14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins

Abstract

Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.

Graphical abstract

Figure S1

All Mammalian 14-3-3 Isoforms Interact with Rnd3 and C-Terminal Rnd3 Residues Are Required for 14-3-3 Protein Interaction, Related to Figure 1 (A) COS7 cells were cotransfected with wild-type (WT) FLAG-Rnd3 and each of the 7 mammalian HA-14-3-3 isoforms. Cell lysates were immunoprecipitated (IP) with anti-FLAG (top panels), anti-HA (middle panels) antibodies, or nonimmune immunoglobulins (IgG) as a control, then immunoblotted (IB) for HA and FLAG. (B) Expression vectors encoding FLAG-Rnd3 and HA-14-3-3 isoforms were transfected into COS7 cells. Cells were treated with staurosporine (to inhibit kinases) or calyculin A (to inhibit phosphatases) for 2 hr or 20 min respectively prior to lysis. Cell lysates were immunoprecipitated then western blotted with the indicated antibodies. The space in the gel image marks the position of lanes that were not germane to these results and were thus removed during figure preparation for clarity. (C) Rnd3-AllA does not bind to 14-3-3 proteins. COS7 cells were transfected with FLAG-Rnd3 or FLAG-Rnd3-AllA. Cell lysates were incubated with GST-14-3-3β or GST as a control, and GST pull-downs were immunoblotted (IB) for GST and FLAG. (D) Schematic showing 3 Rnd3 deletion mutants. (E) The indicated FLAG-Rnd3 mutants were transfected into COS7 cells. Cells lysates were immunoprecipitated with anti-FLAG antibody. Endogenous 14-3-3 proteins bound to FLAG-Rnd3 were detected with anti-14-3-3 antibody. (F) COS7 cells were transfected with the indicated FLAG-Rnd3 constructs and HA-14-3-3β. Cell lysates were immunoprecipitated (IP) with anti-HA or anti-FLAG antibody, then western blotted (IB) for HA and FLAG. (G) Alignment of Rnd3 C-terminal sequences from representative species. Sequences are shown in single letter amino acid code, with amino acid numbers for Homo sapiens (Hs) Rnd3 shown above. Amino acids equivalent to Hs Rnd3 S210, S218 and S240 are underlined. Ss, Sos Scrofus; Gg, Gallus gallus; Dr, Danio rerio; Ca, Crotalus adamanteus; Xl, Xenopus laevis; Oa, Ornithorhynchus anatinus. Asterisk (^*) indicates IgG light chain.

Figure 1

Rnd3 Binds to 14-3-3 Proteins via C-Terminal Phosphorylation Sites (A) Expression vectors encoding FLAG-Rnd3 and HA-14-3-3 isoforms were transfected into COS7 cells. Cell lysates were immunoprecipitated (IP) with FLAG antibody, then treated with or without CIP for 1 hr. Immunoprecipitates were then incubated with cell lysates containing HA-14-3-3 proteins and immunoblotted (IB) with antibodies against FLAG and HA. (B) Schematic showing 7 Rnd3 phosphorylation sites (S/T), mutated to A in AllA. (C–E) The indicated FLAG-Rnd3 mutants were transfected into COS7 cells. Cells were lysed and immunoprecipitated with FLAG antibody. Endogenous 14-3-3 proteins bound to FLAG-Rnd3 were detected with 14-3-3 antibody. In (D), the space in the gel image marks the position of lanes that were not germane to these results and were thus removed during figure preparation for clarity. Asterisk (^*) indicates IgG light chain; S: Serine, T: Threonine, A: Alanine. See also Figure S1 and Table S1.

Figure 2

Rnd3 C-Terminal Phosphorylation by ROCK and PKC Is Required for 14-3-3 Interaction (A–C) The indicated constructs were transfected into COS7 cells. Cell lysates were immunoprecipitated with FLAG antibody. (A) Immunoprecipitates were subjected to an in vitro kinase assay with either ROCK1 or PKCζ kinase domains. (B) FLAG-Rnd3 was co-overexpressed with ROCK1^1–420 where indicated. Cells were treated with chemical inhibitors (staurosporine, calyculin A, H-1152 [ROCK], BIM1 [PKC]) or the chemical activator PMA (PKC) for 2 hr (20 min for calyculin A) prior to lysis. Cell lysates were stained with Pro-Q Diamond reagent or immunoblotted with the indicated antibodies. The space in the gel image marks the position of lanes that were not germane to these results and were thus removed during figure preparation for clarity. (C) Endogenous 14-3-3 proteins were detected with anti-14-3-3 antibody. Membranes were also stained with Pro-Q Diamond reagent or immunoblotted with the indicated antibodies. (D) Alignment of C-terminal regions of three human Rnd proteins, using Rnd3 protein sequence numbering. Sequences are shown in single letter amino acid code, with underlined S/T denoting known phosphorylated residues in Rnd3. (E and F) COS7 cells transfected with the indicated constructs encoding wild-type or mutated Rnd1, Rnd2, or Rnd3 were treated with Calyculin A (Cal. A) for 20 min. Cell lysates were immunoprecipated with FLAG antibody and stained with Pro-Q Diamond reagent or immunoblotted with antibodies to FLAG and 14-3-3. Asterisk (^*) indicates IgG light chain. See also Figure S2.

Figure S2

C-Terminal Phosphorylation of Rnd3, Related to Figure 2 (A) Representative MS² spectrum indicating phosphorylation of S218 in the sequence ²¹⁶R-IpSHMPSRPELSAVATDLR-²³⁴ detected in FLAG immunoprecipitates from COS7 cells cotransfected with FLAG-Rnd3 and myc-ROCK1^1-420, or transfected with FLAG-Rnd3 and incubated with calyculin A for 20 min. Precursor m/z: 554.79⁴⁺, mass error 17 ppm. Data were acquired using an Ultimate LC platform (Dionex, Camberley, UK) coupled online to a QToF-micro mass spectrometer (Waters, Manchester, UK) for data-dependent LC-MS². (B) Representative (i) MS² and (ii) MS³ spectra indicating phosphorylation, farnesylation and carboxymethylation of the C-terminal peptide of Rnd3 KDKAKpSfC-OMe, detected in FLAG immunoprecipitates from COS7 cells cotransfected with FLAG-Rnd3 and myc-ROCK1^1-420, or transfected with FLAG-Rnd3 and incubated with calyculin A for 20 min. Precursor ion for MS²: m/z 539.29248²⁺, mass error −0.1 ppm (30,000 resolution scan); precursor for MS³: m/z 873.38391¹⁺, mass error −6.9 ppm (7,500 resolution scan). Data were acquired using an HP1200 nanoLC platform (Agilent, Wokingham, UK) coupled online to a LTQ Velos Orbitrap mass spectrometer (ThermoFisher Scientific, Hemel Hempstead, UK) for data-dependent LC-MS² and LC-MS³ with all mass measurements performed in the Orbitrap mass analyzer. Annotations are: M – parent peptide mass, f – loss of farnesyl (204.1878 Da) and p – loss of phosphate (97.9769 Da). (C) Schematic showing Rnd3 C-terminal sequence and phosphorylation sites, indicating the 3 sites involved in 14-3-3 binding and their respective kinases. Sequence is shown in single letter amino acid code, with underlined S denoting phosphorylated serine.

Figure S3

Rnd3 Phosphorylation Is Not Required for Morphological Changes or p190RhoGAP Interaction, Related to Figure 3 (A) Cell morphology and FLAG-Rnd3 localization in NIH 3T3 cells transfected with the indicated constructs. Bar, 20 μm. (B) The indicated FLAG-Rnd3 constructs and Myc-p190RhoGAP-B deletion mutant (Δp190B) were transfected into COS7 cells. Cell lysates were immunoprecipitated with anti-FLAG antibody. Myc-p190RhoGAP protein bound to FLAG-Rnd3 was detected with anti-Myc antibody. (C) The indicated FLAG-Rnd3 constructs, HA-14-3-3β and Δp190B were transfected into COS7 cells with or without ROCK1^1-420 cotransfection or calyculin A (cal. A) treatment where indicated. Cell lysates were immunoprecipitated with anti-Myc (upper panels) or anti-HA (middle panels) antibodies followed by immunoblotting with the indicated antibodies. (D–F) NIH 3T3 cells were transfected with the indicated constructs. (D) Single confocal images (maximum intensity z stack projections shown in merge) illustrate cell morphology together with GFP and HA (14-3-3β) localization. Arrowheads (normal phenotype) indicate GFP and HA-14-3-3β-expressing cells (F-actin images). (E) Cell morphology together with FLAG (Rnd3) and F-actin localization. Cells transfected with wild-type Rnd3 show the typical Rnd3-induced rounded phenotype with long thin protrusions (upper panels, arrow), whereas some cells have a distinct phenotype (lower panels, arrowheads). (F) Confocal images of cells expressing FLAG-Rnd1 or FLAG-Rnd2 with or without HA-14-3-3β, and stained for FLAG. Bars, 20 μm. (G) FLAG-Rnd3 WT was transfected into COS7 cells, prior to cell lysis and biochemical fractionation. The different fractions were immunoblotted with the indicated antibodies. T: Total extracts; N: Nuclear fraction; C: Cytosolic fraction; M: Membrane fraction. Asterisk (^*) indicates IgG light chain, double asterisk (^**) indicates a nonspecific band.

Figure 3

14-3-3 Binding Inhibits Rnd3 by Inducing Translocation from the Plasma Membrane to the Cytosol (A and B) NIH 3T3 cells were cotransfected with the indicated constructs. (A) Cell morphology quantification. Graph shows pooled results from technical triplicates in three independent experiments (n = 300 cells/condition/experiment). Conditions with the same number (1, 2, 3) are not statistically significantly different from each other. Error bars indicate mean ± SEM; Table S2 shows statistical analysis between the conditions. (B) Single confocal images (maximum intensity z stack projections in merge) showing cell morphology and FLAG-Rnd3 localization. Arrows: FLAG-Rnd3-expressing cells (F-actin images) with a rounded phenotype with protrusions. Arrowheads: normal phenotype. Scale bar, 20 μm. (C) The indicated constructs were transfected into COS7 cells prior to cell lysis and biochemical fractionation. Fractions were immunoblotted with indicated antibodies. ERK1/2 and transferrin receptor are markers of cytosolic and membrane fractions, respectively. As controls (bottom panels), cells were transfected with the Rnd3 nonisoprenylated mutant S241 or Rnd3 WT with staurosporine (stau.), PMA, or calyculin A (cal. A) treatment. β, 14-3-3β; ROCK1, myc-ROCK1^1-420; T, total extracts; N, nuclear fraction; C, cytosolic fraction; M, membrane fraction. See also Figure S3 and Table S2.

Figure 4

Rnd Farnesylation Is Required for Interaction with 14-3-3 Proteins (A–C) The indicated constructs were transfected into COS7 cells. Cell lysates were immunoprecipitated with FLAG antibody. Endogenous 14-3-3 proteins bound to FLAG-Rnd3 were detected with 14-3-3 antibody. Membranes were stained with Pro-Q Diamond reagent or probed with the indicated antibodies. (B) COS7 cells were treated ± FTI overnight prior to cell lysis. The space in the gel image marks the position of lanes that were not germane to these results and were thus removed during figure preparation for clarity. (C) COS7 cells were treated with calyculin A (Cal. A) for 20 min ± FTI overnight prior to cell lysis. Asterisk (^*) indicates IgG light chain. See also Figure S4.

Figure S4

Farnesylation Is Required for Rnd3 Binding to 14-3-3 Proteins, Related to Figure 4 (A) Cell morphology together with FLAG (Rnd3) and F-actin localization in NIH 3T3 cells transfected with the indicated Rnd3 constructs and treated with FTI or DMSO as a control for 16-18 hr. Bar, 20 μm. (B) FLAG-Rnd3-AllA-S210+218+240 mutant ± ROCK1^1-420 were transfected into COS7 cells. Cells were treated ± FTI for 16–18 hr and PMA for 2 hr where indicated prior to cell lysis. Cell lysates were immunoprecipitated with anti-FLAG antibody. Endogenous 14-3-3 proteins bound to FLAG-Rnd3 were detected with anti-14-3-3 antibody. Membranes were also stained with Pro-Q Diamond reagent or immunoblotted with the indicated antibodies. Asterisk (^*) indicates light chain IgG.

Figure 5

Ser240 Phosphorylation and Cys241 Farnesylation Are Both Essential for Rnd3/14-3-3 Interaction (A) Farnesylated and/or phosphorylated peptides corresponding to the Rnd3 C terminus (see Table S3) were used as competitors for GST-14-3-3β pull-down experiment with lysates from COS7 cells transfected with FLAG-Rnd3. Bound (peptide competition) and unbound (input) fractions were immunoblotted with the indicated antibodies. A PKCε (PKC) peptide corresponding to a high affinity 14-3-3-binding sequence was used as a positive control. (B) Biotinylated peptides were incubated with recombinant 14-3-3β, followed by immunoblotting for 14-3-3. (C) Isothermal titration calorimetry analysis of 14-3-3ζ binding to Rnd3 peptides. Top: raw data; bottom: fitted curves. −, no peptide; −P−F, no modification; +P−F, with phosphorylation; −P+F, with farnesylation; +P+F, with phosphorylation and farnesylation. See also Figure S5 and Table S3.

Figure S5

Rnd3 Farnesylation and Phosphorylation Are Required for Interaction with 14-3-3 Proteins, Related to Figure 5 Isothermal titration calorimetry analysis showing the absence of 14-3-3ζ binding to unphosphorylated Rnd3 peptides with or without farnesylation. Raw data are shown in the top panels with fitted curves below. −P-F: no modification; −P+F: with farnesylation; ND: not detectable.

Figure S6

Structure of Rnd3 C-Terminal Peptide with 14-3-3ζ, Related to Figure 6 Crystal structure of 14-3-3ζ dimer (green ribbon) with one Rnd3 phosphorylated and farnesylated peptide bound to each 14-3-3 monomer (solid rendering, colored by atom type). The two-fold axis of the 14-3-3 dimer is vertical in this view ensuring that each Rnd3 peptide traverses C-N or N-C toward the viewer presenting either the prenyl moieties (Pr1 + Pr2) and Cys241 (left hand peptide, black and yellow) or pSer240 (right hand peptide, red), both are labeled for clarity.

Figure 6

Recognition of Phosphorylated and Farnesylated Rnd3 C-Terminal Peptide by 14-3-3 Reveals a Hybrid III/IV-Binding Mode (A) Structure of the Rnd3 C-terminal farnesylated phosphopeptide bound to 14-3-3ζ shown with the SIGMAA-weighted 2F_o − F_c electron density omit map (σ = 1.0). Electron density is observed for the first two isoprenyl units (Pr1 and Pr2) extending from Cys241. (B) Schematic view of the Rnd3 C-terminal farnesylated phosphopeptide bound to 14-3-3. Selected side chains from 14-3-3 making contact with Rnd3 are shown. (C) Sequence alignment of representative 14-3-3-binding motifs indicating how the Rnd family C-terminal motif is a hybrid III/IV motif with features of both a type III and IV motif. Addition of a farnesyl group effectively extends the length of Rnd3 similar to the ExoS-binding interaction. Red ball indicates the phosphoresidue (except for ExoS which has an alanine). The carboxyl terminus of Rnd and H⁺ATPase are shown as CO2H and the peptide chain direction of 14-3-3 motif sequences is indicated by an N and C subscript. Note the ExoS peptide is oriented in the opposite direction to all other 14-3-3 motifs (N-C rather than C-N). (D) Superposition of the Rnd3 C-terminal farnesylated phosphopeptide (black sticks) with the exoenzyme S peptide (ExoS, blue sticks, PDB code 2O02) and PKCε (salmon sticks, PDB code 2WH0), superposed through their respective 14-3-3 partners (gray surface). Hydrophobic residues from 14-3-3 (green sticks and green surface) contacting Rnd3 isoprenyl units 1 and 2 are labeled. Note the ExoS peptide is oriented in an opposing direction (N-C rather than C-N). Atoms are colored according to standard conventions, red, oxygen; blue, nitrogen; yellow, sulfur; gray, carbon; and green, phosphorus. (A) and (D) were prepared using the graphics program PYMOL (http://www.pymol.org). See also Figure S6 and Table S4.

Figure 7

Interaction of Phosphorylated and Prenylated Proteins with 14-3-3 (A) Biotinylated peptides corresponding to the Rnd3 C terminus modified with different prenyl groups were incubated with recombinant 14-3-3ζ, followed by immunoblotting for 14-3-3. (B) Alignment of C-terminal sequences of prenylated candidate proteins with Rnd3 C-terminal sequence. Sequences are shown in single letter amino acid code; underlined S denotes phosphorylated S240 in Rnd3. The CAAX box residues are represented in italic. (C) Binding of 14-3-3ζ to immobilized biotinylated Rap1A peptides. Biolayer interferometry was used to assess the binding of a phosphorylated Rap1A peptide with or without the geranylgeranyl modification. The geranylgeranylated peptide has a standard-binding profile. The nonmodified phosphopeptide showed little binding, whereas a negative control with no immobilized peptide gave no response. (D) Constructs encoding GFP-Rap1A and GFP were transfected into COS7 cells. Endogenous 14-3-3 proteins bound to immunoprecipitated GFP-Rap1A were detected with 14-3-3 antibody. The membrane was probed with the indicated antibodies. See also Table S5.