Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain.

Barfod, Elisabeth T.; Zheng, Yi; Kuang, Wun Jing; Hart, Matthew J.; Evans, Tony; Cerione, Richard A.; Ashkenazi, Avi

doi:10.1016/s0021-9258(19)74277-x

Cited by 114 publications

(20 citation statements)

References 37 publications

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“…Previous studies have demonstrated a requirement for Rac activity but not Cdc42 activity in myoblast fusion during Drosophila embryogenesis (Luo et al, 1994). The roles of Rho proteins in cell fusion have not been investigated in other organisms; however, mammalian cells have been reported to respond to Cdc42 viE by making huge multinucleated masses (Barfod, 1993). Cell fusion is accompanied by changes in cell adhesion and rearrangement of adherens junctions (Podbilewicz and White, 1994), so it is possible that Rho proteins induce changes in cell adhesion that lead to fusion in some cells.…”

Section: Rho Proteins and Nurse Cell Fusionmentioning

confidence: 99%

Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis.

Murphy

Montell

1996

The Journal of Cell Biology

200

134

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Abstract. The Rho subfamily of GTPases has beenshown to regulate cellular morphology. We report the discovery of a new member of the Rho family, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary caused nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimicked this effect. Expression of constitutively active RhoL led to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity was specifically required for migration of a subset of follicle cells called border cells. All three activities were necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis.

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Section: Rho Proteins and Nurse Cell Fusionmentioning

confidence: 99%

Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis.

Murphy

Montell

1996

The Journal of Cell Biology

200

134

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“…Conditional bem2 mutants incubated at 37°C exhibit uniform cell surface growth, disorganization of the actin cytoskeleton, and they become arrested as large, round, multinucleate, unbudded cells that are osmotically fragile. The carboxyl-terminal 203 residues of the predicted Bem2 protein is homologous to sequences found in a large family of eukaryotic proteins, some of which have been shown to function in vitro as GAPs for members of the Rho-subfamily of Ras-related small GTP-binding proteins (Diekmann et al, 1991;Settleman et al, 1992a;Barfod et al, 1993;Hall et al, 1993;Ridley et al, 1993;. Bem2p most likely also functions as a GAP in vivo because BEM2 function required for polarized cell growth (at 26°C and 37°C) can be fulfilled by simply expressing the GAP-domain of Bem2p or that of human Bcr, which is the protein most homologous to Bem2p identified so far.…”

Section: Discussionmentioning

confidence: 99%

“…The functional significance of this is unknown, but it is interesting to note that human Bcr described below also contains a region rich in these two amino acids. Second, the carboxyl-terminal 203 residues of Bem2p is homologous to sequences found in a large family of proteins (Boguski and McCormick, 1993), including human Bcr (Heisterkamp et al, 1985;Hariharan and Adams, 1987;Lifshitz et al, 1988), chimaerin (Hall et al, 1990(Hall et al, , 1993, CDC42GAP (Barfod et al, 1993), rho-GAP (Lancaster et al, 1994), the rat RasGAP-associated protein p190 (Settleman et al, 1992b), and yeast Bem3p ), Lrglp (Miiller et al, 1994, and Ybr1728p (Doignon et al, 1993). The sequence homology with human Bcr is highest (35 % identity) and that with yeast Bem3p, Lrglp, and Ybr1728p is lower (26%, 25 %, and 29% identity, respectively).…”

Section: Gtpase-activating Proteinsmentioning

confidence: 99%

“…The sequence homology with human Bcr is highest (35 % identity) and that with yeast Bem3p, Lrglp, and Ybr1728p is lower (26%, 25 %, and 29% identity, respectively). The Bem2p-related domains from five of these eight proteins have been shown to function in vitro as GTPase-activating proteins that are specific for members of the Rho-subfamily of Ras-related small GTPbinding proteins (Diekmann et al, 1991;Settleman et al, 1992a;Barfod et al, 1993;Chen et al, 1993a, b;Hall et al, 1993;Ridley et al, 1993;Zheng et al, , 1994Lancaster et al, 1994). Thus, Bem2p may also serve as a GTPase-activating protein (GAP) in vivo.…”

Section: Gtpase-activating Proteinsmentioning

confidence: 99%

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Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation.

Kim

Francisco

Chen

et al. 1994

The Journal of Cell Biology

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Abstract. The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bern2 mutants are defective in bud site selection at 26°C and localized cell surface growth and organization of the actin cytoskeleton at 37°C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bern2 protein (Bera2p) or human Bcr can functionally substitute for Bem2p. The Rhol and Rho2 GTPases are the likely in vivo targets of Bem2p because bern2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RH01 or RH02.CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR/ gene can suppress bem2 mutations, grrl and cdc55 mutants are both elongated in shape and cold-sensitive for growth, and cells lacking both GRR/and CDC55 exhibit a synthetic lethal phenotype, bern2 mutant phenotypes also can be suppressed by the SSDI-vl (also known as SRK1 ) mutation, which was shown previously to suppress mutations in the protein phosphatase-encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSDI-vl suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.

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“…The anti-Cdc42Hs polyclonal antibodies were raised against the unique carboxyl-terminal sequences of Cdc42Hs as described (Shinjo et al, 1990). GST-Dbl protein was expressed in a baculovirus/Sf9 insect cell system (Hart et al, 1994), and the Cdc42Hs-GTPase-activating protein was expressed and purified from E. coli (Barfod et al, 1993). The ⌬C7 Cdc42Hs truncation mutant was generated by polymerase chain reaction using the plaque-forming unit DNA polymerase (Stratagene), and the resulting sequences were verified through fluorescence automated sequencing.…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylinositol 4,5-bisphosphate Provides an Alternative to Guanine Nucleotide Exchange Factors by Stimulating the Dissociation of GDP from Cdc42Hs

Zheng

Glaven

et al. 1996

Journal of Biological Chemistry

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Members of the Rho subfamily of Ras-related GTP-binding proteins play important roles in the organization of the actin cytoskeleton and in the regulation of cell growth. We have shown previously that the dbl oncogene product, which represents a prototype for a family of growth regulatory proteins, activates Rho subfamily GTP-binding proteins by catalyzing the dissociation of GDP from their nucleotide binding site. In the present study, we demonstrate that the acidic phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), provides an alternative mechanism for the activation of Cdc42Hs. Among a variety of lipids tested, only PIP2 was able to stimulate GDP release from Cdc42Hs in a dose-dependent manner, with a half-maximum effect at approximately 50 microM. Unlike the Dbl oncoprotein, which requires the presence of (free) guanine nucleotide in the medium to replace the GDP bound to Cdc42Hs, PIP2 stimulates GDP release from Cdc42Hs in the absence of free guanine nucleotide. PIP2, when incorporated into phosphatidylcholine carrier vesicles, binds tightly to the guanine nucleotide-depleted form of Cdc42Hs and weakly to the GDP-bound form of the GTP-binding protein but does not bind to GTP-bound Cdc42Hs, similar to what was observed for the Dbl oncoprotein. However, mutational analysis of Cdc42Hs indicates that the site that is essential for the functional interaction between PIP2 and Cdc42Hs is distinct from the Dbl-binding site and is located at the positively charged carboxyl-terminal end of the GTP-binding protein. The GDP-releasing activity of PIP2 is highly effective toward Cdc42Hs and Rho (and is similar to the reported effects of PIP2 on Arf (Terui, T., Kahn, R. A., and Randazzo, P. A., (1994) J. Biol. Chem. 269, 28130-28135)), is less effective with Rac, and is not observed with Ras, Rap1a, or Ran. The ability of PIP2 to activate Cdc42Hs (or Rho) and Arf provides a possible point of convergence for the biological pathways regulated by these different GTP-binding proteins and may be related to the synergism observed between Arf and Rho-subtype proteins in the stimulation of phospholipase D activity (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950).

show abstract

Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain.

Cited by 114 publications

References 37 publications

Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis.

Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis.

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation.

Phosphatidylinositol 4,5-bisphosphate Provides an Alternative to Guanine Nucleotide Exchange Factors by Stimulating the Dissociation of GDP from Cdc42Hs

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