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Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membran...

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Autores principales: Zhang, Si-Cai, Gremer, Lothar, Heise, Henrike, Janning, Petra, Shymanets, Aliaksei, Cirstea, Ion C., Krause, Eberhard, Nürnberg, Bernd, Ahmadian, Mohammad Reza
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094549/
https://www.ncbi.nlm.nih.gov/pubmed/25014207
http://dx.doi.org/10.1371/journal.pone.0102425
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author Zhang, Si-Cai
Gremer, Lothar
Heise, Henrike
Janning, Petra
Shymanets, Aliaksei
Cirstea, Ion C.
Krause, Eberhard
Nürnberg, Bernd
Ahmadian, Mohammad Reza
author_facet Zhang, Si-Cai
Gremer, Lothar
Heise, Henrike
Janning, Petra
Shymanets, Aliaksei
Cirstea, Ion C.
Krause, Eberhard
Nürnberg, Bernd
Ahmadian, Mohammad Reza
author_sort Zhang, Si-Cai
collection PubMed
description Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.
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spelling pubmed-40945492014-07-15 Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1 Zhang, Si-Cai Gremer, Lothar Heise, Henrike Janning, Petra Shymanets, Aliaksei Cirstea, Ion C. Krause, Eberhard Nürnberg, Bernd Ahmadian, Mohammad Reza PLoS One Research Article Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes. Public Library of Science 2014-07-11 /pmc/articles/PMC4094549/ /pubmed/25014207 http://dx.doi.org/10.1371/journal.pone.0102425 Text en © 2014 Zhang et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Zhang, Si-Cai
Gremer, Lothar
Heise, Henrike
Janning, Petra
Shymanets, Aliaksei
Cirstea, Ion C.
Krause, Eberhard
Nürnberg, Bernd
Ahmadian, Mohammad Reza
Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1
title Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1
title_full Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1
title_fullStr Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1
title_full_unstemmed Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1
title_short Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1
title_sort liposome reconstitution and modulation of recombinant prenylated human rac1 by gefs, gdi1 and pak1
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094549/
https://www.ncbi.nlm.nih.gov/pubmed/25014207
http://dx.doi.org/10.1371/journal.pone.0102425
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