Critical Role of the Pleckstrin Homology and Cysteine-rich Domains in Vav Signaling and Transforming Activity

Palmby, Todd R.; Abe, Karon; Der, Channing J.

doi:10.1074/jbc.m202641200

Cited by 26 publications

(26 citation statements)

References 51 publications

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“…If only RhoA activation is considered, these studies therefore mirror our findings with Tiam-1. Der and co-workers have also reported similar results for Vav (54). The PH domain of Vav was found to be critical for its transforming activity but appeared to contribute little (if at all) to membrane association of the protein.…”

Section: Discussionmentioning

confidence: 58%

Loss of Phosphatidylinositol 3-Phosphate Binding by the C-terminal Tiam-1 Pleckstrin Homology Domain Prevents in Vivo Rac1 Activation without Affecting Membrane Targeting

Baumeister¹,

Martinu²,

Rossman³

et al. 2003

Journal of Biological Chemistry

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Dbl family guanine nucleotide exchange factors (GEFs) for Rho family small GTPases invariably contain a pleckstrin homology (PH) domain that immediately follows their Dbl homology (DH) domain. Although the DH domain is responsible for GEF activity, the role of the PH domain is less clear. We previously reported that PH domains from several Dbl family members bind phosphoinositides with very low affinity (K d values in the 10 M range). This suggests that, unlike several other PH domains, those from Dbl proteins will not function as independent membrane-targeting modules. To determine the functional relevance of low affinity phosphoinositide binding, we mutated the corresponding PH domain from Tiam-1 to abolish its weak, specific binding to phosphatidylinositol 3-phosphate. We first confirmed in vitro that phosphoinositide binding by the isolated DH/PH domain was impaired by the mutations but that intrinsic GEF activity was unaffected. We then introduced the PH domain mutations into full-length Tiam-1 and found that its ability to activate Rac1 or serum response factor in vivo was abolished. Immunofluorescence studies showed that membrane targeting of Tiam-1 was essentially unaffected by mutations in the C-terminal PH domain. Our studies therefore indicate that low affinity phosphatidylinositol 3-phosphate binding by the C-terminal PH domain may be critical for in vivo regulation and activity of Tiam-1 but that the PH domain exerts its regulatory effects without altering membrane targeting. We suggest instead that ligand binding to the PH domain induces conformational and/or orientational changes at the membrane surface that are required for maximum exchange activity of its adjacent DH domain.Tiam-1 (T-lymphoma invasion and metastasis 1) was first identified in a screen for genes that, when amplified, can induce invasiveness of normally noninvasive lymphoma cells (1). Its 1591-amino acid protein product, Tiam-1, is myristoylated at the N terminus (2) and contains two N-terminal PEST sequences (3), a PDZ (postsynaptic density-96/Discs large/Zona occludens-1) domain (4), a Ras-binding domain (5), two pleckstrin homology (PH) 1 domains (6), plus a Dbl (diffuse B-cell lymphoma) homology (DH) domain (7) (Fig.

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Section: Discussionmentioning

confidence: 58%

Loss of Phosphatidylinositol 3-Phosphate Binding by the C-terminal Tiam-1 Pleckstrin Homology Domain Prevents in Vivo Rac1 Activation without Affecting Membrane Targeting

Baumeister¹,

Martinu²,

Rossman³

et al. 2003

Journal of Biological Chemistry

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“…Several oncogenic versions of Vav have been identified (Δ1-66, Δ1-186, Δ1-186 + Δ608-845, and Y142,160,174F) which relieve autoinhibition [11]. A number of reports have also indicated that PI3 kinase provides an additional control mechanism for Vav family members via binding of PIP3 to the PH domain, which leads to an increase in association with its cognate GTPase [12,13].…”

Section: Introductionmentioning

confidence: 99%

Remedial strategies in structural proteomics: Expression, purification, and crystallization of the Vav1/Rac1 complex

Brooun

Foster

Chrencik

et al. 2007

Protein Expression and Purification

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The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay, and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D-NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/ Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.

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“…7) Several reports show that the PH domain regulates the functions of some RhoGEFs. [8][9][10][11] P-Rex1/KIAA1415 and P-Rex2 are phosphoinositide-and Gβγ subunit-dependent, Rac-specific RhoGEFs whose activities are inhibited by wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K). 12,13) On the other hand, wortmannin does not attenuate FLJ00018-induced SRE-dependent gene transcription.…”

mentioning

confidence: 99%

The Importance of Interaction with Membrane Lipids through the Pleckstrin Homology Domain of the Guanine Nucleotide Exchange Factor for Rho Family Small Guanosine Triphosphatase, FLJ00018

Kimura

Sato

Banno

et al. 2013

Biological & Pharmaceutical Bulletin

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FLJ00018, a heterotrimeric guanosine 5′-triphosphate (GTP)-binding protein (G protein) Gβγ subunitactivated guanine nucleotide exchange factor for Rho family small GTPases, regulates cellular responses, including cell morphological changes and gene transcriptional regulation, and targets the cellular membranes. FLJ00018 contains a Dbl homology (DH) domain in addition to a pleckstrin homology (PH) domain. Here we show that the PH domain of FLJ00018 is required for FLJ00018-induced, serum response element-dependent gene transcription. Although the PH domain of KIAA1415/P-Rex1, another Gβγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, binds to phosphatidylinositol 3,4,5-triphosphate and phosphatidylinositol 3,4-bisphosphate, the PH domain of FLJ00018 binds to polyphosphoinositides including phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid. These results suggest that FLJ00018 is targeted via its PH domain to cellular membranes. Key words Rho family; pleckstrin homology domain; polyphosphoinositideBy cycling between an inactive guanosine 5′-diphosphate (GDP)-bound form and an active guanosine 5′-triphosphate (GTP)-bound form, Rho family small GTPases (Rho), including RhoA, Rac, and Cdc42, act as nucleotide-dependent molecular switches to induce changes in the organization of the actin cytoskeleton to promote a variety of cell responses, including morphogenesis, chemotaxis, axonal guidance, and cell-cycle progression.1,2) The turning on of this cycle is controlled by a large family of Rho-specific guanine nucleotide exchange factors (RhoGEFs) that stimulate the exchange of GDP for GTP. Typically, RhoGEFs are large multidomain proteins that are tightly regulated to control their function. RhoGEF can be subdivided into two main subfamilies. First, they possess a Dbl homology (DH) domain that is found in tandem with a pleckstrin homology (PH) domain. This family is currently represented by 69 members in mammalian genomes.3,4) Second, Dock180-related proteins containing the Dock homology region (DHR)-2 domain (also known as the Docker-ZH2 domain) form a subfamily of 11 mammalian members.5) The DH domain is responsible for catalytic activity, and the PH domain directs the subcellular localization and can modulate the DH domain function.3,4) FLJ00018/ PLEKHG2 is a RhoGEF and contains DH and PH domains. FLJ00018 possesses a region that binds to heterotrimeric GTP-binding protein Gβγ subunits.6) The binding activates the RhoGEF activity of FLJ00018, thereby enhancing serum response element (SRE)-dependent gene transcription and affecting the spreading of cells.It has been shown that PH domains determine the subcellular localization and/or activity of PH domain-containing proteins by interacting with particular phospholipids, including phosphoinositide. For example, the PH domain of Asef, a RhoGEF, binds to phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5) P 3 ) and targets Asef to the cell-cell adhesion sites in Madin-Darby canine kidney II (MDCK II) cells.7) Several repor...

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Critical Role of the Pleckstrin Homology and Cysteine-rich Domains in Vav Signaling and Transforming Activity

Cited by 26 publications

References 51 publications

Loss of Phosphatidylinositol 3-Phosphate Binding by the C-terminal Tiam-1 Pleckstrin Homology Domain Prevents in Vivo Rac1 Activation without Affecting Membrane Targeting

Loss of Phosphatidylinositol 3-Phosphate Binding by the C-terminal Tiam-1 Pleckstrin Homology Domain Prevents in Vivo Rac1 Activation without Affecting Membrane Targeting

Remedial strategies in structural proteomics: Expression, purification, and crystallization of the Vav1/Rac1 complex

The Importance of Interaction with Membrane Lipids through the Pleckstrin Homology Domain of the Guanine Nucleotide Exchange Factor for Rho Family Small Guanosine Triphosphatase, FLJ00018

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