Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes.

Kikuchi, Akira; Yamashita, Toshikazu; Kawata, Masahito; Yamamoto, Katsuhiko; Ikeda, Keisuke; Tanimoto, Tetsuji; Takai, Yoshimi

doi:10.1016/s0021-9258(18)69153-7

Cited by 196 publications

(16 citation statements)

References 41 publications

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“…The K d values for GDP or GTP ␥ S of, dissociation rate of GDP (K Ϫ 1 ) from, and the steady-state rate (K ss ) of GTP hydrolysis of the mutant forms of DRal were determined as described previously (Kikuchi et al, 1988;Shoji et al, 1989).…”

Section: Other Biochemical Assays Of Dralmentioning

confidence: 99%

“…These results indicate that Ser-25 of DRal is important for the action of RalGDS, that Gly-20 is important for the action of Ral-GAP, and that DRal G20V and DRal S25N are constitutively active and dominant negative forms of DRal, respectively. HCl, pH 7.5, 5 mM MgCl 2 , 1 mM DTT, and 1 mg/ml BSA; Kikuchi et al, 1988). To determine the RalGDS activity for DRal mutants, the [ Ϫ3 H]GDP-bound form of DRal mutants (8 pmol) were incubated with or without 200 nM RalGDS for various periods of time at 30°C.…”

Section: Biochemical Characterization Of the Constitutively Active And Dominant Negative Mutants Of The Dral Proteinmentioning

confidence: 99%

“…K Ϫ1 was determined as described (Shoji et al, 1989). K ss of the GTPase activity was determined by incubating DRal mutants with ␥[ 32 P]GTP for various periods of time at 30°C and expressed as the turnover number (Kikuchi et al, 1988). K cat of GTPase activity was determined in the presence or absence of RalGAP (7 g of protein) as described by Higashijima et al (1987).…”

Section: Biochemical Characterization Of the Constitutively Active And Dominant Negative Mutants Of The Dral Proteinmentioning

confidence: 99%

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The Drosophila Ral GTPase Regulates Developmental Cell Shape Changes through the Jun NH2-terminal Kinase Pathway

Sawamoto

Winge

Koyama

et al. 1999

The Journal of Cell Biology

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The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRalG20V and DRalS25N, were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRalG20V and DRalS25N act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRalG20V and DRalS25N mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH2-terminal kinase kinase (JNKK) and Jun NH2-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.

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Section: Other Biochemical Assays Of Dralmentioning

confidence: 99%

Section: Biochemical Characterization Of the Constitutively Active And Dominant Negative Mutants Of The Dral Proteinmentioning

confidence: 99%

Section: Biochemical Characterization Of the Constitutively Active And Dominant Negative Mutants Of The Dral Proteinmentioning

confidence: 99%

See 1 more Smart Citation

The Drosophila Ral GTPase Regulates Developmental Cell Shape Changes through the Jun NH2-terminal Kinase Pathway

Sawamoto

Winge

Koyama

et al. 1999

The Journal of Cell Biology

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“…Modified 1 Abbreviations: G-protein, a member of the family of heterotrimeric GTP-binding proteins that serves as an intermediary in transmembrane signaling processes; the family consists of G, and G¡ that regulate adenylyl cyclase activity, G, or transducin that couples photolyzed rhodopsin to activation of cGMP phosphodiesterase in the vertebrate retinal rods and cones, and G0, the most abundant G-protein in brain; Gp, predominant GTP-binding protein identified in extracts of human placental membranes with an approximate mass of 21 kDa; DTT, dithiothreitol; GTP-yS, guanosine 5'-0-(3-thiotriphosphate); GDP0S, guanosine 5'-0-(2-thiodiphosphate); Gpp(NH)p, guanosine 5'-(/3,7-imidotriphosphate); PAGE, polyacrylamide gel electrophoresis; HEPES, 4-(2hydroxyethyl)-1 -piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate; PEI, poly(ethylenimine). strategies for purification resulted in the isolation of a number of low molecular weight GTP-binding polypeptides from bovine brain (Kikuchi et al, 1988). The identified members of this set of proteins include the rho (Yamamoto et al, 1988), c- Ki-ras (Yamashita et al, 1988), rap-1 (Pizon et al, 1988), and rab-3 (Matsui et al, 1988) gene products.…”

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confidence: 99%

Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp

Tamir

Fawzi²,

Northup³

1990

Biochemistry

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Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…smg p21A was purified from bovine aortic smooth muscle membranes (Kawata, et al, unpublished data). c-Ki-ras p21, rhoA p21, rhoB p20, and smg p25A were purified from bovine brain membranes as described previously (7,12,34,35). v-Ki-ras p21 and c-Ha-ras p21 were generous gifts from H. Nakano (Kyowa Hakko Kogyo Co., Tokyo, Japan) and S. Hattori (University of Tokyo, Tokyo, Japan), respectively.…”

Section: Methodsmentioning

confidence: 99%

Tissue and Subcellular Distributions of the smg-21/rap1/ Krev-1 Proteins Which Are Partly Distinct from Those of c-ras p21s

Kim

Mizoguchi

Kikuchi

et al. 1990

Molecular and Cellular Biology

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We have made a specific antiserum recognizing both smg p21A (the rap1A/Krev-1 protein) and -B (the rap1B protein), ras p21-like GTP-binding proteins having the same putative effector domain as ras p21s and have used this antiserum to study the tissue and subcellular distributions of smg p21s by immunoblot and immunocytochemical analyses. By immunoblot analysis, smg p21s were detected in various rat tissues and at the highest level in brain. By light microscopic immunocytochemical analysis, smg p21s were also detected in various rat tissues. Particularly, smg p21s in brain were found abundantly in the cytoplasmic region of most types of neuronal cell bodies and moderately in neuropil, whereas c-ras p21s were found more abundantly in neuropil than in the cytoplasmic region of most types of neuronal cell bodies. smg p21s in testis were found in spermatogenic cells, in which c-ras p21s were not significantly detected. By subcellular fractionation analysis of cerebrum, smg p21s were detected in all of the particulate fractions but not in the cytosol fraction. Among the particulate fractions, approximately 70% of smg p21s was recovered with the highest specific content in the fraction containing mainly synaptosomes, mitochondria, and myelin. In further fractionation of this fraction, approximately 40% of smg p21s was recovered in each of the synaptosome fraction and the mitochondrial fraction. This subcellular distribution of smg p21s in cerebrum was partly distinct from that of c-ras p21s, which were mainly recovered in the synaptosome and microsome fractions but present at very low levels in the mitochondrial fraction. These tissue and subcellular distributions of smg p 21s together with the fact that smg p21s have the same putative effector domain as ras p21s exert their own specific actions in addition to the actions similar or antagonistic to those of c-ras p21s.

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Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes.

Cited by 196 publications

References 41 publications

The Drosophila Ral GTPase Regulates Developmental Cell Shape Changes through the Jun NH2-terminal Kinase Pathway

The Drosophila Ral GTPase Regulates Developmental Cell Shape Changes through the Jun NH2-terminal Kinase Pathway

Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp

Tissue and Subcellular Distributions of the smg-21/rap1/ Krev-1 Proteins Which Are Partly Distinct from Those of c-ras p21s

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