Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1

Zhang, Si-Cai; Gremer, Lothar; Heise, Henrike; Janning, Petra; Shymanets, Aliaksei; Cirstea, Ion Cristian; Krause, Eberhard; Nürnberg, Bernd; Ahmadian, Mohammad Réza

doi:10.1371/journal.pone.0102425

Cited by 13 publications

(17 citation statements)

References 72 publications

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“… 7,8,37 Similarly, the presence of PIP 3 alone is insufficient to stimulate a robust association of the purified iDHPH domains of P-Rex1 with liposomes. 59 …”

Section: Regulation Of Subcellular Localizationmentioning

confidence: 99%

Regulation and function of P-Rex family Rac-GEFs

Welch

2015

Small GTPases

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The P-Rex family are Dbl-type guanine-nucleotide exchange factors for Rac family small G proteins. They are distinguished from other Rac-GEFs through their synergistic mode of activation by the lipid second messenger phosphatidyl inositol (3,4,5) trisphosphate and the Gbg subunits of heterotrimeric G proteins, thus acting as coincidence detectors for phosphoinositide 3-kinase and G protein coupled receptor signaling. Work in geneticallymodified mice has shown that P-Rex1 has physiological importance in the inflammatory response and the migration of melanoblasts during development, whereas P-Rex2 controls the dendrite morphology of cerebellar Purkinje neurons as well as glucose homeostasis in liver and adipose tissue. Deregulation of P-Rex1 and P-Rex2 expression occurs in many types of cancer, and P-Rex2 is frequently mutated in melanoma. Both GEFs promote tumor growth or metastasis. This review critically evaluates the P-Rex literature and tools available and highlights exciting recent developments and open questions.Rac family small G proteins (Rac1, Rac2, Rac3 and RhoG; a branch of the Rho family) control a myriad of essential cell responses, including adhesion, migration, reactive oxygen species formation and gene expression.1-3 They can be activated by 2 types of guanine-nucleotide exchange factors (GEFs), Dbl or DOCK, which are characterized by the structure of their catalytic domains. Rac-GEFs promote the release of GDP from Rac, allowing excess free cellular GTP to bind, and thus induce the active conformation of Rac which is able to engage downstream targets and stimulate cell responses [4][5][6] (Fig. 1).The P-Rex family are Dbl-type Rac-GEFs. The founding member, PIP 3 -dependent Rac exchanger 1, P-Rex1 (PREX1), was discovered during a search for factors that activate Rac when stimulated by the lipid second messenger phosphatidyl inositol (3,4,5) trisphosphate (PIP 3 ) which is generated by class I phosphoinositide 3-kinase (PI3K).7 P-Rex2 (PREX2) and P-Rex2b were discovered on the basis of their sequence homology with PRex1.8,9 P-Rex family proteins differ from other Dbl-type RacGEFs such as Vav, Tiam or Trio 4-6 in their domain structure and mechanisms of regulation. They have a number of physiological functions that are described in section "Physiological Function", an emerging role in metabolic disease that is discussed in section "Insulin Resistance and Type 2 Diabetes", and important roles in cancer progression that are described in section "Cancer". Genes and ProteinsThe coding sequence of the 2 P-Rex genes is 49% identical and conserved throughout vertebrates but absent from invertebrates.8 In humans, the P-Rex1 gene (PREX1; NM_020820) is located in chromosome 20 (20q13.13), near a region associated with type 2 diabetes, and the P-Rex2 gene (PREX2; NM_024870) on chromosome 8 (8q13.2), in a region linked to aggressive cancers and metastasis. PREX1 encodes the protein P-Rex1 (185 kDa, 1659 amino acids (aa); NP_065871), and PREX2 encodes 2 proteins, full-length P-Rex2 (also known as P-Rex2a; 183 k...

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“… 7,8,37 Similarly, the presence of PIP 3 alone is insufficient to stimulate a robust association of the purified iDHPH domains of P-Rex1 with liposomes. 59 …”

Section: Regulation Of Subcellular Localizationmentioning

confidence: 99%

Regulation and function of P-Rex family Rac-GEFs

Welch

2015

Small GTPases

View full text Add to dashboard Cite

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“…Like all GTPases, the Rac1 is under tight spatial control, which is mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that catalyse GTP exchange and hydrolysis, respectively [23]. Specific GEF allows Rac1 to be activated in specific signal transduction pathways and coordinate more elaborate responses to specific demands at localized cellular sites [26, 42]. In this study, we identified the exchange factor Vav2 as mediating autocrine VEGF and IL-8 signaling to Rac1.…”

Section: Discussionmentioning

confidence: 99%

Autocrine VEGF and IL-8 Promote Migration via Src/Vav2/Rac1/PAK1 Signaling in Human Umbilical Vein Endothelial Cells

Li¹,

Zhou²,

Jiang³

et al. 2017

Cell Physiol Biochem

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Background/Aims: Pro-angiogenic factors VEGF and IL-8 play a major role in modulating the migratory potential of endothelial cells. The goal of this study was to investigate the effect of autocrine VEGF and IL-8 in the form of self-conditioned medium (CM) on human umbilical vein endothelial cells (HUVECs). Methods: Enzyme-linked immunosorbent assay (ELISA) examined the automatic secretion of VEGF and IL-8 protein by HUVECs. Western blot, small interfering RNA (siRNA), pulldown and Transwell assays were used to explore the role and the mechanism of autocrine VEGF and IL-8 in migration of HUVECs. Results: Neutralizing VEGF and IL-8 in CM significantly abrogated CM-induced migration of HUVECs. Autocrine VEGF and IL-8 increased Src phosphorylation, Rac1 activity and PAK1 phosphorylation in a time dependent manner. Additionally, blocking Rac1 activity with Rac1 siRNA largely abolished autocrine VEGF and IL-8-induced cell migration. Vav2 siRNA suppressed autocrine VEGF and IL-8-induced Rac1 activation and cell migration. Furthermore, blocking Src signaling with PP2, a specific inhibitor for Src, markedly prevented autocrine VEGF and IL-8-induced Vav2 and Rac1 activation as well as consequently cell migration. PAK1 siRNA also significantly abolished autocrine VEGF and IL-8-induced cell migration. Conclusions: We demonstrated for the first time that autocrine VEGF and IL-8 promoted endothelial cell migration via the Src/Vav2/Rac1/PAK1 signaling pathway. This finding reveals the molecular mechanism in the increase of endothelial cell migration induced by autocrine growth factors and cytokines, which is expected to provide a novel therapeutic target in vascular diseases.

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“…This is achieved by a hypervariable region (HVR) (66) and a lipid anchor in their C-terminal tail at a distinct cysteine residue in the C AAX motif (C is cysteine, A is any aliphatic amino acid, and X is any amino acid) (67). RHOGDI is known to dislodge RHO proteins from the plasma membrane (68). As IQGAP1 also binds RHOGDI (20), it would be interesting to know whether IQGAP1 is a displacement factor for the RHOGDI complex with RAC1 or CDC42.…”

Section: Discussionmentioning

confidence: 99%

IQGAP1 Interaction with RHO Family Proteins Revisited

Nouri

Fansa

Amin

et al. 2016

Journal of Biological Chemistry

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IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.

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Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1

Cited by 13 publications

References 72 publications

Regulation and function of P-Rex family Rac-GEFs

Regulation and function of P-Rex family Rac-GEFs

Autocrine VEGF and IL-8 Promote Migration via Src/Vav2/Rac1/PAK1 Signaling in Human Umbilical Vein Endothelial Cells

IQGAP1 Interaction with RHO Family Proteins Revisited

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