scholarly journals ADP-ribosylation of the GTP-binding protein Rho by Clostridium limosum exoenzyme affects basal, but not N-formyl-peptide-stimulated, actin polymerization in human myeloid leukaemic (HL60) cells

1994 ◽  
Vol 299 (3) ◽  
pp. 775-779 ◽  
Author(s):  
G Koch ◽  
J Norgauer ◽  
K Aktories
Keyword(s):  
Actin Polymerization ◽  
Binding Protein ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Hl60 Cells ◽  
Adp Ribosylation ◽  
Gtp Binding ◽  
Long Term Treatment ◽  
Formyl Peptide

Treatment of human myeloid leukaemic (HL60) cells with Clostridium limosum exoenzyme, which inactivates the small GTP-binding protein Rho by ADP-ribosylation, decreased the basal F-actin content. Inhibition of F-actin occurred after long-term treatment (24 h) of intact HL60 cells or after introduction of the toxin by electropermeabilization in a toxin-concentration-dependent manner. Concomitantly with the decrease in the basal F-actin content, the GTP-binding protein Rho was ADP-ribosylated in intact cells. However, Clostridium limosum toxin had no inhibitory effect on N-formyl-peptide-induced actin polymerization. Moreover, the relative N-formyl-peptide-stimulated polymerization was substantially enhanced in cells treated with Clostridium limosum transferase. In contrast with Clostridium limosum exoenzyme, component C21 of the Clostridium botulinum C2 toxin, which ADP-ribosylates G-actin, depolymerized basal F-actin and inhibited N-formyl-peptide-induced actin polymerization in electropermeabilized HL60 cells. These findings indicate that Rho proteins are involved in the basal, but not the ligand-evoked, actin polymerization in HL60 cells.

scholarly journals Specific role for p85/p110β in GTP-binding-protein-mediated activation of Akt

2005 ◽  
Vol 392 (3) ◽  
pp. 607-614 ◽  
Author(s):  
Hiroshi Kubo ◽  
Kaoru Hazeki ◽  
Shunsuke Takasuga ◽  
Osamu Hazeki
Keyword(s):  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Point Mutations ◽  
Chinese Hamster ◽  
Middle Part ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Ovary Cells ◽  
Gtp Binding

We prepared CHO (Chinese hamster ovary) cells expressing both IR (insulin receptor) and A1R (A1 adenosine receptor). Treatment of the cells with insulin or PIA [N6-(2-phenylisopropyl)adenosine], a specific A1R agonist increased Akt activity in the cells in a PI3K- (phosphoinositide 3-kinase) dependent manner. Transfection of p110β into the cells augmented the action of PIA with little effect on insulin. Introduction of a pH1 vector producing shRNA (short hairpin RNA) that targets p110β abolished PIA-induced Akt activation. By contrast, an shRNA probe targeting p110α did not impair the effects of PIA. The effect of PIA in p110α-deficient cells was attenuated effectively by both Δp85 and βARK-CT (β-adrenergic receptor kinase-C-terminal peptide). A Δp85-derived protein possessing point mutations in its two SH2 domains did not impair PIA action. These results suggest that tyrosine-phosphorylated proteins and Gβγ (βγ subunits of GTP-binding protein) are necessary for the specific function of p110β in intact cells. The p110β-middle (middle part of p110β) may play an important role in signal reception from GPCRs (GTP-binding-protein-coupled receptor), because transfection of the middle part impaired PIA sensitivity.

scholarly journals ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.

1992 ◽  
Vol 267 (17) ◽  
pp. 11677-11680
Author(s):  
P Lang ◽  
L Guizani ◽  
I Vitté-Mony ◽  
R Stancou ◽  
O Dorseuil ◽  
...  
Keyword(s):  
Binding Protein ◽  
Gtp Binding Protein ◽  
Adp Ribosylation ◽  
Gtp Binding

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin

1993 ◽  
Vol 13 (4) ◽  
pp. 2061-2068
Author(s):  
H Shirataki ◽  
K Kaibuchi ◽  
T Sakoda ◽  
S Kishida ◽  
T Yamaguchi ◽  
...  
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Target Protein ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Reading Frame ◽  
Gtp Binding ◽  
Putative Target

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Effects of lithium ion on ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin, islet-activating protein

1991 ◽  
Vol 206 (1) ◽  
pp. 33-37 ◽  
Author(s):  
Hideo Kawamoto ◽  
Yasuhiro Watanabe ◽  
Taro Imaizumi ◽  
Tadaaki Iwasaki ◽  
Hiroshi Yoshida
Keyword(s):  
Pertussis Toxin ◽  
Binding Protein ◽  
Lithium Ion ◽  
Gtp Binding Protein ◽  
Adp Ribosylation ◽  
Gtp Binding

scholarly journals Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.

1994 ◽  
Vol 126 (3) ◽  
pp. 801-810 ◽  
Author(s):  
K Jalink ◽  
E J van Corven ◽  
T Hengeveld ◽  
N Morii ◽  
S Narumiya ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Binding Protein ◽  
Neuronal Cell ◽  
Force Generation ◽  
Gtp Binding Protein ◽  
Adp Ribosylation ◽  
Neurite Retraction ◽  
Gtp Binding ◽  
Shape Changes

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

scholarly journals Pertussis toxin substrate is a guanosine 5′-[β-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein

1985 ◽  
Vol 232 (1) ◽  
pp. 191-197 ◽  
Author(s):  
S K Wong ◽  
B R Martin ◽  
A M Tolkovsky
Keyword(s):  
Pertussis Toxin ◽  
Binding Protein ◽  
Alkylating Agents ◽  
Guanine Nucleotides ◽  
Temperature Sensitive ◽  
Gtp Binding Protein ◽  
Adp Ribosylation ◽  
Gtp Binding ◽  
Rat Brain And Liver ◽  
Liver Membranes

We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5′-[γ-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5′-[β-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guanosine 5′-[β, γ-imido]triphosphate (p[NH]ppG). The order of labelling intensity was GDP[S] greater than GTP greater than no addition greater than GTP[S] = p [NH]ppG. Mg2+ increased labelling by CT, but decreased labelling by IAP. In addition, Mg2+ potentiated the effects of the guanine nucleotides, increasing the inhibitory effects of GTP[S] and the activatory effects of GDP[S] or GTP. Preincubating liver membranes at 30 degrees C in the presence of 10 mm-MgCl2 inhibited labelling by IAP irreversibly. Pretreatment of liver membranes with 4.95 mM-N-ethylmaleimide decreased labelling by CT by approximately 15%, but almost completely blocked labelling by IAP. These results suggest that the undissociated, GDP-bound, conformation of Ni, the inhibitory GTP-binding protein of adenylate cyclase, is the preferred substrate for ADP-ribosylation by IAP. This conformation, which is prevalent in native membranes, is sensitive to temperature, Mg2+ ions and alkylating agents such as N-ethylmaleimide. At 30 degrees C, Mg2+ may cause dissociation and denaturation of Ni in native membranes.

scholarly journals Developmentally regulated GTP-binding protein 1 modulates ciliogenesis via an interaction with Dishevelled

2019 ◽  
Vol 218 (8) ◽  
pp. 2659-2676 ◽  
Author(s):  
Moonsup Lee ◽  
Yoo-Seok Hwang ◽  
Jaeho Yoon ◽  
Jian Sun ◽  
Adam Harned ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Binding Protein ◽  
Basal Bodies ◽  
Apical Surface ◽  
Gtp Binding Protein ◽  
Developmentally Regulated ◽  
Rhoa Activity ◽  
Gtp Binding ◽  
Xenopus Laevis Embryo ◽  
Multiciliated Cells

Cilia are critical for proper embryonic development and maintaining homeostasis. Although extensively studied, there are still significant gaps regarding the proteins involved in regulating ciliogenesis. Using the Xenopus laevis embryo, we show that Dishevelled (Dvl), a key Wnt signaling scaffold that is critical to proper ciliogenesis, interacts with Drg1 (developmentally regulated GTP-binding protein 1). The loss of Drg1 or disruption of the interaction with Dvl reduces the length and number of cilia and displays defects in basal body migration and docking to the apical surface of multiciliated cells (MCCs). Moreover, Drg1 morphants display abnormal rotational polarity of basal bodies and a decrease in apical actin and RhoA activity that can be attributed to disruption of the protein complex between Dvl and Daam1, as well as between Daam1 and RhoA. These results support the concept that the Drg1–Dvl interaction regulates apical actin polymerization and stability in MCCs. Thus, Drg1 is a newly identified partner of Dvl in regulating ciliogenesis.

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