Recombinant Antibodies to the Small GTPase Rab6 as Conformation Sensors

Nizak, Clément; Monier, Solange; Nery, Elaine Del; Moutel, Sandrine; Goud, Bruno; Perez, Franck

doi:10.1126/science.1083911

Cited by 124 publications

(96 citation statements)

References 22 publications

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“…In tumors, a severalfold overexpression multiplied by severalfold increased activation state might yield a dramatic increase in total signaling output, and we propose to address this question in future reports using Ral activation assays on frozen tumor extracts. Alternatively, recent techniques using antibody phage display have yielded recombinant antibodies that detect specifically the GTP-bound conformation of the related Rab6 small GTPase (46), and such technology could be applied to Ral.…”

Section: Discussionmentioning

confidence: 99%

Expression of Ral GTPases, Their Effectors, and Activators in Human Bladder Cancer

Smith

Oxford

Baras³

et al. 2007

Clinical Cancer Research

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Purpose: The Ral family of small G proteins has been implicated in tumorigenesis, invasion, and metastasis in in vitro and animal model systems; however, a systematic evaluation of the state of activation, mutation, or expression of these GTPases has not been reported in any tumor type. Experimental Design: We determined the activation state of the RalA and RalB paralogs in 10 bladder cancer cell lines with varying Ras mutation status. We sequenced RalA and RalB cDNAs from 20 bladder cancer cell lines and functionally evaluated the mutations found. We determined the expression of Ral, Ral activators, and Ral effectors on the level of mRNA or protein in human bladder cancer cell lines and tissues. Results: We uncovered one E97Q substitution mutation of RalA in 1 of 20 cell lines tested and higher Ral activation in cells harboring mutant HRAS. We found overexpression of mRNAs for RalA and Aurora-A, a mitotic kinase that activates RalA, in bladder cancer (both P < 0.001), and in association with tumors of higher stage and grade. RalBP1, a canonical Ral effector, mRNA and protein was overexpressed in bladder cancer (P < 0.001), whereas Filamin A was underexpressed (P = 0.004). We determined that RalA mRNA levels correlated significantly with protein levels (P < 0.001) and found protein overexpression of both GTPases in homogenized invasive cancers. Available data sets suggest that RalA mRNA is also overexpressed in seminoma, glioblastoma, and carcinomas of the liver, pancreas, and prostate. Conclusion: These findings of activation and differential expression of RalA and RalB anchor prior work in model systems to human disease and suggest therapeutic strategies targeting both GTPases in this pathway may be beneficial.

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Section: Discussionmentioning

confidence: 99%

Expression of Ral GTPases, Their Effectors, and Activators in Human Bladder Cancer

Smith

Oxford

Baras³

et al. 2007

Clinical Cancer Research

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“…11/Rab11BP, myosin Vb, Rip11, RCP, Rab11-FIP1, Rab11-FIP2, Rab11-FIP3, Rab11-FIP4 and Rab6IP1 Lapierre et al, 2001;Mammoto et al, 1999;Nizak et al, 2003;Prekeris et al, 2001;Prekeris et al, 2000;Wallace et al, 2002;Zeng et al, 1999). Rip11, RCP, Rab11-FIP2, Rab11-FIP1, Rab11-FIP3 and Rab11-FIP4 have been grouped into a family of proteins called the Rab11-FIPs.…”

Section: Discussionmentioning

confidence: 99%

The C2 domains of the class I Rab11 family of interacting proteins target recycling vesicles to the plasma membrane

Lindsay

McCaffrey

2004

Journal of Cell Science

104

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The Rab11 family of interacting proteins (Rab11-FIP) is a recently identified protein family composed of, to date, six members that interact with Rab11. They all share a highly homologous Rab11-binding domain (RBD) at their C-termini. However, apart from the RBD, they vary in their domain organization. Rab11-FIP3 and Rab11-FIP4 possess an ezrin-radixin-moesin (ERM) domain in their C-terminal half and EF hands in their N-terminal region. They have been termed class II Rab11-FIPs. The class I Rab11-FIPs, Rab coupling protein (RCP), Rip11 and Rab11-FIP2, each have a C2 phospholipid-binding domain near their N-termini. Although they are still membrane associated, truncation mutants of the class I Rab11-FIPs that lack their C2 domains display an altered subcellular distribution in vivo, indicating that this domain plays an important role in specifying their correct intracellular localization. To determine the phospholipids to which they bind, a protein phospholipid overlay assay was performed. Our results indicate that the class-I Rab11-FIPs bind preferentially to phosphatidylinositol-(3,4,5)-trisphosphate [PtdIns(3,4,5)P3] and the second messenger phosphatidic acid. Stimulation of PtdIns(3,4,5)P3 or phosphatidic acid synthesis results in the translocation of the Rab11-FIPs from a perinuclear location to the periphery of the cell. By contrast, the transferrin receptor does not translocate to the plasma membrane under these conditions. This translocation is dependent on the presence of the C2 domain, because class I Rab11-FIP green-fluorescent-protein fusions that lack the C2 domain cannot translocate to the plasma membrane. We propose that the C2 domains of the class I Rab11-FIPs function to target these proteins to `docking sites' in the plasma membrane that are enriched in PtdIns(3,4,5)P3 and phosphatidic acid.

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“…Dans d'autres études, nous avons montré que les méthodes de crible par la technique du phage display permettent de sélectionner des scFv dits conformationnels ne détectant que certains états tridimensionnels de la protéine ciblée. Nous avons ainsi réussi à produire des anticorps dirigés contre les formes liées au GTP de Rab6 [11] ou de la tubuline [12]. Ces anticorps utilisés comme intrabodies nous ont permis d'obtenir des résultats originaux : nous avons ainsi mis en évidence la dynamique endogène de cette protéine [11] et de l'anti-tubuline et avons proposé un nouveau modèle expliquant la dynamique instable des microtubules [12].…”

Section: Revuesunclassified

“…Nous avons ainsi réussi à produire des anticorps dirigés contre les formes liées au GTP de Rab6 [11] ou de la tubuline [12]. Ces anticorps utilisés comme intrabodies nous ont permis d'obtenir des résultats originaux : nous avons ainsi mis en évidence la dynamique endogène de cette protéine [11] et de l'anti-tubuline et avons proposé un nouveau modèle expliquant la dynamique instable des microtubules [12]. La sélection d'intrabodies a aussi été réalisée en utilisant des anticorps de lamas qui semblent montrer une meilleure résistance à la réduction cytoplasmique [13] (➜).…”

Section: Revuesunclassified

Utilisation desintrabodies: de l’étude des protéines intracellulaires à l’immunisation thérapeutique

Moutel

Perez

2009

Med Sci (Paris)

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Recombinant Antibodies to the Small GTPase Rab6 as Conformation Sensors

Cited by 124 publications

References 22 publications

Expression of Ral GTPases, Their Effectors, and Activators in Human Bladder Cancer

Expression of Ral GTPases, Their Effectors, and Activators in Human Bladder Cancer

The C2 domains of the class I Rab11 family of interacting proteins target recycling vesicles to the plasma membrane

Utilisation desintrabodies: de l’étude des protéines intracellulaires à l’immunisation thérapeutique

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