14-3-3 interacts with Regulator of G Protein Signaling proteins and modulates their activity

(RGS) proteins (GAPs) that stimulate the inactivation of heterotrimeric G proteins. We have recently shown that RGS proteins may be regulated on a posttranslational level ( Benzing, T., ). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding of 14-3-3 is mediated by a conserved phosphoserine located in the G α -interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a conserved residue within the RGS domain, and results in inhibition of GAP function. Collectively, these data indicate that phosphorylation-dependent binding of 14-3-3 may act as molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant state. In Vitro Phosphorylation and Interaction. In vitro phosphorylation of GST.RGS7 315-469 and MBP.RGS7 315-469 was performed for 30 min at 37°C in a 100- µ l reaction in a buffer containing 20 mM HEPES, pH 7.4, 10 mM MgCl 2 , 0.1 mM CaCl 2 , 100 µ M ATP, 20 µ g/ml DAG, 100 µ g/ml phosphatidylserine, 0.03% Triton X-100, and the indicated amount of recombinant RGS7 protein. The phosphorylation was initiated by the addition of 0.5 U of recombinant protein kinase C α (1850 U/mg, Panvera) in enzyme dilution buffer or enzyme dilution buffer alone (control). To monitor the incorporation of phosphate, the unlabeled ATP was supplemented with 10 µ Ci [ γ 32 P]ATP, and radiolabeled MBP.RGS7 or GST.RGS7 315-469 was visualized by SDS-PAGE and autoradiography or spotted on nitrocellulose filter and counted in a scintillation counter. in vitro interaction studies, purified recombinant protein (1 µ g of phosphorylated or unphosphorylated GST.RGS7 or GST alone) was immobilized to glutathione sepharose beads and incubated with bacterial lysates containing 2.5 µ g/ml of recombinant MBP.14-3-3 for 90 min. in 450 µ l of binding buffer containing 50 mM potassium phosphate, pH 7.5, 150 mM KCl, 1 mM MgCl 2 , 10% (v/v) glycerol, 1% Triton X-100, and protease inhibitors. The washed precipitate was separated on a 10% SDS acrylamide gel. Bound MBP.14-3-3 τ was detected by immunoblotting using an anti-MBP rabbit antiserum (New England Biolabs). in 3 separate proteins were serially diluted (0-223 µ M) to a final volume of 150 µ l in PBS in 6 x 50 mm borosilicate glass tubes. Fluorescein-tagged peptide was added (59 nM final concentration), mixed, and fluorescence polarization measured at 22 o C after a 120 s delay with a 16 s integration. Background fluorescence was measured for each sample prior to peptide addition. Binding data was analyzed by assuming that fluorescence polarization was a linear function of ligand binding (21), and that each 14-3-3 monomer contained a single peptide binding site (19). Curves were fit to the equation: L B /L tot = L f /(k D +L f ) where L B is bound ligand, L tot is total ligand, L f is free ligand and k D is the dissociation constant, in closed form using non-linear regression analysis (Kaleidograph).

Pharmacia Biotech). Bound proteins were separated by 10% SDS-PAGE and RGS3 was visualized with anti-Flag antibody. Equal loading of GST.14-3-3τ was confirmed by coomassie blue staining of the gels.
In Vivo Co-Immunoprecipitation from Brain. For preparation of brain protein extracts whole brains of female BALB/c mice (20 g in body weight, Charles River) were removed and homogenized in 4 ml of brain lysis buffer (20 mM Tris, pH 7.5, 0.1% Triton X-100, 40 mM NaCl, 50 mM NaF, 15 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 1 mM EDTA, containing protease inhibitor mix and 44 µg/ml PMSF) for 15 min. on ice. Following centrifugation and ultracentrifugation (100,000 x g, 4°C, 30 min.), the supernatant was divided into 2 fractions and immunoprecipitated with specific anti-RGS7 antiserum and control antibody followed by incubation with protein Gsepharose. Resulting precipitates were subjected to immunoblot analysis with anti-14-3-3 mAb (Santa Cruz) followed by incubation with HRP-coupled secondary antiserum and enhanced chemiluminescence.
Pull-Down of Native RGS7 from Brain. GST and GST.14-3-3τ fusion protein was immobilized on affinity chromatography mini columns (BioRad) using glutathione sepharose beads; columns were washed extensively and pre-equilibrated by three washes with lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM NaF, 15 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 1 mM EDTA, and protease inhibitors) at 4°C. Whole brains of healthy adult WKY rats were frozen in liquid nitrogen, homogenized in 4 ml of lysis buffer, incubated on ice for 20 minutes, and centrifuged at 20,000 x g for 20 min. Supernatants were loaded on a GST or a GST.14-3-3τ column. Flow-through (identical volumes) was subjected SDS-PAGE and immunoblot analysis with anti-RGS7 antiserum, PKCα and β-catenin antibodies.
To monitor the incorporation of phosphate, the unlabeled ATP was supplemented with 10 µCi [γ 32 P]ATP, and radiolabeled MBP.RGS7 315-469 or GST.RGS7 315-469 was visualized by SDS-PAGE and autoradiography or spotted on nitrocellulose filter and counted in a scintillation counter.

Fluorescence Polarization Studies.
For fluorescence polarization assays GST.14-3-3τ fusion proteins were expressed in bacteria and purified on glutathione sepharose beads as described previously (19,20). Fluorescent peptides were synthesized using N-α-FMOC-protected amino acids and standard BOP/HOBt coupling chemistry on an ABI 431A Peptide BioSynthesizer, with fluorescein isothiocyanate connected to the peptide amino-terminus via a β-alanine linker.
Fluorescence polarization anisotropy was measured using a Panvera Beacon 2000 Variable Temperature Fluorescence Polarization System. Low fluorescence buffers and reagents (Panvera Corp) were used throughout. Binding curves were measured independently in 3 separate experiments. 14-3-3 proteins were serially diluted (0-223 µM) to a final volume of 150 µl in PBS in 6 x 50 mm borosilicate glass tubes. Fluorescein-tagged peptide was added (59 nM final concentration), mixed, and fluorescence polarization measured at 22 o C after a 120 s delay with a 16 s integration. Background fluorescence was measured for each sample prior to peptide addition. Binding data was analyzed by assuming that fluorescence polarization was a linear function of ligand binding (21), and that each 14-3-3 monomer contained a single peptide binding site (19). Curves were fit to the equation: L B /L tot = L f /(k D +L f ) where L B is bound ligand, L tot is total ligand, L f is free ligand and k D is the dissociation constant, in closed form using nonlinear regression analysis (Kaleidograph).

GTP Hydrolysis Assays.
Single turnover GTPase activity measurements were carried out as described (22,23,24). Recombinant myristoylated Gα i1 subunits (Calbiochem, 250 nM) were loaded with [γ− 32 P]GTP (1.0 µM) for 20 min. at 30°C in 500 µl of buffer containing 50 mM HEPES, pH 8.0, 5 mM EDTA, 2 mM DTT, and 0.1% lubrol. The stoichiometry of GTP binding of Gα i1 subunits was 25-40%. For zero time point a 12.5 µl aliquot was removed and added to 375 µl of 5% (w/v) Norit in 50 mM NaH 2 PO 4 . GTP hydrolysis was initiated at 4°C by adding 150 µl of the loaded Gα i1 subunits on ice to MgCl 2 (15 mM final concentration) and unlabelled GTP (150 µM final concentration), with or without purified, phosphorylated or unphosphorylated MPB.RGS7 315-469 (1.0 µM final concentration), that was preincubated with GST or GST.14-3-3τ (final concentration 5 µM, 30 min. on ice) as indicated. Aliquots of 25 µl were removed from the hydrolysis reaction, mixed with 375 µl of 5% (w/v) Norit in 50 mM NaH 2 PO 4 on ice, centrifuged at 10,000 rpm for 5 min. and counted by liquid scintillation spectrometry. Zero time values were subtracted from all experimental points. Statistical analysis was performed using the statistical and curve fitting functions of SigmaPlot 4.01 (Jandel Scientific). Hydrolysis rate constants were calculated according to Wang et al. (27). To demonstrate statistical significance of differences in GTP hydrolysis, hydrolysis rate constants were normalized, expressed as fold increase of basal hydrolysis rate constant, and averages of these constants were depicted in a table.
ERK1/2 phosphorylation. For the determination of ERK1/2 phosphorylation HEK293T cells were transfected with the plasmid DNA as indicated. After transfection cells were serum-starved overnight and incubated in the absence/presence of carbachol. After 15 min. the stimulation was stopped by placing the cells on ice and exchange of the media with ice-cold phosphate-buffered saline. Cells were harvested, lysed in a 1% Triton X-100 lysis buffer containing 20 mM Tris, pH 7.5, 50 mM NaCl, 50 mM NaF, 15 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 1 mM EDTA, protease inhibitors. The lysate was cleared by centrifugation and equal amounts of protein were separated by 12% SDS-PAGE. Dually phosphorylated ERK1/2 was visualized with phosphospecific antisera (New England Biolabs) that detects ERK1/2 only when phosphorylated at threonine 202 and tyrosine 204 (pT E pY motif). Equal loading was confirmed by reprobing the membrane with β-actin and amidoblack staining. The degree of dual phosphorylation of ERK1/2 was quantitated by densitometric analysis of non-saturated radiographs with the NIH Image software.

Staurosporine nearly abrogated the interaction between RGS3 and 14-3-3 in vivo and in vitro.
Only trace amounts of 14-3-3τ were co-immunoprecipitated in staurosporine-treated HEK 293T cells (Fig. 2b). Similarly, treatment of RGS3-expressing HEK 293T cells with staurosporine dramatically reduced binding of RGS3 to immobilized 14-3-3τ in vitro (Fig. 2c). Note that the staurosporine treatment did not cause a nonspecific reduction in the cellular amounts of either RGS3 or 14-3-3τ ( Fig. 2 b and c, lower panels). Similar experiments with RGS7 were precluded by the destabilization of RGS7 by staurosporine treatment. Both RGS7 and 14-3-3 are highly abundant in mouse brain. When we examined their endogenous interaction by coimmunoprecipitation of mouse brain lysates (Fig. 3a), 14-3-3 specifically co-precipitated with RGS7 indicating an endogenous in vivo interaction (Fig. 3b). To quantitatively assess the capacity of endogenous RGS7 to interact with 14-3-3, we determined the fraction of endogenous RGS7 retained by a recombinant glutathione-S-transferase-14-3-3 fusion protein immobilized to a glutathione sepharose column. Unbound RGS7 was measured in the flow-through by immunoblotting; PKCα and ß-catenin levels were used to correct for unspecific binding and equal loading (Fig. 3c). More than 50% of the RGS7 contained in brain lysates was retained on a GST.14-3-3τ column (Fig. 3d). Collectively, these data indicate that RGS3 and RGS7 interact with 14-3-3 in a phosphorylation-dependent manner. This interaction does not only occur in transfected cells but can also be demonstrated with endogenous proteins. Furthermore, the data suggest that a substantial fraction of RGS proteins is bound to 14-3-3 in vivo.
Mutational analysis revealed that serine 434 of RGS7 was critical for binding to 14-3-3.
Fluorescence polarization measurements confirmed that 14-3-3τ rapidly binds to a 14-mer phosphopeptide containing the serine 434 14-3-3 binding site of RGS7 with a k D of 15.9 µM (Fig.   4 b and c). Most published affinities for 14-3-3 interacting peptide sequences range from 0.1-2 µM, using surface plasmon resonance (Biacore); however, the interpretation of these affinities is complicated by an avidity effect since dimeric 14-3-3 may simultaneously bind to two phosphopeptides immobilized on the Biacore chip (19), while the fluorescence polarization experiments were performed with solubilized molecules. Given the strong interaction of 14-3-3 with RGS7 and RGS3 in the co-immunoprecipitation and pull-down experiments, this moderate k D suggests that additional factors such as dimerization of RGS proteins or tandem binding of 14-3-3 may contribute to the interaction of RGS proteins with 14-3-3 in vivo. Indeed, both RGS3 and RGS7 contain at least two additional potential 14-3-3 binding sites in close proximity within the RGS domain. Although our data clearly implicate serine 434 as a critical 14-3-3 binding residue in RGS7, it is conceivable that simultaneous binding to additional sites increases the affinity and stability of this interaction. Several 14-3-3 binding proteins such as c-Raf-1, Cbl, and BAD contain two 14-3-3 binding sequences separated by polypeptide segments of various length, and tandem binding to adjacent 14-3-3 sites has been shown to facilitate the formation of a highaffinity, bidentate complex (19).
Based on the resolution crystal structure of RGS4 complexed with activated Gα i subunits (26) the 14-3-3 binding site at serine 434 in RGS7 aligns with one of the three putative domains required for Gα i1 interaction and stimulation of GTPase activity of Gα i1 (Fig. 5c). To test whether binding of 14-3-3 interferes with the GTPase accelerating activity of RGS7, we measured the GAP activity of RGS7 in single-turnover GTP hydrolysis assays (22). GTP hydrolysis follows a single exponential time course equivalent to a first order reaction which can be expressed by means of the rate constant of a first order reaction (27). Addition of 14-3-3 reduced the hydrolysis rate constant of phosphorylated RGS7 from 10-fold to 3.5-fold (Fig. 5a), while addition of 14-3-3 to unphosphorylated RGS7 or Gα i1 had no effect on the hydrolysis rate constant (data not shown). In order to more quantitatively assess the effect of phosphorylation and/or 14-3-3 interaction on GAP activity of RGS7 hydrolysis rate constants of several experiments were averaged and expressed as fold increase of basal hydrolysis rate ( Table 1).
Addition of 14-3-3 to phosphorylated RGS7 almost completely abrogated the GTPasestimulatory effect, suggesting that phosphorylation of serine 434 and the subsequent interaction with 14-3-3 dramatically interferes with binding of activated Gα i1 . These findings suggest that a phosphorylation-dependent interaction between 14-3-3 and the RGS domain may regulate the GAP activity of RGS proteins; however, the kinase responsible for this phosphorylation in vivo has yet to be determined. RGS3 impairs MAP kinase activation by mammalian G protein-linked receptors in human embryonic kidney (HEK) cells (28). To illustrate the functional consequences of the interaction between 14-3-3 and RGS proteins, we co-transfected the Gα i -linked m2 cholinergic receptor with and without RGS3 and 14-3-3 into HEK cells expressing Rap1 and Rap1GAPII. Stimulation with carbachol (30 µM, 15 min) resulted in a strong dual phosphorylation of ERK1 and ERK2 on threonine 202 and tyrosine 204 (Fig. 5b). Dual phosphorylation of ERK1/2 within the T E Y motif leads to the activation of the kinase and represents a sensitive measure of ERK1/2 activity. RGS3 inhibited the carbachol-mediated MAP kinase phosphorylation. Co-expression of 14-3-3 rescued the Gα i -induced MAP kinase phosphorylation from this inhibitory effect of RGS3 (Fig. 5b), but did not affect MAP kinase activation in the absence of RGS3 (data not shown). Equal protein loading was ensured by reprobing the blot against actin and by amidoblack staining. These data provide further evidence that binding of 14-3-3 counteracts the inhibitory effect of RGS proteins on G protein-initiated signaling.
Alignment of the sequence bordering serine 434 in RGS7 with other RGS members reveals a putative 14-3-3 binding motif: K/E K/R D pS Y P (Fig. 5c) (19,20). Since the 14-3-3 binding site in RGS7 is conserved in other RGS members, we speculate that 14-3-3 binding may similarly regulate the GAP activity of other RGS proteins. It is unclear whether the reduction of RGS GAP activity depends upon a conformation change induced by 14-3-3 binding or on the physical impedance of the association of RGS and Gα. Our data suggest that phosphorylation-dependent interaction of RGS proteins with regulatory proteins such as 14-3-3 may rapidly and dynamically control RGS GAP activity without altering their expression.      Fluorescence polarization was used to determine the affinity between RGS7 and 14-3-3τ. A fluorescein-tagged RGS7 phosphopeptide CLMKSDpSYPRFIRS, derived from the putative 14-3-3 binding site of RGS7, was incubated with GST.14-3-3τ.   Table 1. Phosphorylation and interaction of RGS7 with 14-3-3 inhibits GAP activity. Hydrolysis rate constants were determined as described (27), and expressed as fold increase of basal GTP hydrolysis rate constant to compare three independent experiments. The average of the relative hydrolysis rate constants of these three experiments is shown in Table 1.