Liposome reconstitution and modulation of recombinant prenylated human Rac1 by GEFs, GDI1 and Pak1

Si-Cai Zhang; Lothar Gremer; Henrike Heise; Petra Janning; Aliaksei Shymanets; Ion C Cirstea; Eberhard Krause; Bernd Nürnberg; Mohammad Reza Ahmadian

doi:10.1371/journal.pone.0102425

Liposome reconstitution and modulation of recombinant prenylated human Rac1 by GEFs, GDI1 and Pak1

PLoS One. 2014 Jul 11;9(7):e102425. doi: 10.1371/journal.pone.0102425. eCollection 2014.

Authors

Si-Cai Zhang¹, Lothar Gremer², Henrike Heise³, Petra Janning⁴, Aliaksei Shymanets⁵, Ion C Cirstea⁶, Eberhard Krause⁷, Bernd Nürnberg⁵, Mohammad Reza Ahmadian¹

Affiliations

¹ Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany.
² Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany; Institute of Physical Biology, Heinrich-Heine University, Düsseldorf, Germany; Institute of Complex Systems, ICS-6, Research Center Jülich GmbH, Jülich, Germany.
³ Institute of Physical Biology, Heinrich-Heine University, Düsseldorf, Germany; Institute of Complex Systems, ICS-6, Research Center Jülich GmbH, Jülich, Germany.
⁴ Department of Chemical Biology, Max-Planck Institute of Molecular Physiology, Dortmund, Germany.
⁵ Institute of Experimental and Clinical Pharmacology and Toxicology, Tübingen Medical School, Tübingen, Germany.
⁶ Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany; Leibniz Institute for Age Research, Jena, Germany.
⁷ Laboratory of Mass Spectrometry, Leibniz Institute of Molecular Pharmacology, Berlin, Germany.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Baculoviridae / genetics
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Guanine Nucleotide Dissociation Inhibitors / chemistry*
Guanine Nucleotide Dissociation Inhibitors / genetics
Guanine Nucleotide Dissociation Inhibitors / metabolism
Guanine Nucleotide Exchange Factors / chemistry*
Guanine Nucleotide Exchange Factors / genetics
Guanine Nucleotide Exchange Factors / metabolism
Humans
Liposomes / chemistry*
Liposomes / metabolism
Models, Biological
Protein Prenylation
Protein Processing, Post-Translational*
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Spodoptera
p21-Activated Kinases / chemistry*
p21-Activated Kinases / genetics
p21-Activated Kinases / metabolism
rac1 GTP-Binding Protein / chemistry*
rac1 GTP-Binding Protein / genetics
rac1 GTP-Binding Protein / metabolism

Substances

GDP dissociation inhibitor 1
Guanine Nucleotide Dissociation Inhibitors
Guanine Nucleotide Exchange Factors
Liposomes
RAC1 protein, human
Recombinant Proteins
PAK1 protein, human
p21-Activated Kinases
rac1 GTP-Binding Protein

Grants and funding

This research was gratefully supported in part by the German Research Foundation (Deutsche Forschungsgemeinschaft or DFG) through the Collaborative Research Center 974 (SFB 974) “Communication and Systems Relevance during Liver Injury and Regeneration”, and the International NRW Research School BioStruct, granted by the Ministry of Innovation, Science and Research of the State North Rhine-Westphalia, the Heinrich-Heine-University of Düsseldorf, and the Entrepreneur Foundation at the Heinrich-Heine-University of Düsseldorf. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.