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

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

doi:10.1371/journal.pone.0102425

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.

Highlights

The Rho family GTPases are known to play an important role in diverse cellular processes and progression of different diseases, such as cardiovascular diseases, developmental and neurological disorders, as well as in tumor invasion and metastasis [1]
Subcellular localization of human Rac1 overexpressed in insect cells
A striking characteristic of baculovirus-infected Sf9 cells is the so-called cytopathic effect, which is observed as a reduction of cell numbers and swollen cell size depending on the extent of infection as compared to the non-infected, highly confluent culture (Fig. S1A in File supernatant 1 (S1))

Summary

Introduction

The Rho family GTPases are known to play an important role in diverse cellular processes and progression of different diseases, such as cardiovascular diseases, developmental and neurological disorders, as well as in tumor invasion and metastasis [1]. Subcellular localization of Rho GTPases to different cellular membranes is known to be critical for their biological activity This is achieved by a hypervariable region (HVR) [4] and a lipid anchor in their C-terminal tail at a distinct cysteine residue in the CAAX motif (C is cysteine, A is any aliphatic amino acid, and X is any amino acid) [2,5,6,7]. Membrane-associated Rho GTPases act, with some exceptions [10], as molecular switches by cycling between an inactive GDP-bound state and an active GTP-bound state [10]. This cycle underlies two critical intrinsic functions, the GDP-GTP exchange and GTP hydrolysis [10] and is controlled by at least three classes of regulatory proteins [3]: (i) Guanine nucleotide exchange factors (GEFs), especially those of the diffuse B-cell lymphoma (Dbl) family, which catalyze the exchange of GDP to GTP and activate the GTPase [11,12]; ii)

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Results

Conclusion