A palmitoylation switch mechanism regulates Rac1 function and membrane organization

Abstract

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell-cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol-rich liquid-ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post-translational modification targets Rac1 for stabilization at actin cytoskeleton-linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C-terminal polybasic region and is regulated by the triproline-rich motif. Non-palmitoylated Rac1 shows decreased GTP loading and lower association with detergent-resistant (liquid-ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation-deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1-deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization.

Figure 1

Rac1 incorporates [³H]-palmitic acid and its localization and activity are altered by inhibition of palmitoylation. (A) COS7 cells expressing GFP, GFP–Rac1 or GFP–V12Rac1 were incubated with 25 μM 2-Brp or DMSO (control) for 30 min. Cell recovery was analysed 24 h after 2-Brp washes out. GFP fluorescence was detected by confocal microscopy after excitation at 488 nm (bar, 25 μm). (B) GST–PBD pull-down assay of Rac1 activity. Incubation with GTP-gamma-S was included to confirm that 2-Brp does not interfere with nucleotide binding. The chart shows quantification of GTP–Rac band intensities relative to total Rac1. Values are means±s.e.m. (n=3). (C) Subcellular fractionation of 2-Brp-treated cells. Lysates of GFP–Rac1-expressing COS7 cells treated with vehicle or 2-Brp were separated into soluble (S) or particulate (P) fractions, which were analysed by western blot for GFP–Rac1 content. Caveolin-1 and RhoGDI were detected as fraction-specific markers. (D) Autoradiograph showing [³H]-palmitic acid incorporation by GFP-tagged Rac1 proteins expressed in COS7 cells (top panel). Cells were incubated with radiolabel for 4 h, and Rac1 proteins were immunoprecipitated with anti-GFP. Content of GFP-tagged proteins in immunoprecipitates was confirmed by western blot (bottom panel). (E) For acyl thioester analyses, gels prepared as in (A) were incubated with either Tris (1 M, pH 7.0) or hydroxylamine (1 M, pH 7.0) for 24 h before autoradiography. Where indicated, cells were treated with 2-Brp during the last 30 min of radiolabelling. (F) Effect of PDGF and 2-Brp on the subcellular distribution and palmitoylation of endogenous Rac1. Mouse embryonic fibroblasts (MEFs) were stimulated with 20 ng/ml PDGF during the last 15 min of [³H]-palmitic acid labelling or treated with 2-Brp as previously indicated. Palmitoylation was quantified relative to the control condition (left chart). Changes in Rac1 subcellular distribution were analysed by confocal microscopy (bar, 25 μm). Effective stimulation by PDGF was confirmed by GST–PBD pull-down assay of Rac1 activity. The chart on the right shows quantification of GTP–Rac band intensities relative to total Rac1. Values are means±s.e.m. (n=3) ^*P<0.05, ^**P<0.01 (see also Supplementary Figure S1 and Supplementary Movie S1).

Figure 2

Rac1–GTP binding depends on palmitoylation at cysteine 178. (A) Comparison of amino- and carboxy-terminal sequences of Rac1 with other human Ras and Rho GTPases. CAAX prenylation motifs are in blue. Polybasic residues are underlined. Putative or known palmitoylated cysteines are in red (carboxy-terminal sequences) or green (amino-terminal sequences). The positions of introduced mutations are shaded grey. A scheme of the cysteine substitution mutants of GFP-tagged Rac1 is shown below. (B) Confocal images showing the subcellular localization of GFP-tagged single (C6S or C178S) and double (C6S+C178S) Rac1 mutants (bar, 25 μm). (C) Western blot of plasma membrane-enriched fractions of cells expressing the indicated GFP-tagged Rac1 constructs. Blots were probed with antibodies to GFP (Rac1), the phosphorylated (active) form of the Rac1 effector PAK (p-PAK) and total PAK. Results are representative of three independent experiments. (D) [³H]-palmitic acid incorporation by wt and mutant GFP-tagged Rac1 proteins. iNOS (1–94 aa) was used as a positive control. (E) GST–PBD pull-down assay in cells expressing wt and mutant GFP-tagged Rac1 proteins. Bands were quantified relative to total Rac1. Values are means±s.e.m. (n=3) ^**P<0.01. Incubation with GTP-gamma-S was included to confirm that the C178S mutation does not affect the potential of Rac1 to bind nucleotides.

Figure 3

Absence of palmitoylation impairs recruitment of Rac1 to detergent-resistant membrane fractions and favours its oligomerization. (A) Western blots of sucrose density gradient fractions of cells expressing GFP-tagged wt or C178S Rac1. Red boxes denote caveolin-enriched DRM fractions (8–10). Fractions were analysed for the distribution of Rac1 (GFP), Cav1 RhoGDI, F-actin, tubulin and p-PAK. GM1 was detected with horseradish peroxidase-tagged cholera toxin B subunit. The chart shows the amounts of wt and C178S Rac1 in each fraction, relative to the total amount; mean±s.e.m. (n=3). (B) Recruitment of endogenous Rac1 to DRMs depends on palmitoylation. Lysates of 2-Brp-treated MEFs were separated into soluble (S) and particulate (P) fractions (left) or centrifuged to equilibrium on sucrose density gradients (right). Fractions were analysed by western blot for the content of Rac1. Caveolin-1 and RhoGDI were detected as fraction-specific markers. (C) Confocal images showing p-PAK colocalization (yellow) in cells expressing wt or C178S Rac1 (bar, 50 μm). Plasma membrane regions are enlarged in the right panel. (D) Localized clustering of GM1-containing DRMs promoted by cholera toxin-coated beads. The top panel shows the experimental scheme. COS7 cells expressing GFP–wt or GFP–C178S Rac1 were incubated with 5 μm beads coated with the GM1 marker Alexa 647-cholera toxin B. Representative confocal images show intense GFP signal around beads (bar, 10 μm). Plots show pixel intensities for Rac proteins (green) and GM1 (red) along the lines drawn through the indicated beads. (E) Regulation of Rac1–RhoGDI binding by palmitoylation. Lysates of 2-Brp-treated MEFs (upper panel) or COS7 expressing the indicated GFP-tagged Rac1 proteins (lower panel) were pulled down with recombinant (His)₆-tagged RhoGDI. Pull downs were analysed by western blot. (F) Oligomerization of palmitoylation-deficient Rac1. GFP- or pCherry-tagged wt, C6S or C178S Rac1 was loaded onto non-reducing polyacrylamide gels and analysed by western blot. Oligomerized Rac1 was detected in untreated cells expressing C178S Rac1 or cells expressing wt Rac1 treated with 2-Brp (see also Supplementary Figure S2).

Figure 4

Rac1 palmitoylation requires prenylation and the polybasic region. (A) Incorporation of [³H]-palmitic acid by wtRac1, constitutively active Rac1 (V12Rac1), or derived constructs mutated in cysteine 178 (V12-C178S), the polybasic region (V12-6Q) or the prenylation site (V12-SAAX). Arrow indicates palmitoylated Rac1. (B) Rac1 colocalization of Rac1 proteins with markers of subcellular trafficking. COS7 cells were transfected with V12Rac1, V12-6Q, V12-SAAX and V12-C178S. Confocal images show single staining of Rac1 mutants (GFP) in green (left), subcellular localization markers (the early endosome marker EEA1, the Cis-Golgi marker β-cop, and the endoplasmic reticulum marker calreticulin) in red, and colocalization of Rac1 proteins with all the markers in yellow (middle) (bar, 25 μm). Intensity profiles across the cell diameter are shown for the four Rac1 proteins. (C) Scheme of GFP-tagged fragments corresponding to the C-terminal (Ct) 50 aa of Rac1; the position of the mutation corresponding to C178 is indicated. Images show distribution of the GFP fusions in COS7 cells (bar, 25 μm). (D) Incorporation of [³H]-palmitic by cells expressing the Ct Rac1 constructs. H-Ras was used as a positive control.

Figure 5

Rac1 palmitoylation state regulates plasma membrane domain organization. (A) Absence of Rac1 palmitoylation induces PM restructuring. Images show TIRF microscopy of live COS7 cells expressing GFP-tagged wt or C178S Rac1 (bar, 25 μm). Magnified views of boxed areas show the distinct PM structures observed (arrows). (B) TIRF microscopy analysis of the colocalization of GFP-tagged wt or C178S Rac1 with the actin marker mRFP–Ruby–Lifeact (bar, 10 μm). (C) Effect of cholesterol depletion on wt Rac1-dependent PM structures. Cells were treated with 10 mM methyl-β-cyclodextrin before TIRF microscopy. (D) Differential Rac1-regulated PM organization is driven by distinct actin polymerization assembly. TIRF microscopy analysis of the colocalization of GFP-tagged wt Rac1 with mRFP–Ruby–Lifeact in cells expressing siRNAs against p21Arc, a member of the Arp2/3 complex (bar, 10 μm). (E) TIRF microscopy analysis of the colocalization of Rac1 membrane structures with liquid-ordered domains. Live COS7 cells expressing GFP-tagged wt or C178S Rac1 were incubated with 2.5 μg/ml cholera toxin B-Alexa Fluor 594, a ligand of the liquid-ordered domain marker GM1, with solids arrows indicating ‘worm’-like structures (Rac1 wt) and dashed arrows indicating ‘pond’-like structures (C178S). (F) Distribution of GM1 in non-transfected COS7 cells measured by TIRF microscopy (see also Supplementary Figures S4B and S5 and Supplementary Movies S4–S6).

Figure 6

Rac1 regulates membrane fluidity. (A) COS7 cells expressing mCherry–wtRac1 or the palmitoylation-deficient mutant mCherry–C178SRac1 were labelled with 5 μM Laurdan for 30 min at 37°C. mCherry confocal image (inset) and two-photon Laurdan images were acquired sequentially. Laurdan images were converted into GP images (see Materials and methods). GP images are pseudo-coloured with high GP (ordered membranes) in yellow and low GP (fluid membranes) in blue. The right panels show GP images masked for the mCherry–Rac1 channel and the GP colour scale image merged with the mCherry intensity channel. Plots show mean GP and relative abundance (percentage of coverage) for fluid and ordered membranes. Error bars are standard deviations of two independent experiments with a total of 35 cells per condition. (B) Measurement of cholesterol levels in COS1 cells expressing mCherry–wtRac1 or the palmitoylation-deficient mutant mCherry–C178SRac1. (C) Effect of loss of Rac1 on sucrose gradient partitioning of caveolin 1 and GM1. Western blots of sucrose density gradient fractions of wt and Rac1^−/− MEFs and Rac1^−/− MEFs reconstituted with wt or C178S Rac1. Fractions were analysed for the distribution of Rac1 and Cav1. GM1 was detected with horseradish peroxidase-tagged cholera toxin B subunit. Red boxes denote DRM fractions (7–10). Note the absence of GM1 and Cav1 from DRM fractions of Rac1^−/− cells and the partial recovery upon reexpression of wt or C178S Rac1. (D) Wt and Rac1^−/− MEFs and Rac1^−/− MEFs reconstituted with wt or C178S Rac1 were labelled with 5 μM Laurdan for 30 min at 37°C. GP images are pseudo-coloured with high GP (ordered membranes) in red and low GP (fluid membranes) in blue. The panels show GP images (left inset) masked for the mCherry–Rac1 channel (right inset) and the GP colour scale image merged with the mCherry intensity channel. Plots (right) show mean GP and relative abundance (percentage of coverage) for fluid and ordered membranes.

Figure 7

Role of Rac1 palmitoylation in cell motility. (A) Wound closure assays with COS7 cells expressing GFP–wt or GFP–C178S Rac1. The graph plots relative wound area (n=3) (bar, 100 μm). (B) Morphology of migrating MEFs expressing GFP–wt or GFP–C178S Rac1. Actin and Hoechst staining show cell positions. Inserts in the merged images highlight the different morphologies (bar, 100 μm). (C) Cells expressing GFP–wt or C178S Rac1 were plated at low density on FN and migration recorded by time-lapse video microscopy (bar, 50 μm). Lines show representative migration tracks. The chart shows net distance as a function of total distance travelled (directionality index). Values are means±s.e.m. of 50 cells per condition from two independent experiments. (D) Identical studies were performed with conditionally Rac1^−/− MEFs. The Rac1 allele was deleted by treating Rac1 loxp/loxp MEFs with adeno-Cre. Rac1 expression was rescued by transfection with pCherry–wtRac1 or the C178S mutant. Representative confocal images show cell morphologies and distributions of reexpressed Rac1 proteins (bar, 20 μm). (E) Migration of reconstituted cells on FN (bar, 50 μm). Charts show average velocity (μm/min) and directionality index. Values are means±s.e.m. of 25 cells per condition from two independent experiments (see also Supplementary Figures S4A and S5B and Supplementary Movies S2 and S3).

Figure 8

Working model: A palmitoylation switch mechanism in the regulation of Rac1. (A) Scheme of the three carboxy-terminal motifs of Rac1 involved in association with membranes. A polybasic domain is flanked by two post-translational modifications, prenylation at Cys 189 (blue) and palmitoylation at Cys 178 (red). (B) Proposed model of three-signal membrane targeting mechanism for Rac1 regulation. Palmitoylation would favour signaling downstream of Rac1 by stabilizing its localization to pre-existing L_o domains. L_o -localized Rac1 would initiate spatially confined branched-actin polymerization, which in turn would promote the expansion or coalescence of L_o domains, a process essential for cell migration. Cycles of palmitoylation and depalmitoylation of Rac1 would thus provide a rapid and spatially restricted mechanism for the control of Rac1-dependent cell functions.