FKBP12 binds to acylated H-ras and promotes depalmitoylation

Abstract

A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP loading of Ras in response to growth factors, and promotes H-Ras-dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines.

Figure 1. FKBP12 Regulates H-Ras Palmitoylation

(A) COS-1 cells were co-transfected with mCherry-H-Ras and GFP extended either with the last 10 or 19 amino acids of H-Ras (GFP-Htail) and imaged alive with a confocal microscope. Bars indicate 2μm. A proline rich sequence (PRS) found only in the 19aa tail is shown below. (B-F) Metabolic labeling of COS-1 cells with [³H]palmitic acid. (B) Cells expressing GFP-19aaTail or GFP-10aaTail. Text indicates relative levels of palmitoylation after normalization for protein (mean ± SEM, n=3, p<0.0001). (C, D) Cells expressing GFP-H-Ras labeled in the presence of vehicle (DMSO), CHX (50 ng/ml), DM-CHX (10 μM), FK506 (1 μM), rapamycin (500 nM), AP21967 (2 μM) or 2-bromopalmitate (10 μM). (E) Cells expressing the indicated H-Ras or H-Ras tail constructs were treated with FK506 or vehicle. (F) Cells expressing GFP-H-Ras, GFP-H-RasP179A or GFP-H-RasPP173/4AA and pre-treated for 72 hrs with a control siRNA or siRNA directed to FKBP12 or FKBP38 (immunoblot below shows extent of knockdown). (C-F) Representative fluorograms and immunoblots are shown as are plots of pooled data where fluorogram values were normalized to protein expression as determined by Li-Cor Odyssey quantification of immunoblots. The ratio of the normalized values with/without drug or siRNA is plotted (mean ± SEM, n≥3) such that a value>1 indicates enhanced palmitoylation.

Figure 2. FKBP12 Facilitates H-Ras Depalmitoylation

(A) Palmitoylation of GST-H-Ras30aa tail in vitro using purified DHHC9/GPC16 and [³H]palmitoyl CoA in the presence or absence of FK506 or recombinant FKBP12. (B) [³H]palmitate pulse (1 mCi/ml × 5 min)–chase labeling of GFP-H-Ras expressed in COS-1 cells. (C, D) [³H]palmitate pulse–chase labeling as in (B) for the GFP-H-Ras and GFP-H-RasP179A, with or without FK506 (1 μM). A representative fluorogram and immunoblot are shown (C) and pooled results are plotted as percent ³H remaining after 5 min chase (D) (mean ± SEM, n=3). (E) [³H]palmitate pulse–chase labeling as in (B) in cells expressing the indicated H-Ras constuct and pretreated for 72 hrs with a control siRNA or siRNA directed to FKBP12 or FKBP38.

Figure 3. FKBP12 Binds Palmitoylated Ras

(A, B) Lysates of COS-1 cells expressing GFP-K, N, or H-Ras were immunoprecipitated with anti-Ras Y13-259 antibody and immunoblotted for Ras and FKBP12 (A) or affinity purified with GST-FKBP12 and immunoblotted for Ras (B). (C) Detergent extracts of neonatal mouse brains were immunoprecipitated with Y13-259 or a control rat Ig in the presence or absence of FK506 (1 μM) and precipitates were immunoblotted for endogenous FKBP12 and Ras. (D) Lysates of HeLa S2 cells were affinity purified with GST or GST-FKBP12 and immunoblotted for endogenous Ras. GST binds the secondary antibody non-specifically. (E, F) Lysates of COS-1 expressing GFP, GFP-H-Ras (WT) ± FK506 (1 μM), 2-bromopalmitate (2BP, 50 nM) or simvastatin (statin, 10 μM) or H-Ras palmitoylation-deficient mutants (181S or 184S) were immunoprecipitated with Y13-259 (E) or affinity purified with GST or GST-FKBP12 (F) and immunoblotted for Ras. (G, H) Lysates from COS-1 cells expressing GFP-H-Ras 10aa tail or 19aa tail ± 2BP or simvastatin as in (E, F) were immunoprecipitated with anti-GFP antibody (G) or affinity purified with GST-FKBP12 (H) and immunoblotted for GFP. (I, J) Lysates from COS-1 expressing GFP-H-Ras wild type or single or double proline mutants (179A or 173A/174A) were analyzed as in (G, H). (K) Lysates of COS-1 expressing GFP-H-Ras were immunoprecipitated with anti-GFP antibodies and immunoblotted for GFP and either FKBP12 or FKBP38.

Figure 4. FKBP12 Regulated H-Ras Trafficking

(A) COS-1 cells were co-transfected with mCherry-GalT (Golgi marker) and GFP extended with either the C-terminal 10 or 19 aa of H-Ras (GFP-H-Ras-10aaTail and GFP-H-Ras-19aaTail) or 19 aa in which the equivalent of proline 179 was changed to alanine (GFP-H-Ras-19aaTailP179A). Cells were imaged alive by confocal microscopy following overnight treatment with FK506 (1 μM) or vehicle control (DMSO). (B) COS-1 cells transfected and imaged as in (A) 3 days after transfection with siRNA directed against FKBP12 or a non-targeting control siRNA. Bar represents 10 μm. Each experiment was performed at least 3 times and 5 to 14 cells were imaged on each plate. The patterns shown are representative of 80–100% of the cells examined.

Figure 5. FKBP12 Promotes Retrograde Trafficking of H-Ras from the PM to the Golgi

Confluent monolayers of MDCK cells were co-transfected with mCherry H-Ras and H-Ras wt or P179A tagged at the N-terminus with a photoactivatable GFP (paGFP). Cells were imaged alive in the presence or absence of FK506 (1 μM) before and after photoactivation of paGFP-H-Ras exclusively at the PM compartment marked by mCherry-H-Ras (activation region outlined in white in pre-activation panels). Bar represents 10 μm. Images are representative of 13 to 15 experiments. Accumulation of photoactivated paGFP-H-Ras on the Golgi was observed as follows: WT 8/14; WT + FK506 1/14; P179A 10/13; P179A + FK506 12/15.

Figure 6. FKBP12 Limits H-Ras Activation at the PM

(A) Lysates from HEK293 stably expressing a tetracycline inducible, tandem affinity peptide tagged H-Ras (TAP-H-Ras) were immunoblotted, before and after induction with doxycycline, using antibodies specific for H-Ras, N-Ras or K-Ras. (B) HEK293 expressing TAP-H-Ras or TAP-K-Ras were serum starved overnight in the presence or absence of FK506 (1 μM) and then stimulated with 10 ng/ml EGF for 5 or 30 minutes. GTP-bound Ras was affinity purified from cell lysates with GST-RBD. Ras was detected by immunoblot with a pan-Ras antibody from both GST-RBD pull downs (top panels) and lysates (bottom panels). (C) Quantification of GTP-Ras from (B) with results presented as fold change in GTP-bound Ras normalized to expression. Values plotted are mean ± SEM, n=4. (D) Spatiotemporal activation of H-Ras in live COS-1 cells co-transfected with H-Ras and GFP-RBD. Cells were serum starved, treated with FK506 (1 μM), AP21967 (2 μM) or vehicle and imaged alive before and after stimulation with EGF (50 ng/ml). Arrowheads indicate initial recruitment of GFP-RBD to the PM. Bar represents 10 μm. The experiment shown is representative of eight.

Figure 7. FKBP12 Restricts H-Ras Signaling

(A) PC12 cells were co-transfected with H-Ras17N or vector and GFP and then treated with NGF (50 ng/ml) with or without FK506 (1 μM) or vehicle control (DMSO). After 4 days GFP positive cells were scored for neurite processes with lengths 1.5 times the diameter of the cell soma. Results shown are mean±SEM, n=3, p<0.001 for vector ± FK506. (B) PC12 cells were transiently transfected with either GFP alone or GFP-H-Ras12V, GFP-N-Ras12D, or GFP-K-Ras12V. After 3 days in the presence of FK506 (1 μM) or vehicle control (DMSO), GFP positive cells were scored for neurites. Bar represents 50 μm. (C) Results from (B) are plotted as percent of cells with neurite outgrowth (mean ± SEM, n=4). (D) PC12 cells were transfected with GFP-H-Ras12V or GFP-H-Ras12V179A and treated with or without FK506. Results shown are mean±SEM, n=4.