The Stress-Induced MAP Kinase p38 Regulates Endocytic Trafficking via the GDI:Rab5 Complex

Molecular Cell, Vol. 7, 421–432, February, 2001, Copyright 2001 by Cell Press

The Stress-Induced MAP Kinase p38 Regulates Endocytic Trafficking via the GDI:Rab5 Complex Valeria Cavalli,* Francis Vilbois,† Michela Corti,* Maria J. Marcote,*# Kumiko Tamura,‡ Michael Karin,‡ Steve Arkinstall,§ and Jean Gruenberg*k * Department of Biochemistry Sciences II University of Geneva 30 quai Ernest Ansermet 1211 Geneva 4 Switzerland † Serono Pharmaceutical Research Institute 14 chemin des Aulx 1228 Plan-les-Ouates Geneva Switzerland ‡ Laboratory of Gene Regulation and Signal Transduction Department of Pharmacology University of California, San Diego La Jolla, California 92093 § Serono Reproductive Biology Institute 280 Pond Street Randolph, Massachusetts 02368

Summary Early endocytic membrane traffic is regulated by the small GTPase Rab5, which cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle depends on GDI, which functions as a Rab vehicle in the aqueous environment of the cytosol. Here, we report that formation of the GDI:Rab5 complex is stimulated by a cytosolic factor that we purified and then identified as p38 MAPK. We find that p38 regulates GDI in the cytosolic cycle of Rab5 and modulates endocytosis in vivo. Our observations reveal the existence of a cross-talk between endocytosis and the p38-dependent stress response, thus providing molecular evidence that endocytosis can be regulated by the environment. Introduction After endocytosis into animal cells, cell surface molecules, including receptors, are delivered to early endosomes at the cell periphery. Then, some receptors are recycled back to the cell surface, while others are transported to late endosomes and lysosomes for degradation (Gruenberg and Maxfield, 1995). Rab5 is one of the key regulators of early endocytic traffic and, like other Rab proteins (Martinez and Goud, 1999), coordinates different trafficking events. Rab5 controls homotypic early endosome fusion (Gorvel et al., 1991), internalizak To whom correspondence should be addressed (e-mail: jean.

[email protected]). # Present address: Instituto de Biologia Molecular y Celular de Plantas, UPVA-CSIC Universidad Politecnica, Camino de Vera s/n 46022, Valencia, Spain.

tion (Bucci et al., 1992), clathrin-coated vesicle formation (McLauchlan et al., 1998), and motility of early endosomes on microtubules (Nielsen et al., 1999). The active GTP-bound form of Rab5 interacts with several effectors (Stenmark et al., 1995; Gournier et al., 1998; Christoforidis et al., 1999; Nielsen et al., 1999; Nielsen et al., 2000). EEA1, one of these effectors, interacts with the SNAREs syntaxin-13 in endosome fusion (McBride et al., 1999) and syntaxin-6 (Simonsen et al., 1999), perhaps in TGNendosome transport. In addition to cycling between GDP- and GTP-bound states, Rab5, like other Rab proteins, also cycles between a membrane-bound and a cytosolic state, despite geranyl-geranylation of C-terminal Cys residues (Desnoyers et al., 1996). This cycle depends on the guanylnucleotide dissociation inhibitor (GDI) (Sasaki et al., 1990). GDI has the capacity to extract from membranes the inactive GDP-bound form of most, if not all, prenylated Rab proteins (Novick and Zerial, 1997). Isoforms of GDI are differentially expressed in cells and tissues and do not appear to exhibit major functional differences (Yang et al., 1994). After extraction, the cytosolic GDI:Rab complex is delivered to the appropriate target membrane where the Rab protein is reloaded, presumably via a GDI displacement factor (Dirac-Svejstrup et al., 1997). Previous studies showed that ␣GDI and GDI2 are phosphorylated in the cytosol, suggesting that kinases control the Rab cytosolic cycle (Steele-Mortimer et al., 1993; Shisheva et al., 1999). However, it is still unclear how the GTP–GDP and membrane–cytosol cycles are coordinated functionally. It is generally accepted that endocytosis, much like other housekeeping membrane transport steps, occurs constitutively. In this paper, we provide evidence that the protein kinase p38 MAPK regulates GDI activity in the cytosolic cycle of the small GTPase Rab5. We also find that activation of p38 MAPK modulates endocytic rates in vivo, and this modulation is reduced in p38␣⫺/⫺ fibroblasts. Our data indicate that membrane traffic can be controlled by external stimuli, emphasizing the possible role of trafficking regulation in infection, aging, and a number of degenerative diseases. Results GDI Activity Is Stimulated by a Cytosolic Factor We designed an assay to monitor the capacity of GDI to extract Rab5 present on early endosomal membranes as a GDI:Rab5 complex. Purified early endosomes (Gorvel et al., 1991) were incubated with purified recombinant GST-GDI for 20 min at 30⬚C to allow Rab5 capture by GST-GDI. Membranes were removed by floatation on a sucrose gradient, and then the GST-GDI:Rab5 complex was retrieved onto glutathione beads. Rab5 was extracted by 30 ␮M GST-GDI (not shown; see Figure 4A), as observed by others (Barbieri et al., 1998). However, Rab5 was not extracted by 1 ␮M GDI, a concentration presumably closer to the physiological range, unless cytosol was added (Figures 1A and 1C). Rab5 was

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Figure 1. GDI Is Activated by Cytosol (A) In the assay, GST-GDI or GST was incubated with (⫹) or without (⫺) early endosomes and with (⫹) or without (⫺) HeLa cytosol. After endosome removal, GST-GDI was bound onto glutathione beads. Rab5 bound to GSTGDI was analyzed by SDS gel electrophoresis and Western blotting. (B) As in (A), using 1 ␮M GST-GDI with 100 ␮M AMP-PNP or GTP␥S. (C) As in (A), with 0–1 ␮M GST-GDI and with or without 100 ␮M ATP␥S. Rab5 bound to GST-GDI is compared to Rab5 remaining on endosomes after the experiment. Equal loading of endosomes was confirmed using antibodies against Annexin II. (D) As in (A), with (⫹) or without (⫺) cytosol. When indicated, cytosol was partially denatured for 5 min at 56⬚C or digested with 0.01 mg/ml (1:1000) or 0.1 mg/ml (1:100) trypsin followed by 0.5 mg/ml soybean trypsin inhibitor. (E) In the sequential assay, 1 ␮M GST-GDI was first incubated with (⫹) or without (⫺) cytosol and then bound onto beads. Beads were then washed with buffer or with 0.25–2 M NaCl. Then, washed beads (with bound GDI) were added to 25 ␮g endosomes in the absence of cytosol. Samples were analyzed as in (A). (F) As in (E), GST-GDI (1 ␮M) was incubated with cytosol in the presence of 1 ␮Ci [32P]ATP. GST-GDI was immediately recovered onto beads and analyzed in 2D gels followed by autoradiography and Western blotting with anti-GDI antibodies. Arrowheads indicate GDI. The background in autoradiography is due to the absence of washing steps to limit the action of phosphatases. Results in A–F are representative of at least three independent experiments.

not detected on beads when cytosol alone was used, and GST alone did not bind Rab5 (Figure 1A). Cytosoldependent extraction fulfilled the expected GDI specificity for the GDP-bound form of Rab5 (Sasaki et al., 1990), as the process was sensitive to GTP␥S (Figure 1B). GDI was limiting in the presence of cytosol, since an increase in GDI concentrations (0.3–1 ␮M) increased Rab5 capture by GDI, with a concomitant decrease in endosome-associated Rab5 (Figure 1C). We also found that the activity of the cytosol was reduced after partial heat denaturation (5 min at 56⬚C) and abolished by trypsin (Figure 1D). Our observations thus suggest that GDI is activated by a cytosolic factor, presumably a protein. The GDI-Activating Factor Is a Protein Kinase To further characterize the GDI-activating factor, the assay was carried out sequentially so that GDI activation and Rab5 extraction could be analyzed separately. GSTGDI was first incubated with cytosol alone (in the absence of membranes), and then the protein was retrieved onto glutathione beads. Beads were then washed and added to endosomes in the absence of cytosol. Then, Rab5 capture by GDI was as efficient as observed when mixing all components in the same assay (data not shown and Figure 1E), indicating that the cytosol was probably not acting on Rab5. Thus, GDI could be activated by a cytosolic factor during the first incubation and remained active during the second incubation with membranes. This sustained active state was unlikely to be due to the

formation of a complex with a cytosolic protein. GDI remained activated after treatment with high salt, including 2 M NaCl (Figure 1E), or with detergents, including 1% NP-40 (not shown). In addition, no protein could be detected associated to GDI by SDS gel analysis (not shown). Finally, cytosol that had been pretreated with excess GST-GDI to affinity deplete putative binding partner(s) exhibited the same activity in the assay as native, untreated cytosol (data not shown). It is thus conceivable that a cytosolic enzyme was responsible for GDI activation. In previous studies, we showed that cytosolic GDI is phosphorylated on serine residues and speculated that a kinase controlled GDI functions (Steele-Mortimer et al., 1993). Consistently, we found that both ATP␥S and AMP-PNP, two nonhydrolyzable ATP analogs, inhibited Rab5 binding to GDI (Figures 1C and 1B) and that GDI was phosphorylated under our assay conditions (Figure 1F), suggesting that a kinase is responsible for GDI activation. Purification of the GDI-Activating Factor We next made use of our assay to purify the cytosolic factor responsible for GDI activation. After an (NH4)2SO4 precipitation step, the activity was fractionated by gel filtration on a Superose 12 column and recovered at a mobility corresponding to a high apparent molecular weight (⬇400 kDa), suggesting that the activity is associated to a protein complex (Figures 2A and 2B). Proteins known to interact with Rab5 exhibited different elution

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Figure 2. Purification Factor

of

GDI-Stimulating

(A) The activity was fractionated on a Superose 12, assayed as in Figure 1A, and eluted in fractions 6/7. (B) Total protein was analyzed online during fractionation at OD 280 nm. (C) Indicated fractions were analyzed by gel electrophoresis and Western blotting with the indicated antibodies (P indicates the starting materials, [NH4]2SO4 pellet). Arrows in (B) and (C) point at calibration markers (dextran, 2000 kDa; ferritin, 440 kDa; and aldolase, 158 kDa). (D) Purification from rat liver cytosol (lane 1). One microgram of each fraction containing the activity was analyzed by SDS–PAGE and silver staining. Lane 2, 30% (NH4)2SO4 pellet; lane 3, MonoQ (pH 8.0); lane 4, (NH4)2SO4 pellet 60%; and lane 5, ReactiveGreen19. Molecular weight markers are shown; an arrow points at p38. (E) Cytosol was analyzed by Western blotting with antibodies against phosphorylated p38 or p54/p46 JNK isoforms. Osmotic stress (NaCl 0.2 M, 20 min) caused phosphorylation of JNK and p38, but 50 ␮M H2O2 only activated p38. (F) BHK cells were incubated for 30 min without (control) or with 50 ␮M H2O2 and 0.5 mCi/ ml 32Pi. GDI was then immunoprecipitated, and both immunoprecipitates (IP) and supernatants (Spnt) were analyzed by Western blotting with anti-GDI antibodies (WB) and autoradiography (AR). (G) BHK cells were treated without (control) or with 50 ␮M H2O2, as above. Cytosol was prepared and fractionated on a Superose12. GDI:Rab5 complex in fractions was analyzed by Western blotting using antibodies against Rab5 or GDI.

profiles, including Rabex5 (data not shown), EEA1, Rabaptin5, REP1, and the endogenous HeLa Rab5:GDI complex (Figure 2C). The activity was then further fractionated by two sequential ion-exchange chromatography steps and then by affinity binding to ReactiveGreen19 beads, resulting in a final purification ⬇30,000-fold (see supplemental table at www.molecule.org/cgi/content/full/7/2/ 421/DC1). To identify protein(s) responsible for the activity, the protocol was scaled up (see Experimental Procedures) and resulted in a final enrichment ⬇5000fold. This value may well underestimate enrichment, since the activity decreased during purification with storage time and freezing/thawing. Figure 2D shows the polypeptide composition at the successive steps of purification. The purified fraction contained five major polypeptides and several minor bands, consistent with our observations that the activity copurified with a high molecular weight complex. The polypeptide indicated by an arrow was identified by tandem mass spectrometry as the mitogen-activated protein kinase p38 (SwissProt accession number P70618, O08594; herein referred to as p38).

Subsequent studies will be needed to characterize the other polypeptides present in the fraction and to study their possible role in GDI activation. Here, we concentrated on p38 and tested whether the protein was involved in controlling GDI functions. To be active, p38 itself needs to be phosphorylated (Ono and Han, 2000). Phospho-p38 was detected in the cytosol under our assay conditions, in contrast to the related stressinduced kinase c-jun N-terminal kinase (JNK) (Figure 2E). Thus, p38 but not JNK is to some extent constitutively active in HeLa cells, as shown in 3T3-L1 adipocytes (Jain et al., 1999). H2O2 is a well-established activator of the p38-dependent stress response (Robinson and Cobb, 1997; Ogura and Kitamura, 1998; Blair et al., 1999). Figure 2E shows that p38 was activated by low (50 ␮M) doses of H2O2; the protein was then present in both cytosol and nucleus, but higher doses caused greater p38 phosphorylation and nuclear translocation (not shown), as expected (Robinson and Cobb, 1997). JNK was not activated by H2O2 (Figure 2E). We, therefore, decided to make use of this treatment to study p38 functions in GDI regulation. Treatment with H2O2

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Figure 3. p38 Activates GDI The assay was as in Figure 1A. (A) Cytosol was preincubated for 10 min at room temperature before the assay with PD 169316 or SB 203580. (B) As in (A) but using PD 98059, which inhibits MEK1 activation, or olomoucine, which inhibits cdc2/cyclinB. (C) Cytosol was preincubated for 30 min at 4⬚C with increasing amounts of antibodies against phospho-p38 (anti P-p38), phosphoJNK (anti P-JNK), or buffer alone. (D) The assay was supplemented with 150 nM recombinant GST-p38, 50 nM GST-MKK6(E), or 10 ␮M SB 203580 in the presence (⫹) or absence (⫺) of cytosol. The assay was also carried out sequentially (“sequential”) as in Figure 1E, except that 150 nM His-p38 and 50 nM GST-MKK6(E) were used instead of cytosol during the first incubation. (E) The assay was as in (D) in the presence of 50 nM GST-MKK6(E), 150 nM GST-p38, and 0–100 mM ATP (ATP), or 50 nM GSTMKK6(E), 100 ␮M ATP, but 0–150 nM GSTp38 (p38). Phospho-p38 was analyzed by Western blotting with antibodies against phospho-p38. (F) GST-ATF2 (10 ␮g) was incubated with Hisp38 and GST-MKK6(E) in the presence of 1 ␮Ci [32P]ATP. GST-ATF2 was recovered onto beads and analyzed by SDS–PAGE and autoradiography. An arrowhead indicates His-p38 remaining on beads. Results are representative of at least three independent experiments.

increased both GDI phosphorylation (Figure 2F) and the cytosolic amounts of the Rab5:GDI complex in vivo (Figure 2G). These observations thus support the view that p38 is involved in GDI regulation. p38 Regulates GDI Functions Next, we tested effects of the specific p38 inhibitors SB 203580 (Cuenda et al., 1995) and PD 169316 (Gallagher et al., 1997) in our in vitro assay. Figure 3A shows that a 10 min preincubation at room temperature with either drug reduced the capacity of the cytosol to activate GDI, while p38 remained equally phosphorylated under all conditions. However, PD 98059, which inhibits the activation of the related MAPK/extracellular signal-regulated protein kinase (ERK) MEK1 (Dudley et al., 1995), and olomoucine, which inhibits the cell cycle–control protein kinase cdc2 (Misteli and Warren, 1995), had no effect (Figure 3B). Also, cytosol prepared from cells that had been treated with SB 203580 exhibited reduced activity in our assay compared to control cytosol (data not shown). These observations thus suggest that p38 may be responsible for cytosol-mediated GDI activation. Additional evidence for a role of p38 in GDI activation came from the observations that activation was significantly decreased by the addition of an antibody against phospho-p38 that only recognized phospho-p38 on Western

blots (not shown), whereas an antibody against phospho-JNK used as a control had no effect (Figure 3C). In vivo, p38 can be activated by the upstream p38 kinases MKK6, MKK3, and MKK4 (Cohen, 1997). We found that the activity of our purified fraction (containing p38; see Figure 2D) could be potentiated by purified GST-MKK6(E) (GST-MKK6 S151E-T155E), a constitutively active mutant of MKK6, presumably because partial inactivation occurred during purification via dephosphorylation. This process was also sensitive to SB 203580 (data not shown). We then investigated whether GDI activation could be reconstituted with the purified recombinant proteins GST-p38 and GST-MKK6(E) instead of cytosol. As shown in Figure 3D, addition of both p38 and MKK6(E) was sufficient to activate GDI, and activation was inhibited by SB 203580. GDI could also be activated when the assay was carried out sequentially (Figure 3D). In these experiments, GST-GDI was first incubated with purified His-p38 and MKK6(E) in the absence of membranes and then bound to beads. Beads were then washed and added to endosomes in the absence of p38 and MKK6(E). GST-GDI was not activated by MKK6(E) alone, and activation was dependent on p38 and ATP (Figure 3E). As expected, p38 itself was phosphorylated by MKK6(E) (Figure 3E) and was then activated, as shown by SB 203580-dependent phosphorylation of the transcription factor ATF2 (Figure

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3F), a well-established p38 substrate. These experiments demonstrate that p38, when present together with the active form of its upstream kinase, could substitute for the cytosol in the GDI activation process. GDI Ser-121 Is Required for Activation Since GDI contains 26 Ser residues, we used the established GDI three-dimensional structure (Schalk et al., 1996) to identify residues predicted to face the outer surface of the molecule. Using Swiss-PdbViewer (see http://www.expasy.ch/spdbv) and BRUGEL-version 10 programs, we selected Ser-45, -121, and -213 and mutated them to Ala (GDI S45A, GDI S121A, and GDI S213A). Ser-45 is located in the SCR1 domain, which is part of the Rab binding domain. Ser-121 and Ser-213 are located in the SCR2 and SCR3A domain, respectively. Both domains fold into a compact structure believed to face the membrane and interact with a putative Rab-GDI receptor. We found that GDI S213A at a concentration of 1 ␮M could be activated by the cytosol, much like the wildtype (WT) protein (Figure 4A), indicating that Ser-213 is not required for GDI functions. The GDI S45A mutant could no longer interact with Rab5 at any concentration (Figure 4A), indicating that the Rab5 binding platform was affected. In marked contrast, GDI S121A behaved like WT GDI when used at a concentration sufficiently high (30 ␮M) to bypass cytosol requirements (Figure 4A), indicating that the S121A mutant was properly folded and functional, and that the mutation had not affected the Rab5 binding site. However, at low concentrations the mutant could no longer be activated by the cytosol (Figure 4A), suggesting that Ser-121 is required for GDI activation, perhaps as a p38 target. Consistent with this, p38 and MKK6(E) were unable to activate GDI S121A (Figure 4B). In addition, GDI S121A could not be phosphorylated in the presence of p38 and MKK6(E) (Figure 4C) unlike WT GDI and GDI S213A, suggesting that Ser-121 is indeed the p38 target on GDI. To demonstrate that activation of GDI depended on its phosphorylation state, GDI was first treated with p38 and MKK6(E) and, after washings to remove the kinases, with or without alkaline phosphatase. Figure 4D shows that GDI activation was inhibited by the phosphatase, while the same activation/deactivation sequence had little, if any, effect on the S121A mutant. Together, these observations indicate that phosphorylation by p38, presumably on Ser-121, is required for GDI activation. The p38-Dependent Stress Response Regulates the Rab5 Cycle As Rab5 is a key regulator of endocytic membrane traffic (Novick and Zerial, 1997), our observations suggest that p38 may contribute to the regulation of endocytosis by controlling the activity of GDI. To investigate the possible role of p38 in the in vivo regulation of endocytic trafficking, cells were treated with 1 mM H2O2. Subcellular fractionation then showed that concomitantly with p38 activation, decreased amounts of Rab5 were associated to endosomes compared to untreated controls (Figure 5A). However, Rab5 was clearly still present on endosomes after the treatment (see Discussion). The process was also sensitive to the p38 inhibitor SB

Figure 4. Ser-121 Is Necessary for GDI Activation (A) The sequential assay was as in Figure 1E, using WT GDI or mutants with (⫹) or without (⫺) cytosol. (B) WT GDI (1 ␮M) or the GDI S121A mutant was used in the presence (⫹) or absence (⫺) of 150 nM GST-p38 and 50 nM GST-MKK6(E), as in Figure 3D. (C) WT GST-GDI, GDI S121A, and GDI S213A (1 ␮M) were incubated as in (B) with His-p38 and GST-MKK6(E) in the presence of 1 ␮Ci [32P]ATP, as in Figure 1F. Analysis was done by SDS–PAGE, Coomassie staining, and autoradiography. An arrow indicates phosphoGST-GDI (lanes are from the same autoradigraph). (D) The assay was carried out sequentially as in Figure 1E except that 150 nM GST-p38 and 50 nM GST-MKK6(E) were added instead of cytosol during the first incubation, and 1 ␮M WT GDI or GDI S121A was used. After the first incubation, beads with bound GDI were washed with 100 mM Tris (pH 8.8) containing 5 mM MgCl2 and reincubated with or without 15 U alkaline phosphatase (AP). Samples were analyzed as in Figure 3D (sequential). Results are representative of three independent experiments.

203580 (Figure 5A). These in vivo observations fully support our in vitro findings that activation of GDI by p38 stimulates the cytosolic cycle of Rab5. Rab5 interacts physically with EEA1, one of the Rab5 effectors involved in membrane fusion (Mills et al., 1998; Simonsen et al., 1998; Christoforidis et al., 1999). Much like Rab5, EEA1 was released from purified endosomes by H2O2 in a SB 203580-sensitive manner (Figure 5A). (Differences in amounts of Rab5 and EEA1 associated to endosomes are likely due to differences in mechanisms of membrane association, EEA1 being partially lost during fractionation in contrast to Rab5, which is prenylated). When analyzed by immunofluorescence microscopy, EEA1 exhibited a characteristic punctate distribution in control cells, corresponding to early endosomes (Mu et al., 1995). Addition of 50 ␮M H2O2 caused partial redistribution of EEA1 to the cytosol, as did 100 ␮M menadione (not shown), a potent oxidative stress inducer. Some EEA1, however, clearly remained associ-

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Figure 5. Stress and p38 Inhibitors Affect Rab5 and EEA1 (A) BHK cells (lane 1) or cells treated in vivo with 10 ␮M SB 203580 for 50 min (lane 2), 1 mM H2O2 for 10 min (lane 3), 10 ␮M SB 203580 for 40 min prior to addition of 1 mM H2O2 for 10 min (lane 4) were homogenized, and early endosomes were prepared. Samples were analyzed by Western blotting using antibodies against EEA1, Rab5, and Annexin II (as a control). The bottom panel shows Western blots of the corresponding cytosols with antibodies against phospho-p38. (B) The distribution of EEA1 was analyzed by indirect immunofluorescence in control HeLa cells, or in cells incubated for 10 min with 50 ␮M H2O2, or in cells preincubated for 40 min with 10 ␮M SB 203580 prior to addition of 50 ␮M H2O2. (C) HeLa cells were cotransfected with WT GDI or GDI S121A and myc-NAGT1-GFP to identify transfected cells. After 36 hr, cells were incubated for 10 min at 37⬚C with or without 50 ␮M H2O2 and then processed for indirect immunofluorescence as in (B).

ated to endosomal membranes. In both cases, EEA1 redistribution was inhibited by SB 203580 (Figure 5B and data not shown). More importantly, EEA1 could be protected by overexpression of the S121A mutant but not by WT GDI, demonstrating that stress-induced redistribution of EEA1 was mediated by GDI (Figure 5C). Together, these data show that Rab5 and the Rab5dependent machinery are stimulated by stress, leading to activation of p38. The p38-Dependent Stress Response Regulates Early Endocytic Membrane Traffic Previous studies showed that overexpression of WT Rab5 accelerated endocytic vesicle formation, since the estimated lifetime of a coated pit at the plasma membrane was then decreased from 1 min to ⬇14 s (Bucci et al., 1992). We tested whether stimulation of the Rab5 cycle also accelerated endocytosis. In these experiments, internalization rates were measured over very short time periods in order to limit possible indirect effects of GDI activation on other Rabs, hence on other

trafficking routes (see Discussion). As shown in Figure 6A, 50 ␮M H2O2 caused a marked stimulation of endocytosis, which was abolished by the p38 inhibitor SB 203580. Consistently, endosomes appeared more numerous in H2O2-treated cells after internalization of two sequential pulses of rhodaminee and Oregon Green dextran (Figure 6B). These observations demonstrate that stress accelerates endocytosis, presumably via p38 activation. Recent studies showed that targeted disruption of the p38␣ locus is embryonically lethal in mice (Tamura et al., 2000). We tested whether endocytosis in p38␣⫺/⫺ embryonic fibroblasts could be regulated by stress. While p38 was not detected in (⫺/⫺) cells (Tamura et al., 2000), treatment with UV light, a potent activator of the p38 stress response, caused p38␣ phosphorylation in control (⫹/⫹) embryonic fibroblasts (not shown), as expected (Tamura et al., 2000). Much like H2O2, UV light stimulated endocytosis in control (⫹/⫹) cells (Figure 6C). By contrast, endocytosis stimulation by UV light was markedly reduced in the p38␣⫺/⫺ cells (Figure 6C). More-

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Figure 6. The p38 Stress Response Stimulates Endocytosis (A) BHK cells (open squares), cells treated with 50 ␮M H2O2 for 10 min (closed squares), or cells preincubated for 30 min with 10 ␮M SB 203580 prior to adding 50 ␮M H2O2 (open triangles) were incubated with 2 mg/ml horseradish peroxidase (HRP) for very short time periods. The enzymatic activity of cell-associated HRP was quantified; each point represents the mean of two independent experiments (each in duplicates). Error bars, standard error of the mean. (B) Cells were sequentially incubated for 5 min at 37⬚C with 10 kDa rhodamine dextran (red pulse), for 10 min with or without 50 ␮M H2O2 (chase), and then for 5 min with 10 kDa Oregon Green dextran (green pulse). (C) p38␣⫹/⫹ (square) or (⫺/⫺) (triangle) mouse embryonic fibroblasts were treated without (open symbols) or with (closed symbols) UV light and then incubated with 2 mg/ml HRP. HRP enzymatic activity was quantified as in (A). Since p38␣⫹/⫹ and (⫺/⫺) cells exhibited marked differences in growth rates and endocytosis capacity, data were normalized to the 2 min timepoint. (D) EEA1 distribution was analyzed by indirect immunofluorescence, as in Figure 5, in p38␣⫹/⫹ and (⫺/⫺) cells treated or not treated with UV light.

over, EEA1 was partially redistributed to the cytosol in UV-treated (⫹/⫹) cells, as in H2O2-treated cells, but was not affected by UV light in (⫺/⫺) cells (Figure 6D). These experiments thus demonstrate that p38 is required for the stress-induced activation of endocytosis. Interestingly, basal rates of endocytosis did not appear affected in p38␣⫺/⫺ cells when compared to (⫹/⫹) cells. Hence, the constitutive GDI cycle does not appear to be regulated by p38␣ in this genetic background and/or at this embryonic stage of development, perhaps because p38␤ is involved. Indeed, p38␤ is expressed in (⫺/⫺) cells (Tamura et al., 2000) but is not activated by UV light (not shown). The idea that p38 plays a consitutive role in endocytosis in vivo was supported by observations that EEA1-positive early endosomes often appeared larger in stressed cells, when the p38 inhibitor SB 203580 was present. Large, EEA1-positive endosomes were also evident in cells treated only with the inhibitor in the absence of H2O2 (Figure 7A), and these were functional, as they were accessible to endocytosed rhodamine dextran (Figure 7A). As expected, endocytosis measured over very short time periods was reduced after inhibition of basal p38 activity (Figure 7B); inhibition

being in a range similar to that observed after overexpression of a dominant-negative Rab5 mutant (Stenmark et al., 1994). Our data thus demonstrate that p38 regulates endocytosis via GDI activation, and that this regulatory pathway can also operate under basal conditions in the absence of stress. Discussion We show that the activity of GDI to extract Rab5 from endosomal membranes is regulated by a cytosolic factor. We find this factor to be p38 and confirm that recombinant p38 in the presence of an active mutant of the upstream kinase MKK6 substitutes for complete cytosol in GDI activation. Our observations also indicate that GDI Ser-121 is necessary for p38 activation but not for Rab5 binding and suggest that Ser-121 is the target of p38. Consistently, we find that p38 activation in living cells stimulates the cytosolic cycle of Rab5. We also find that UV light and oxidative stress accelerates endocytosis, while endocytic rates are reduced by inhibition of p38 basal activity. Finally, we find that UV light does not stimulate endocytosis in p38␣⫺/⫺ embryonic fibro-

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cific signal in a given MAPK module requires the additional presence of scaffolding and adaptor proteins (Schaeffer and Weber, 1999). Future work will be required to assess the possible role of other proteins in the p38-dependent regulation of endocytic trafficking. We also find that recombinant p38 together with an active mutant of its upstream kinase MKK6 can substitute for complete cytosol. Similarly, we find that p38 also regulates formation of a complex between GDI and Rab7, which is present on late endosomes (Chavrier et al., 1990), and that this process also depends on Ser-121 (not shown). Conversely, no morphological alterations of the biosynthetic pathway were observed in H2O2stressed cells (not shown). These observations suggest that p38 controls the activity of two critical regulators of the pathway leading to lysosomes. Clearly, it will be important to determine whether this mechanism only controls selective transport steps via a subset of Rabs (e.g., endocytic) or whether its functions are more general in membrane traffic. GDI is phosphorylated in vivo on Ser residues (SteeleMortimer et al., 1993). We now find that Ser-121 is necessary both for cytosol- and p38-dependent GDI activation as well as for phosphorylation by purified p38, but not for Rab5 binding. Ser-121 is located on the surface of the molecule in a compact domain opposing the Rab binding site (Schalk et al., 1996), and, thus, in a region of the protein potentially accessible to p38. The residue faces the recently identified mobile effector loop, which is important for GDI binding to membranes and Rab extraction (Luan et al., 2000). Ser-121 is present within a degenerate phosphorylation site. p38 may well use an unconventional site, but Ser-121 may also be necessary for p38–GDI interactions without being phosphorylated. Altogether, these data further establish the regulatory role of p38 in GDI activation and show that Ser-121 is necessary for this regulated process. Figure 7. p38 Inhibition Affects Endosome and Endocytosis (A) BHK cells were incubated without (control) or with 10 ␮M SB 203580 for 30 min and then with 3 kDa rhodamine dextran during the last 5 min (to visualize early endosomes). Cells were labeled with antibodies against EEA1 and analyzed by double fluorescence emission. (B) Cells preincubated with or without 10 ␮M SB 203580 for 30 min were incubated with 2 mg/ml HRP with or without SB 203580. Cellassociated HRP was quantified; results are expressed as a percentage of the untreated control for each timepoint. The histogram represents the mean of two independent duplicate experiments; the standard error of the mean is shown. Corresponding kinetics are shown in the inset: open circle, control cells; solid squares, SB 203580-treated cells.

blasts, in contrast to p38␣⫹/⫹ control cells. These observations demonstrate that p38 regulates GDI action on the cytosolic cycle of the small GTPase Rab5 and that membrane transport in the endocytic pathway can be modulated by the signal transduction cascade triggered by stress. GDI Regulation by p38 We find that GDI is activated by p38 MAPK, perhaps associated to a larger protein complex. Indeed, it is becoming increasingly clear that propagation of a spe-

The Rab5 Cycle A generally accepted view is that Rab proteins control transport directionality (Bourne, 1988): Rab proteins in the GTP-bound state are transported by vesicles from donor to target membranes, and then, after GAP-mediated GTP hydrolysis, are recycled via GDI through the cytosol and reloaded back onto donor membranes where nucleotide exchange occurs. According to this view, vectorial cycling and functions are contingent on the differential localization of GAP, exchange factor, as well as perhaps GDI-displacement factor. However, several lines of evidence question the validity of this model when applied to Rab5. The protein controls both vectorial transport of clathrin-coated vesicles from the plasma membrane to early endosomes (Bucci et al., 1992) and homotypic fusion of early endosomes (Gorvel et al., 1991). In the latter case, donor and target membranes do not exhibit known functional differences, so it is difficult to envision how vectorial Rab5 cycling can occur. Nor is it clear how vectorial cycling can be maintained between plasma membrane and endosomes. Indeed, Rab5 can be reloaded by GDI onto Rab5-depleted endosomes (Ullrich et al., 1994), thereby restoring their docking/fusion ability, and also onto clathrin-coated vesicles where GDP/GTP exchange can occur (Horiuchi et al., 1995).

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We now find that p38 stimulates formation of the GDI:Rab5 complex both in vivo and in vitro and accelerates endocytosis, while p38 inhibition has the opposite effects. Excess GDI has been used previously as a sink to sequester Rab proteins, causing an inhibition of Rabdependent transport reactions in vitro. In vivo, however, GDI is a necessary component of the Rab cycle (Garrett et al., 1994). Our observations using low GDI concentrations now suggest that availability of cytosolic GDI:Rab5 is limiting in endocytic transport. Previous studies showed that Rab5 itself is a rate-limiting component of the transport machinery (Gorvel et al., 1991; Bucci et al., 1992). Yet, a minor fraction only of the protein (5%– 10%) is cytosolic and associated to GDI at steady state, while the bulk is membrane associated (Gorvel et al., 1991; Stenmark et al., 1994). This distribution raises the issue of whether the minor cytosolic form or the abundant membrane-bound form is rate limiting at each round of transport. Recent studies showed that only 10%–20% of the normal membrane-associated pool of Rab proteins is sufficient to sustain growth in yeast (Alory and Balch, 2000), indicating that membrane-associated forms of Rab proteins are not limiting. These observations may also help to explain the comparatively mild pathological consequences of a defect in the Rab escort protein component of the geranylgeranyl transferase (choroideremia; Seabra et al., 1992). After GDI activation, we find that Rab5 and EEA1 are both still present on early endosomal membranes in amounts presumably sufficient to support transport. It thus seems reasonable to conclude that cytosolic GDI:Rab5 is limiting under steady-state conditions, and that p38 accelerates endocytosis by stimulating formation of the complex. GDI:Rab5 may act at two levels in order to stimulate endocytosis. The complex may play a direct role in the formation of endocytic vesicles at the plasma membrane. GDI:Rab5 was indeed identified as a component of the machinery controlling endocytic vesicle formation (McLauchlan et al., 1998), in agreement with more recent observations (Seachrist et al., 2000). Similarly, it has been proposed that Rab1 (Nuoffer et al., 1994) and ypt31/ypt32 (Jedd et al., 1997) are required for vesicle budding from the endoplasmic reticulum and Golgi apparatus, respectively. In addition, GDI may also facilitate the delivery of Rab5:GDP to active regions of the membrane involved in docking/fusion and interactions with the cytoskeleton (McBride et al., 1999; Nielsen et al., 1999; Sonnichsen et al., 2000). Indeed, a fraction of membrane-associated Rab5 may not be associated to active complexes at steady state but may be engaged in futile GTPase cycles (Rybin et al., 1996). Similarly, a fraction of EEA1, and perhaps other effectors, may be transiently associated with Rab5 outside active regions. GTP hydrolysis, whether triggered by rounds of transport or by futile GTPase cycles, generates the substrate of GDI, Rab5:GDP. Reactivation of Rab5:GDP by GDP:GTP exchange may thus depend on its delivery via GDI to the active oligomeric complexes, since these also contain the Rab5 exchange factor Rabex5 (McBride et al., 1999). We propose that p38, presumably together with a phosphatase, controls Rab5 functions by controlling formation of the GDI:Rab5 complex.

p38 in Health and Stress In agreement with others (Ben et al., 1998; Jain et al., 1999), we find that active, phosphorylated p38 is present in the cytosol of resting cells under steady-state conditions, corresponding to the activity we purified. Since p38 regulates the cytosolic cycle of Rab5, a key endocytic regulator, via GDI activity, it seems reasonable to conclude that the kinase also controls constitutive endocytic membrane transport. Observations that basal endocytosis was not affected in p38␣⫺/⫺ embryonic fibroblasts may reflect a role of the ␤ isoform at this stage of development or in this genetic background. In addition, other regulatory pathways may cross the p38 pathway, and this cross-talk may depend on proteins that copurify with p38. In HeLa or baby hamster kidney (BHK) cells, evidence for a constitutive role of p38 in endocytosis comes from the observations that in the absence of stress, specific p38 inhibitors cause both an inhibition of endocytosis and the formation of large endosomes. These are reminiscent of endosomes observed after overexpression of WT Rab5 or a Rab5 GTP mutant that cannot bind GDI (Bucci et al., 1992; Stenmark et al., 1994). Inhibition of basal p38 activity thus appears to mimic both overexpression of active (GTP) and inactive (GDP) Rab5 mutants on endosome morphology (enlargement) and endocytosis (inhibition), respectively. These observations may reflect, at least in part, conflicting effects of p38 inhibition on different Rabs. However, Rab5 is limiting in transport, and thus Rab5-dependent functions are likely to be differentially affected by changes in the Rab5 membrane/cytosol ratio after p38 activation or by overexpression of Rab5, particularly when using mutants that cannot cycle. Conflicting effects of WT Rab5 and its active Q79L mutant, which does not cycle, have been observed previously, suggesting that inhibition of the GTPase activity in the mutant may prevent disassembly of the docking machinery and thereby block assembly of the machinery required for vesicle formation (Stenmark et al., 1994). In fact, p38 inhibition may cause endosome enlargement by stabilizing Rab5 on endosomes but also inhibit endocytosis by reducing availability of the cytosolic Rab5:GDI complex. In addition to constitutive functions, our data indicate that activation of p38 stimulates the Rab5 cycle and endocytosis. Previous studies have established that p38 is activated in vivo by the upstream MAPK kinases MKK6, MKK3, and MKK4, reflecting the existence of a network of signal transduction pathways depending on different regulators (Cohen, 1997). Our in vitro data show that GDI can indeed be activated by a constitutively active mutant of MKK6 together with p38. When activated, phosphorylated p38 becomes translocated to the nucleus, and then MKK3 and MKK6 are found both in the nucleus and cytoplasm (Ben et al., 1998), but we find that active phospho-p38 remains partially cytosolic after stress. Under conditions of high stress, a temporal sequence may control the action of active phosphop38 on GDI before transit to the nucleus. Alternatively, cytosolic functions of p38 after stress may depend, at least in part, on nucleo-cytoplasmic transport. Indeed, MAPKAP kinase-2, the kinase substrate MAP-kinaseactivated protein kinase-2, serves both as a p38 effector and as a determinant of p38 cellular localization (Ben

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et al., 1998). Nuclear export of phosphorylated p38 mediated by MAPKAP kinase-2 may thus allow phosphorylation of cytosolic substrates. In conclusion, our data reveal the existence of an unsuspected layer of membrane traffic regulation directly dependent on the signal transduction cascade triggered by the p38-dependent stress response. These findings emphasize the possibility that stress stimuli contribute to the regulation of endocytosis and perhaps other transport steps via the p38-GDI pathway. It is attractive to speculate that the stressed-induced increase in endocytosis allows more efficient internalization of cell surface components for repair, storage, or degradation. Experimental Procedures Reagents and Antibodies Alpha minimal essential media (␣-MEM), GMEM, fetal calf serum, and antibiotic solutions were from Life Technologies, Inc. (Merelbeke, Belgium). JOKLIK medium was from Seromed (Biochrom, Berlin). PGEX-2t containing the cDNA encoding bovine ␣GDI was a gift from M. Zerial (EMBL, Heidelberg). The cDNA coding for myc-tagged NAGTI-GFP was a gift of G. Warren (Yale University, New Haven, Connecticut). Oligonucleotides were from MWG Biotech (Ebersberg, Germany), Pfu polymerase and BL21(DE3)plysS were from Stratagene Europe (Amsterdam), restriction enzymes and plasmid purification kit were from Promega (Madison, Wisconsin), alkaline phosphatase were from Roche Diagnostics (Rotkreuz, Switzerland), glutathione Sepharose 4B and 32P-␥ATP were from Amersham Pharmacia Biotech Europe (Du¨bendorf, Switzerland), protein assay reagents and chemicals for SDS–PAGE electrophoresis were from Bio-Rad Laboratories (Hercules, California), kinase inhibitors were from Calbiochem (La Jolla, California), 35SMet-35SCys-Express labeling mix were from NEN Life Science Products (Zaventem, Belgium), and dextran-rhodaminee and Oregon Green were from Molecular Probes (Leiden, The Netherlands). Other chemicals were from Sigma Chemicals Co. (Buchs, Switzerland). Antibody against EEA1 was from Transduction Laboratories (Lexington, Kentucky); antibodies against p38, phospho-p38, JNK, and phospho-JNK were from New England Biolabs Inc. (Bioconcept, Allschwil, Switzerland). The monoclonal antibodies against Rab5 and GDI were gifts from R. Jahn (Tuebingen, Germany). Antibodies against Rabex5, REP1, Rabaptin5, and AnnexinII were gifts from M. Zerial, M. Seabra (London), S. Catsicas (Lausanne, Switzerland), and V. Gerke (Muenster, Germany), respectively.

GDI Mutagenesis Generation of GDI point mutants was carried out with the QuickChange Site-Directed Mutagenesis kit (Stratagene). The plasmid PGEX-2t containing the cDNA encoding bovine ␣GDI was used as a template for the PCR reaction. GDI S45A was obtained by PCR using 5⬘-GGGGGCGAGAGCTCCGCCATCACCCCCCTGGAGG-3⬘ sense primer and 5⬘-CCTCCAGGGGGGTGATGGCGGAGCTCTCG CCCCc-3⬘ antisense primer. GDI S121A was obtained by PCR using the 5⬘-GATCTACAAAGTACCAGCTACTGAGACTGAAGCC-3⬘ sense primer and 5⬘-GGCTTCAGTCTCAGTAGCTGGTACTTTGTAGATC-3⬘ antisense primer. GDI S213A was obtained by PCR using 5⬘-CAACC GCATCAAGTTGTACGCCGAATCCCTGGCTCGGTATGG-3⬘ sense primer and 5⬘-CCATACCGAGCCAGGGATTCGGCGTACAACTTGA TGCGGTTG-3⬘ antisense primer. Triplets underlined in the oligonucleotide sequences encode the mutagenized alanine residue. The PCR products were used to transform the Epicurian coli supercompetent cells. Analysis of the mutants was carried out to ensure that sequences were correct. Induction and purification of WT GDI and mutants was as described by the manufacturer (Amersham-Pharmacia Biotech). The cDNA of GDI or GDI S121A was inserted into the expression plasmid pCB6 and cotransfected with NAGT1-GFP to identify transfected cells.

Early Endosome and Cytosol Preparation Early endosomal fractions were prepared from BHK-21 cells using a step flotation gradient (Gorvel et al., 1991). HeLa cytosol was prepared from cells grown in JOKLIK medium (Seromed) containing 10% newborn calf serum (GIBCO Life Technology), 1% NaHCO3, 1% MEM nonessential amino acids (GIBCO) in a 5 l spinner flask, up to a density of 6 ⫻ 105 cells/ml. Cells were collected by centrifugation in a Sorvall GSA rotor (Dupont) and homogenized with a potter in HB (250 mM sucrose, 3 mM imidazole [pH 7.4]) containing 1 mM DTT and an inhibitor cocktail (10␮g/ml aprotinin, 10 ␮M leupeptin, 10 ␮g/ml trypsin inhibitor, 0.1 mM vanadate, 50 mM ␤-glycerophosphate in HB). Cytosol was collected after centrifugation at 200,000 ⫻ g in a TLS 55 rotor (Beckman Instruments, Fullerton, California) and desalted on a PD10 column using HB as elution buffer. Rat liver cytosol was prepared as in Aniento et al. (1993). Analysis of the Factor Stimulating GDI Activity In the assay, 25 ␮g early endosomes was incubated for 20 min at 30⬚C in EE buffer (30 mM HEPES, 75 mM K-acetate, 5 mM MgCl2) containing 100 ␮M ATP, 500 ␮M GDP, GST-GDI, and 100 ␮g HeLa cytosol, or fractions obtained after purification, or recombinant proteins. Then, the mixture was adjusted to 40.6% sucrose, overlaid with 0.5 ml 35% sucrose and 0.5 ml HB in a TLS55 tube, and centrifuged for 1 hr at 50,000 rpm. Early endosomes were recovered at the 35% sucrose/HB interface. GST-GDI was recovered from the load with glutathione beads. When indicated, the assay was also carried out sequentially. Then, GST-GDI was incubated for 20 min at 30⬚C with 100 ␮g HeLa cytosol in EE buffer containing 500 ␮M ATP, 100 ␮M GDP, and the inhibitor cocktail and then bound onto beads. Beads were washed two times and mixed with 25 ␮g early endosomes. The mixture was adjusted to 1 mM GDP, incubated for 20 min at 30⬚C, and processed as above. Purification of the Factor Stimulating GDI Activity Cytosol was prepared from HeLa cells metabolically labeled with 35S-Met. The activity was sequentially precipitated at 40% and 60% (NH4)2SO4 and recovered by centrifugation at 15,000 ⫻ g for 30 min. The 60% pellet (⬇14 mg) was solubilized and dialyzed with EE buffer and fractionated on a Superose 12 using the SMART system (Amersham-Pharmacia Biotech). The activity (fractions 6 and 7) was applied to a MonoQ after dialysis against Tris8 (25 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 mM DTT). The activity eluted at 0.1 M NaCl and was applied to a MonoQ equilibrated in 25 mM Tris-HCl (pH 7.0), 0.5 mM EDTA, 1 mM DTT. The activity eluted at 0.5 M NaCl and was applied to a 100 ␮l ReactiveGreen19-agarose column after dialysis with Tris7.5 (50 mM Tris [pH 7.5], 50 mM NaCl, 1 mM EDTA). The activity eluted with Tris-ATP (500 ␮M ATP in 150 mM Tris [pH 7.5], 50 mM NaCl), containing 1 mM EDTA. Purification was also scaled up using 3 g rat liver cytosol. Then, the activity was precipitated with 10 g (NH4)2SO4 (30% final concentration) after gentle stirring for 60 min at 4⬚C. After centrifugation for 20 min at 15,000 rpm in a Sorvall SS-34 rotor, the pellet was resuspended in 6 ml EE buffer containing the inhibitor cocktail and dialyzed overnight in Tris8. The sample (0.3 g) was filtered through a 0.45 ␮m Millipore filter and fractionated by HPLC (Gilson) on a MonoQ. The activity eluted at 0.5 M NaCl (22 mg) and was sequentially precipitated at 30% and then at 60% (NH4)2SO4 for 1 hr at 4⬚C. The 60% pellet (6 mg) was resuspended in 0.6 ml EE containing protease inhibitors, dialyzed against Tris7.5 containing 1 ␮M ATP, and applied to the ReactiveGreen19 column. Elution was above. Analysis of Cells HeLa and BHK cells were processed for immunofluorescence as in Kobayashi et al. (1998). Fluid phase uptake was measured using 2 mg/ml HRP (Gruenberg et al., 1989). When needed, BHK cells were pretreated with 50 ␮M H2O2 for 10 min or 10 ␮M SB 203580 for 40 min at 37⬚C, and reagents remained present during internalization. UV treatment of mouse embryonic fibroblasts was as in Tamura et al. (2000). Tandem Mass Spectrometry One microgram of the purified fraction was resolved by 10% SDS– PAGE. The protein bands were excised from the silver-stained gel

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and digested in-gel with trypsin (Shevchenko et al., 1996). The extracted peptide mixture was analyzed by tandem mass spectrometric sequencing (Wilm et al., 1996) using a Q-TOF mass spectrometer (Micromass, Manchester, UK) equipped with a nano-electrospray ion source.

Cohen, P. (1997). The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol. 7, 353–361.

Other Methods Western blotting was with peroxidase-conjugated secondary antibodies (Bio-Rad Labs, Hercules, California). Chemiluminescence was detected using the SuperSignal reagent (Pierce Chemical Co., Rockford, Illionois) and quantified with a scanner with NIH image 1.59/fat software. Production and purification of GST-GDI, GSTp38, His-p38, GST-MKK6, and GST-ATF2 (E) was according to the manufacturer’s instructions (Amersham-Pharmacia Biotech). In vivo analysis of GDI phopshorylation and immunoprecipitation were as described in Steele-Mortimer et al. (1993). For in vitro analysis, 15 ␮g GDI or ATF2 was incubated for 20 min at 30⬚C in EE buffer with 100 ␮g cytosol or recombinant kinases, 1 ␮Ci 32P␥ATP, 30 ␮M ATP, the inhibitor cocktail, and 1 ␮M okadaic acid. Then, GDI or ATF2 was recovered onto glutathione beads and analyzed in 1D or 2D gels.

Desnoyers, L., Anant, J.S., and Seabra, M.C. (1996). Geranylgeranylation of Rab proteins. Biochem. Soc. Trans. 24, 699–703.

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