scholarly journals The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity

2006 ◽  
Vol 400 (3) ◽  
pp. 563-572 ◽  
Author(s):  
Mark A. Baumeister ◽  
Kent L. Rossman ◽  
John Sondek ◽  
Mark A. Lemmon
Keyword(s):  
Rho Gtpases ◽  
Regulatory Role ◽  
Pleckstrin Homology Domain ◽  
Membrane Targeting ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Dh Domain

Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent membrane targeting modules. In the present study, we show that dimerization of a Dbs (Dbl's big sister) DH/PH domain fragment is sufficient to drive it to the plasma membrane through a mechanism involving PH domain–phosphoinositide interactions. Thus, the Dbs PH domain could play a significant role in membrane targeting if it co-operates with other domains in the protein. We also show that mutations that prevent phosphoinositide binding by the Dbs PH domain significantly impair cellular GEF activity even in chimaeric proteins that are robustly membrane targeted by farnesylation or by the PH domain of phospholipase C-δ1. This finding argues that the Dbs PH domain plays a regulatory role that is independent of its ability to aid membrane targeting. Thus, we suggest that the PH domain plays dual roles, contributing independently to membrane localization of Dbs (as part of a multi-domain interaction) and allosteric regulation of the DH domain.

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

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

scholarly journals A mammalian Rho-specific guanine-nucleotide exchange factor (p164-RhoGEF) without a pleckstrin homology domain

2002 ◽  
Vol 366 (3) ◽  
pp. 721-728 ◽  
Author(s):  
Ulrich RÜMENAPP ◽  
Andrea FREICHEL-BLOMQUIST ◽  
Burkhard WITTINGHOFER ◽  
Karl H. JAKOBS ◽  
Thomas WIELAND
Keyword(s):  
Gene Transcription ◽  
Rho Gtpases ◽  
Serum Response Factor ◽  
Full Length ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Pleckstrin Homology ◽  
Guanine Nucleotide Exchange ◽  
Dh Domain

Rho GTPases, which are activated by specific guanine-nucleotide exchange factors (GEFs), play pivotal roles in several cellular functions. We identified a recently cloned human cDNA, namely KIAA0337, encoding a protein containing 1510 amino acids (p164). It contains a RhoGEF-specific Dbl homology (DH) domain but lacks their typical pleckstrin homology domain. The expression of the mRNA encoding p164 was found to be at least 4-fold higher in the heart than in other tissues. Recombinant p164 interacted with and induced GDP/GTP exchange at RhoA but not at Rac1 or Cdc42. p164-ΔC and p164-ΔN are p164 mutants that are truncated at the C- and N-termini respectively but contain the DH domain. In contrast with the full-length p164, expression of p164-ΔC and p164-ΔN strongly induced actin stress fibre formation and activated serum response factor-mediated and Rho-dependent gene transcription. Interestingly, p164-ΔN2, a mutant containing the C-terminus but having a defective DH domain, bound to p164-ΔC and suppressed the p164-ΔC-induced gene transcription. Overexpression of the full-length p164 inhibited M3 muscarinic receptor-induced gene transcription, whereas co-expression with Gβ1γ2 dimers induced transcriptional activity. It is concluded that p164-RhoGEF is a Rho-specific GEF with novel structural and regulatory properties and predominant expression in the heart. Apparently, its N- and C-termini interact with each other, thereby inhibiting its GEF activity.

scholarly journals Hormones Secretion and Rho GTPases in Neuroendocrine Tumors

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1859
Author(s):  
Laura Streit ◽  
Laurent Brunaud ◽  
Nicolas Vitale ◽  
Stéphane Ory ◽  
Stéphane Gasman
Keyword(s):  
Neuroendocrine Tumors ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Secretory Activity ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins

Neuroendocrine tumors (NETs) belong to a heterogeneous group of neoplasms arising from hormone secreting cells. These tumors are often associated with a dysfunction of their secretory activity. Neuroendocrine secretion occurs through calcium-regulated exocytosis, a process that is tightly controlled by Rho GTPases family members. In this review, we compiled the numerous mutations and modification of expression levels of Rho GTPases or their regulators (Rho guanine nucleotide-exchange factors and Rho GTPase-activating proteins) that have been identified in NETs. We discussed how they might regulate neuroendocrine secretion.

scholarly journals Requirements for Vav Guanine Nucleotide Exchange Factors and Rho GTPases in FcγR- and Complement-Mediated Phagocytosis

Immunity ◽  
2006 ◽  
Vol 24 (3) ◽  
pp. 305-316 ◽  
Author(s):  
Amy B. Hall ◽  
M. Angelica Martinez Gakidis ◽  
Michael Glogauer ◽  
Julie L. Wilsbacher ◽  
Sizhen Gao ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

scholarly journals Function of the N-terminus of zizimin1: autoinhibition and membrane targeting

2007 ◽  
Vol 409 (2) ◽  
pp. 525-533 ◽  
Author(s):  
Nahum Meller ◽  
M. Jody Westbrook ◽  
John D. Shannon ◽  
Chittibabu Guda ◽  
Martin A. Schwartz
Keyword(s):  
Limited Proteolysis ◽  
Small Gtpases ◽  
Guanine Nucleotide Exchange Factors ◽  
Pleckstrin Homology Domain ◽  
Membrane Targeting ◽  
Nucleotide Exchange ◽  
Rho Proteins ◽  
Cellular Functions ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.

scholarly journals Critical but Distinct Roles for the Pleckstrin Homology and Cysteine-Rich Domains as Positive Modulators of Vav2 Signaling and Transformation

2002 ◽  
Vol 22 (8) ◽  
pp. 2487-2497 ◽  
Author(s):  
Michelle A. Booden ◽  
Sharon L. Campbell ◽  
Channing J. Der
Keyword(s):  
Membrane Association ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Guanine Nucleotide Exchange ◽  
Ph Domains ◽  
Transforming Activity ◽  
Dh Domain ◽  
Exchange Activity

ABSTRACT Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo.

scholarly journals Human Ect2 Is an Exchange Factor for Rho Gtpases, Phosphorylated in G2/M Phases, and Involved in Cytokinesis

1999 ◽  
Vol 147 (5) ◽  
pp. 921-928 ◽  
Author(s):  
Takashi Tatsumoto ◽  
Xiaozhen Xie ◽  
Rayah Blumenthal ◽  
Isamu Okamoto ◽  
Toru Miki
Keyword(s):  
Signaling Pathways ◽  
Rho Gtpases ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Animal Cells ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. Here we show that the human ECT2 is involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH2-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.

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