle Elongation ◽

Chaperone Complex

Systematic affinity purification combined with mass spectrometry analysis of N- and C-tagged cytoplasmic Hsp70/Hsp110 chaperones was used to identify new roles of Hsp70/Hsp110 in the cell. This allowed the mapping of a chaperone–protein network consisting of 1,227 unique interactions between the 9 chaperones and 473 proteins and highlighted roles for Hsp70/Hsp110 in 14 broad biological processes. Using this information, we uncovered an essential role for Hsp110 in spindle assembly and, more specifically, in modulating the activity of the widely conserved kinesin-5 motor Cin8. The role of Hsp110 Sse1 as a nucleotide exchange factor for the Hsp70 chaperones Ssa1/Ssa2 was found to be required for maintaining the proper distribution of kinesin-5 motors within the spindle, which was subsequently required for bipolar spindle assembly in S phase. These data suggest a model whereby the Hsp70–Hsp110 chaperone complex antagonizes Cin8 plus-end motility and prevents premature spindle elongation in S phase.

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