Elsevier

Methods in Enzymology

Volume 403, 2005, Pages 367-381
Methods in Enzymology

Capture of the Small GTPase Rab5 by GDI: Regulation by p38 MAP Kinase

https://doi.org/10.1016/S0076-6879(05)03032-6Get rights and content

Abstract

The small GTPase Rab5 is one of the key regulators of early endocytic traffic and, like other GTPases, cycles between GTP‐ and GDP‐bound states as well as between membrane and cytosol. The latter cycle is controlled by a guanine nucleotide dissociation inhibitor (GDI), which functions as a Rab vehicle in the cytosol. GDI extracts from membranes the inactive GDP‐bound form of the Rab. Then, the cytosolic GDI:Rab complex is delivered to the appropriate target membrane, where the Rab protein is reloaded, presumably via a GDI displacement factor (Pfeffer and Aivazian, 2004). We previously reported that the formation of the GDI:Rab5 complex is stimulated by the mitogen‐activated protein kinase p38 (Cavalli et al., 2001). Mol. Cell7, 421–432.]. Selective activation of p38 MAPK increases endocytic rates in vivo, presumably allowing more efficient internalization of cell surface components for repair, storage, or degradation. These observations emphasize the possibility that external stimuli contribute to the regulation of membrane traffic. Here, we describe how to monitor the ability of GDI to extract Rab5 from early endosomal membranes in vitro and the role of p38 MAPK in this process. In addition, we detail how to investigate the possible role of p38 MAPK in the regulation of endocytosis in vivo.

Introduction

Internalization of cell surface receptors into animal cells occurs mainly via clathrin‐dependent endocytosis, although other pathways, mediated by caveolae, rafts, or macropynosomes, have been described (Conner and Schmid, 2003). Endocytosed molecules are delivered to the early endosomes, where sorting occurs. Molecules that need to be reutilized, like receptors with housekeeping functions, are transported back to the plasma membranes. In contrast, molecules that need to be degraded, like the epidermal growth factor receptor (EGFR) and other down‐regulated receptors, are sorted to late endosomes and finally delivered to lysosomes (Gruenberg, 2001).

Endocytosis has long been considered as a mean to terminate signaling by internalizing active receptors after ligand binding. However, studies have uncovered the existence of new roles for endocytosis in the regulation of signaling events (Felberbaum‐Corti 2002, Felberbaum‐Corti 2003, Le Roy 2005). In particular, targeting of ligand–receptor complexes to different endocytic compartments is proposed to directly regulate signal transduction through localized assembly of specific signaling complexes (Miaczynska et al., 2004b). The observations that after ligand addition, the majority of activated EGFRs and their downstream signaling factors such as Shc, mSos, and Grb2 are found on early endosomes led to the notion that receptor activation and thus perhaps signaling can occur intracellularly (Di Guglielmo et al., 1994). Consistently, endocytosis of activated EGFR appears necessary for full‐scale mitogen‐activated protein kinase (MAPK) activation (Vieira et al., 1996). Furthermore, ligand‐bound EGFRs that are specifically activated in early endosomes can presumably recruit signaling molecules and elicit biological responses (Wang et al., 2002). In the same line, Teis et al. (2002) reported that the late endosomal protein p14 interacts with MP1—a scaffold protein that binds MEK1 and facilitates ERK1 activation—and that late endosomal localization of the p14/MP1–MAPK complex is required for efficient signaling in the ERK cascade after EGF stimulation. EGFR signaling might also occur from specialized endosomes primarily devoted to signaling. In fact, an endocytic structure, distinct from early endosomes and bearing Rab5 with its two effectors APPL1 and APPL2, was identified as an intermediate in signaling between the plasma membrane and the nucleus (Miaczynska et al., 2004a).

Tumor growth factor‐β (TGF‐β)‐regulated signaling also requires targeting to early endosomes. Indeed, the interaction of TGF‐β receptor with SARA, FYVE finger protein specifically recruited to early endosomes, is essential for Smad2 phosphorylation and subsequent signal propagation. A study proposes that targeting of TGF‐β receptor to different endocytic pathways can influence signaling output, Clathrin‐dependent internalization to early endosomes mediates signaling, while raft‐dependent uptake into caveolin‐positive structure leads to degradation (Di Guglielmo et al., 2003). Endosomes can also mediate transport of signaling molecules to their target sites. Particularly illustrative is the case of neurons, where signaling endosomes carrying Nerve growth factor (NGF) bound to its receptor TrkA and signaling‐competent complexes undergo retrograde trafficking from the presynaptic terminals to the cell bodies that are far away located (Delcroix et al., 2003). Finally, studies in Drosophila indicate that morphogens can disperse through developing target tissues by trafficking through the cells rather than by free diffusion. As a result, the signaling range—that is, the distance over which the ligand can travel and signal—is controlled by adjusting the rate of recycling and degradation of the morphogens in the target cells (Gonzalez‐Gaitan, 2003).

If membrane trafficking influences signal transduction, the reverse is also true. Indeed, while endocytosis has long been considered a constitutive function, evidence shows that signaling can modulate it. In particular, the small GTPase Rab5, which modulates early endocytic events, is a key regulator of this coupling. Rab5 can cycle not only between an active GTP‐, and an inactive GDP‐bound form, as well as between membrane and cytosol (Fig. 1). Specific factors modulate Rab5 GDP/GTP exchange (GEFs) and GTP hydrolysis (GAPs), while GDI, which is a generic Rab vehicle in the cytosol, controls the membrane–cytosol cycle. Evidence is accumulating that signaling influences each step of the Rab5 GTPase cycle, thus regulating transport.

It has long been known that binding of EGF to EGFR triggers both receptor endocytosis and fluid‐phase uptake. EGF stimulation also activates Rab5, which appears essential for internalization (Barbieri et al., 2000). Activation of Rab5 is mediated, at least in part, by the ability of Ras to directly potentiate the Rab5 nucleotide exchange activity of Rin1 (Tall et al., 2001). Conversely, Rin1 probably recruits active Rab5 and Ras to the activated receptor (Barbieri et al., 2003), leading to a complex regulating both receptor signaling and trafficking. It has also been proposed that the stimulatory effect of Ras on endocytosis and Rab5 activation involves protein kinase B (PKB/Akt), a kinase involved in growth factor receptor signal transduction (Barbieri et al., 1998). EGF stimulation can also negatively regulate endocytosis by modulating the GTPase activity of Rab5 through the sequential recruitment of Eps8 and RN‐Tre (Lanzetti et al., 2000). Eps8 is a substrate of EGFR kinase, while RN‐Tre is a Rab5 GAP. Therefore, it inactivates Rab5, thus inhibiting receptor internalization and, consequently, prolonging EGFR signaling at the plasma membrane. Furthermore, RN‐Tre diverts Eps8 from its Rac‐activating function, resulting in attenuation of Rac signaling. Recently, the Grb2 adaptor protein was shown to mediate RN‐Tre recruitment to activated receptor in an Eps8‐independent manner (Martinu et al., 2002). Thus, Grb2 can potentially contribute to both the activation of Rab5 (via Sos‐mediated activation of Ras, leading to Rin1 recruitment) and to its inactivation (via RN‐Tre).

A key component of the Rab5 cycle is Rab–GDI. GDI can be phosphorylated when complexed to Rab proteins (Steele‐Mortimer et al., 1993), suggesting that kinases control the Rab:GDI cycle and thus Rab activity. We found that p38 MAPK mediates GDI phosphorylation and its activation in the cytosolic cycle of Rab5, leading to enhanced formation of the GDI:Rab5 complex (Cavalli et al., 2001). We also observed that p38 MAPK activation by stress stimuli modulates endocytosis, probably through a net increase in Rab5 cycling (Fig. 1). Recently, Huang et al. (2004) reported that p38 MAPK activation during metabotropic glutamate receptor (mGluR)‐dependent long‐term depression (LTD) also accelerates AMPA receptor (AMPAR) endocytosis by stimulating the formation of the GDI:Rab5 complex. Moreover, N‐methyl‐d‐aspartate (NMDA) receptor‐dependent LTD induction produces a rapid and transient activation of Rab5, which in turns drives the specific removal of synaptic AMPAR in a clathrin‐dependent manner (Brown et al., 2005). Interestingly, NMDAR opening also triggers the activation of p38 MAPK (Zhu et al., 2002), which would correlate with Rab5 activation at the plasma membrane, either through regulation of the GDI:Rab5 cycle or through other unknown mechanisms.

Here, we describe the experimental system we developed to monitor in vitro the ability of GDI to capture Rab5 from early endosomal donor membranes and the role of p38 MAPK in this process (see Rab Capture Assay, and Fig. 2, Fig. 3). In addition, we detail how to investigate the possible role of p38 in the in vivo regulation of endocytic trafficking (see Activation of p38 MAPK Modulates Endocytosis In Vivo, and Fig. 3).

Section snippets

Rab Capture Assay

This assay monitors the capacity of GDI to extract Rab5 from early endosomal membranes as a GDI:Rab5 complex. The formation of the complex is stimulated by a cytosolic factor that we purified and identified as p38 MAPK. Accordingly, purified recombinant forms of p38 MAPK and MKK6(E)—a constitutively active mutant of p38 MAPK upstream kinase—can substitute for the cytosol in the GDI activation process (Fig. 3A). This activation process is inhibited by a p38‐specific inhibitor, SB 203580 (Fig. 3A

Conclusions and Perspectives

Assays described in this chapter are useful to monitor how a stress stimulus contributes to the regulation of endocytosis. Indeed, we show that the stress‐induced MAP kinase p38 regulates GDI functions in the cycle of the small GTPase Rab5 and thus modulates endocytic rates in vivo. Preliminary results suggest that p38 also regulates formation of a complex between GDI and Rab7, which is present on late endosomes and regulates transport to the degradative pathway, but perhaps not between GDI and

Acknowledgments

We would like to thank Marie‐Hélène Beuchat and Marie‐Claire Velluz for expert technical assistance. We also wish to thank Zeina Chamoun and Julien Chevallier for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation and the International Human Frontier Science Program.

References (29)

Cited by (0)

View full text