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

Baumeister, Mark A.; Rossman, Kent L.; Sondek, John; Lemmon, Mark A.

doi:10.1042/bj20061020

Cited by 45 publications

(35 citation statements)

References 48 publications

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“…Similarly, a Dbl mutant that is defective for oligomerization does not affect in vitro GEF activity (27). Our observations that mutants that are oligomerization-defective and chemically induced Cdc24p oligomerization both affect the localization of this exchange factor are consistent with this recent study on Dbs (42). Oligomerization-defective or constitutively oligomerized Cdc24p mutants catalyzed Cdc42p GDP-GTP exchange in vitro similar to wild-type Cdc24p.…”

Section: Discussionsupporting

confidence: 80%

“…Recently Baumeister et al (42) have shown that chemically induced dimerization of the Dbs DHPH domain is sufficient to alter its subcellular distribution and drive it to the plasma membrane. This effect is presumably because of an increase in avidity of the PH domain interactions with the phosphoinositide phosphatidylinositol 4,5-bisphosphate.…”

Section: Discussionmentioning

confidence: 99%

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Oligomerization Regulates the Localization of Cdc24, the Cdc42 Activator in Saccharomyces cerevisiae

Mionnet¹,

Bogliolo

Arkowitz

2008

Journal of Biological Chemistry

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Guanine nucleotide exchange factor activation of Rho G-proteins is critical for cytoskeletal reorganization. In the yeast Saccharomyces cerevisiae, the sole guanine nucleotide exchange factor for the Rho G-protein Cdc42p, Cdc24p, is essential for its site-specific activation. Several mammalian exchange factors have been shown to oligomerize; however, the function of this homotypic interaction is unclear. Here we show that Cdc24p forms oligomers in yeast via its catalytic Dbl homology domain. Mutation of residues critical for Cdc24p oligomerization also perturbs the localization of this exchange factor yet does not alter its catalytic activity in vitro. Chemically induced oligomerization of one of these oligomerization-defective mutants partially restored its localization to the bud tip and nucleus. Furthermore, chemically induced oligomerization of wild-type Cdc24p does not affect in vitro exchange factor activity, yet it results in a decrease of activated Cdc42p in vivo and the presence of Cdc24p in the nucleus at all cell cycle stages. Together, our results suggest that Cdc24p oligomerization regulates Cdc42p activation via its localization.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Oligomerization Regulates the Localization of Cdc24, the Cdc42 Activator in Saccharomyces cerevisiae

Mionnet¹,

Bogliolo

Arkowitz

2008

Journal of Biological Chemistry

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“…The majority of Dbl family GEFs are located in the cytosol of resting cells, and membrane association is generally accepted to be a prerequisite for nucleotide exchange on Rac. In the case of GEFs homologous to Dbl, the isolated DH-PH tandem alone is sufficient for PM localization and biological activity in vivo (24,25,(51)(52)(53)(54). Therefore, to investigate the possible role of Dbl-related GEFs in Rac1(GDP)⅐ RhoGDI dissociation, we designed a system consisting of DOPC/DOPS or PtdIns(3,4,5)P 3 -enriched DOPC/DOPS liposomes (representing the PM of resting and stimulated phagocytes, respectively), DH-PH tandems of GEF, Rac1(GDP)⅐ RhoGDI complexes, and guanine nucleotides.…”

Section: Discussionmentioning

confidence: 99%

Dissociation of Rac1(GDP)·RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP

Ugolev

Berdichevsky

Weinbaum

et al. 2008

Journal of Biological Chemistry

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“…For example, proto-Dbl and Dbs both require phosphoinositide binding to the PH domain for membrane recruitment and to maintain their transforming abilities (39,40). The emerging consensus is that the PH domain in the DH/PH tandem often plays a dual role; it can participate in membrane recruitment, and it regulates GEF activity through allosteric interaction with the DH domain and/or the GTPase (41).…”

Section: Discussionmentioning

confidence: 99%

Membrane Translocation of P-Rex1 Is Mediated by G Protein βγ Subunits and Phosphoinositide 3-Kinase

Barber

Donald

Thelen

et al. 2007

Journal of Biological Chemistry

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P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac that is directly activated by the ␤␥ subunits of heterotrimeric G proteins and by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), which is generated by phosphoinositide 3-kinase (PI3K). G␤␥ subunits and PIP 3 are membrane-bound, whereas the intracellular localization of P-Rex1 in basal cells is cytosolic. Activation of PI3K alone is not sufficient to promote significant membrane translocation of P-Rex1. Here we investigated the subcellular localization of P-Rex1 by fractionation of Sf9 cells co-expressing P-Rex1 with G␤␥ and/or PI3K. In basal, serum-starved cells, P-Rex1 was mainly cytosolic, but 7% of the total was present in the 117,000 ؋ g membrane fraction. Co-expression of P-Rex1 with either G␤␥ or PI3K caused only an insignificant increase in P-Rex1 membrane localization, whereas G␤␥ and PI3K together synergistically caused a robust increase in membrane-localized P-Rex1 to 23% of the total. PI3K-driven P-Rex1 membrane recruitment was wortmannin-sensitive. The use of P-Rex1 mutants showed that the isolated Dbl homology/pleckstrin homology domain tandem of P-Rex1 is sufficient for synergistic G␤␥-and PI3K-driven membrane localization; that the enzymatic GEF activity of P-Rex1 is not required for membrane translocation; and that the other domains of P-Rex1 (DEP, PDZ, and IP4P) contribute to keeping the enzyme localized in the cytosol of basal cells. In vitro Rac2-GEF activity assays showed that membrane-derived purified P-Rex1 has a higher basal activity than cytosol-derived P-Rex1, but both can be further activated by PIP 3 and G␤␥ subunits.The small GTPase Rac (isoforms Rac1, Rac2, and Rac3) is a member of the Rho family of GTPases that regulates a range of important cellular functions, including gene expression, cytoskeletal structure, and reactive oxygen species formation (1). Like all small GTPases, Rac is directly activated by guaninenucleotide exchange factors (GEFs) 3 (2). The P-Rex family of Rac-GEFs is composed of P-Rex1, P-Rex2, and P-Rex2b (3-5). P-Rex1 is expressed in white blood cells and brain (3), P-Rex2 expression is more widespread but low or absent in leukocytes (4), and P-Rex2b, a splice variant of P-Rex2 that lacks the C-terminal half, is expressed mainly in the heart (5). Studies in P-Rex1-deficient mice have shown that P-Rex1 is involved in the regulation of G protein-coupled receptor (GPCR)-dependent Rac2 activation in neutrophils, production of reactive oxygen species, chemotaxis, and neutrophil recruitment to inflammatory sites (6, 7). In the neuronal PC12 cell line, RNA interference-mediated down-regulation of P-Rex1 leads to impaired neurotrophin-stimulated cell migration (8). In the endothelial HUVEC cell line, suppression of P-Rex2b levels by RNA interference results in reduced Rac1 activation and cell migration in response to sphingosine 1-phosphate (9). P-Rex family GEFs are directly and synergistically activated in vitro and in vivo by the ␤␥ subunits of heterotrimeric...

show abstract

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

Cited by 45 publications

References 48 publications

Oligomerization Regulates the Localization of Cdc24, the Cdc42 Activator in Saccharomyces cerevisiae

Oligomerization Regulates the Localization of Cdc24, the Cdc42 Activator in Saccharomyces cerevisiae

Dissociation of Rac1(GDP)·RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP

Membrane Translocation of P-Rex1 Is Mediated by G Protein βγ Subunits and Phosphoinositide 3-Kinase

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