14-3-3:Shc scaffolds integrate phosphoserine and phosphotyrosine signaling to regulate phosphatidylinositol 3-kinase activation and cell survival

Abstract

Integrated cascades of protein tyrosine and serine/threonine phosphorylation play essential roles in transducing signals in response to growth factors and cytokines. How adaptor or scaffold proteins assemble signaling complexes through both phosphotyrosine and phosphoserine/threonine residues to regulate specific signaling pathways and biological responses is unclear. We show in multiple cell types that endogenous 14-3-3zeta is phosphorylated on Tyr(179) in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3zeta can function as an intermolecular bridge that couples to phosphoserine residues and also directly binds the SH2 domain of Shc via Tyr(179). The assembly of these 14-3-3:Shc scaffolds is specifically required for the recruitment of a phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN cell survival in response to cytokine. The biological significance of these findings was further demonstrated using primary bone marrow-derived mast cells from 14-3-3zeta(-/-) mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3zeta(-/-) mice when reconstituted with wild type 14-3-3zeta, but the Akt phosphorylation and survival response was reduced in cells reconstituted with the Y179F mutant. Together, these results show that 14-3-3:Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses.

FIGURE 1.

14-3-3 proteins undergo tyrosine phosphorylation in response to GM-CSF stimulation. A, primary human mononuclear cells from peripheral blood (left panel) or CTL-EN cells (right panel) were stimulated with GM-CSF. Endogenous 14-3-3 was precipitated with a Ser(P)⁵⁸⁵ peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG; to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-Shc and anti-14-3-3 pAbs. B, factor-dependent CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight in medium containing 0.5% FCS and then stimulated with GM-CSF. The cells were lysed, and pulldown experiments were performed using either GST-SH2^Shc (to precipitate tyrosine-phosphorylated endogenous 14-3-3) or the Ser(P)⁵⁸⁵ peptide (to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-14-3-3 pAb. C, HEK 293T cells were transfected with a construct for the expression of wt 14-3-3ζ-Myc. After 48 h, the cells were lysed and 14-3-3ζ-Myc immunoprecipitated using the 9E10 mAb. Immunoprecipitates were subjected to immunoblot analysis with either the 4G10 mAb (Millipore) (top panel), an anti-phosphotyrosine pAb (Biomol) (middle panel) or the 9E10 mAb (bottom panel). D, the crystal structure of human 14-3-3ζ indicates that it is composed of 2 monomers that dimerize to form a large central groove that binds to phosphoserine peptides (PS-peptide) (16). The location of tyrosines 19, 118, 178, 179, and 211 of 14-3-3ζ are indicated. E and F, recombinant purified 14-3-3ζ and 14-3-3ζ-T179F were phosphorylated in vitro using c-Src and then subjected to immunoblot analysis using the 4G10 anti-phosphotyrosine mAb (anti-PY) (E, top panel), anti-Tyr(P)¹⁷⁹-14-3-3ζ pAb (anti-PY179) (F, top panel) or anti-14-3-3ζ pAb to indicate loading (E and F, bottom panels). G, HEK-293T cells were transfected with constructs for the expression of wt 14-3-3ζ-Myc or the T179F mutant. After 48 h, the cells were stimulated for 15 min with sodium pervanadate (VO₄) (+) or left unstimulated (-). The cells were then lysed and subjected to immunoprecipitation using the 9E10 mAb. Immunoprecipitated proteins were subjected to immunoblot analysis using the anti-Tyr(P)¹⁷⁹-14-3-3ζ pAb (G, top panel) and the 9E10 mAb (G, bottom panel). H, CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight. The cells were then stimulated with 50 ng/ml GM-CSF or sodium pervanadate (VO₄), following which the cells were lysed, and endogenous 14-3-3 was precipitated (PD) using either a Ser(P)⁵⁸⁵ peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG) or an Ser⁵⁸⁵ (non-phospho-Ser⁵⁸⁵) control peptide (Biotin-NHS-KGGFDFNGPYLGPPHSRSLPDGG) adsorbed to streptavidin-Sepharose resin. Pulldowns were subjected to immunoblot analysis using the anti-Tyr(P)¹⁷⁹-14-3-3ζ pAb or anti-14-3-3 pAb. I, primary human mononuclear cells were stimulated with GM-CSF and total endogenous 14-3-3 was precipitated as in A. Precipitates were blotted with anti-Tyr(P)¹⁷⁹-14-3-3ζ pAb or anti-14-3-3 pAb. J, the UT7 factor-dependent cell line was factor-deprived overnight and then stimulated with 50 ng/ml GM-CSF, following which the cells were lysed, and the βc subunit of the GM-CSF receptor was immunoprecipitated using the 8E4 and 1C1 mAbs. Immunoprecipitates were then subjected to immunoblot analysis with either anti-Tyr(P)¹⁷⁹ pAb (top panel) or 1C1 anti-βc mAb (bottom panel). The results are typical of at least two experiments. WB, Western blotting.

FIGURE 2.

Tyr¹⁷⁹ of 14-3-3ζ is necessary for GM-CSF-mediated recruitment of Shc. A, structural analysis of 14-3-3ζ and the SH2 domain of Src shows the solvent-accessible surface of 14-3-3ζ (i) and the location of the ¹⁷⁹YEIL is indicated as a patch of red on the surface, the ¹⁷⁹YEIL motif (cyan) from 14-3-3 in the orientation required to bind to the SH2 domain of Shc (ii), and a cartoon of the SH2 domain of Shc (green) with phosphopeptide (blue) bound (iii). B, purified recombinant GST or GST-SH2^Shc bound to glutathione-Sepharose was incubated with either purified recombinant 14-3-3ζ phosphorylated on Tyr¹⁷⁹ or a nonphosphorylated 14-3-3ζ control for 1 h in Nonidet P-40 lysis buffer. Pulldowns were washed and subjected to immunoblot analysis with 4G10 mAb. The diffuse gray band corresponds to GST-SH2^Shc (bracket), and tyrosine-phosphorylated 14-3-3ζ (arrowhead) lies immediately below. C, HEK 293T cells were transfected with constructs expressing either the wt 14-3-3ζ-Myc or the indicated mutants. After 48 h, the cells were lysed, and lysates were incubated with either the GST-SH2^Shc or a GST control. Pulldowns (PD) and whole cell lysates (WCL) were subjected to immunoblot analysis using the 9E10 mAb. D, CTL-EN cells expressing the human GM-CSF receptor were electroporated with constructs for the expression of either wt 14-3-3ζ-Myc or the indicated mutants and factor-deprived overnight. The cells were then stimulated with 50 ng/ml GM-CSF followed by lysis and anti-Shc immunoprecipitation. Immunoprecipitates (IP) were subjected to immunoblot analysis using the 9E10 mAb or anti-Shc pAb. Tyrosine phosphorylation of the βc subunit of the GM-CSF receptor in response to GM-CSF was examined in lysates (WCL) using the 4G10 mAb (PY-βc). WCL were also immunoblotted using the 1C1 anti-βc mAb and the 9E10 mAb to demonstrate equal loading. The results are typical of two experiments.

FIGURE 3.

The 14-3-3ζ-T179F mutant is able to bind phosphoserine/threonine target proteins and heterodimerize with other isoforms of 14-3-3. HEK 293T cells were lysed and whole cell lysates subjected to pulldown experiments using either GST-Sepharose, GST-14-3-3ζ-Sepharose, or GST-14-3-3ζ T179F-Sepharose. Pulldowns were examined by immunoblot analysis using anti-Raf-1 and anti-BAD pAb (A) or a phospho-specific antibody that recognizes phosphorylated 14-3-3 binding motifs (Cell Signaling) (B). C, recombinant purified 14-3-3ζ was phosphorylated with c-Src (Tyr(P)¹⁷⁹-14-3-3ζ) or left nonphosphorylated (non-phospho-14-3-3ζ) and then incubated with either GST-SH2^Shc or GST alone bound to glutathione-Sepharose resin. The resin was washed to remove unbound 14-3-3ζ, and the assembled complexes (the complex assembled in lane 1 and used in the pulldown experiment is illustrated) were then used to pulldown 14-3-3-binding proteins from HEK 293T cell lysates. The pulldowns were washed in Nonidet P-40 lysis buffer and subjected to immunoblot analysis with phospho-specific antibodies that recognize phosphorylated 14-3-3 binding motifs or anti-Raf-1 pAb. D, HEK-293T cells were either mock transfected or transfected with constructs for the expression of wt 14-3-3ζ-Myc or the T179F mutant. After 48 h, the cells were lysed in Nonidet P-40 lysis buffer and subjected to immunoprecipitation using the 9E10 mAb. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblot analysis with phospho-specific antibodies that recognize phosphorylated 14-3-3 binding motifs (top panel) or the 9E10 mAb (bottom panel). E, HEK-293T cells were co-transfected with the indicated combinations of 14-3-3 isoforms. After 48 h, the cells were lysed, and lysates were immunoprecipitated (IP) with the 9E10 mAb. Immunoprecipitates and whole cell lysates (WCL) were subjected to SDS-PAGE and immunoblotted using the 9E10 mAb or the 12CA5 mAb. The 14-3-3ζ-Myc-T179F mutant was able to co-immunoprecipitate with 14-3-3γ-EE (data not shown), 14-3-3ζ-HA, and 14-3-3τ-HA. WB, Western blotting.

FIGURE 4.

Tyr¹⁷⁹ of 14-3-3ζ is necessary for the GM-CSF-mediated recruitment and activation of PI 3-kinase. A, CTL-EN cells expressing the human GM-CSF receptor were electroporated with constructs for the expression of either the wt 14-3-3ζ-Myc or the indicated mutants, and cells were factor-deprived overnight. The cells were then stimulated with 50 ng/ml GM-CSF for 5 min and lysed, and 14-3-3ζ-Myc was immunoprecipitated using the 9E10 mAb. Immunoprecipitates were then washed, and PI 3-kinase activity was measured in vitro using ³²P-γATP and phosphatidylinositol (PIP) as substrates. Extracted ³²P-lipids were subjected to thin layer chromatography, and indicated are ³²P-PIP and the origin. Quantification of these PhosphorImager signals (Typhoon) is shown in B. C, CTL-EN cells were electroporated without plasmid (mock), wt 14-3-3ζ-Myc, or the T179F mutant, following which the cells were factor-deprived and stimulated with GM-CSF as for A. 14-3-3ζ-Myc was immunoprecipitated with the 9E10 mAb with ⅓ of the immunoprecipitate used for PI 3-kinase assays (D) and 2/3 used for immunoblot analysis with anti-p85 pAb or 9E10 mAb (C). E, UT7 factor-dependent cells were electroporated with the indicated plasmids and PI 3-kinase assays were performed as in A and D. Quantification of PhosphorImager signals from two experiments is shown, and the error bars indicate the standard deviations (F). PI 3-kinase activity associated with the T179F mutant (♦) at 5 min was significantly decreased in comparison to the activity associated with wt 14-3-3ζ (▪) (^*, p < 0.03). Mock transfected cells are indicated by the ▴.

FIGURE 5.

14-3-3ζ can simultaneously bind phosphoserine residues and Shc. CTL-EN cells were electroporated with either wt 14-3-3ζ-Myc or the T179F mutant following which the cells were factor-deprived overnight and then stimulated with 50 ng/ml GM-CSF for 5 min. The cells were then lysed and pulldowns (PD) were performed using either a Ser(P)⁵⁸⁵ peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG) or a non-phospho-Ser⁵⁸⁵ control peptide (Biotin-NHS-KGGFDFNGPYLGPPHSRSLPDGG) adsorbed to streptavidin-Sepharose. PD and whole cell lysates (WCL) were subjected to SDS-PAGE and immunoblot analysis using anti-Shc pAb and the 9E10 mAb. The results are typical of two experiments.

FIGURE 6.

Tyr¹⁷⁹ of 14-3-3ζ required for the phosphorylation of Akt and is necessary and sufficient for regulating GM-CSF-mediated cell survival. CTL-EN cells expressing the GM-CSF receptor were co-electroporated with constructs expressing Akt-HA (A) or ERK-HA (B) and either wt 14-3-3ζ-Myc or the indicated 14-3-3ζ mutants. Co-transfection of Akt-HA or ERK-HA reporter plasmids allowed the specific examination of Akt and ERK phosphorylation in response to GM-CSF in transfected cells only. The cells were factor-deprived overnight and then stimulated with GM-CSF. The cells were then lysed, and the lysates were subjected to immunoblot analysis using the anti-phospho-Akt pAb (Thr³⁰⁸ and Ser⁴⁷³) (A), anti-active-ERK pAb (B) or the 9E10 or 12CA5 mAb. C, CTL-EN cells expressing the βcS585G mutant of GM-CSF receptor were co-electroporated with constructs expressing EGFP (to allow detection of transfected cells) and either wt 14-3-3ζ-Myc or the indicated mutants. After 24 h, the cells were washed and plated in medium containing either no factor (-) or 50 ng/ml GM-CSF (+). After a further 48 h, the cells were stained with annexin V-PE and analyzed by flow cytometry. Shown is the percentage of EGFP-positive cells that are annexin V-PE-positive. Cell proliferation was also examined (D). CTL-EN cells expressing the βcS585G mutant of GM-CSF receptor were co-electroporated with constructs expressing enhanced cyan fluorescent protein and either wt 14-3-3ζ-Myc or the T179F mutant. After 24 h, cyan fluorescent protein-positive cells were purified by fluorescence-activated cell sorting and plated in either no factor (-) or 50 ng/ml GM-CSF (+) for 24 h with a BrdUrd pulse for the last 4 h. The cells were then fixed, stained for BrdUrd incorporation, and analyzed by flow cytometry. E, primary bone marrow-derived mast cells from 14-3-3ζ^-/- mice were electroporated with either wt 14-3-3ζ-Myc or the T179F mutant. After 24 h, the cells were washed and plated at 1.5 × 10⁵/ml in serum-free DMEM containing 0.1% bovine serum albumin overnight and then stimulated with 1000 pm IL-3 and Akt phosphorylation was examined as in A. F, primary bone marrow-derived mast cells from wt and 14-3-3ζ^-/- mice were electroporated with either wt 14-3-3ζ-Myc or the T179F mutant together with EGFP to allow identification of transfected cells. After 24 h, the cells were washed and plated at 1.5 × 10⁵/ml in serum-free DMEM containing 0.1% bovine serum albumin and 1000 pm IL-3. The number of viable GFP-positive cells was determined by flow cytometry and forward/side scatter characteristics of mast cells at 48 h as a percentage of the initial GFP-positive population to account for differences in transfection efficiency. A significant reduction in IL-3-mediated survival of 14-3-3ζ^-/- bone marrow-derived mast cells expressing the T179F mutant was observed when compared with knock-out cells expressing the wt 14-3-3ζ-Myc (^*, p < 0.0007 from two separate experiments). All of the results are representative of at least two experiments.

FIGURE 7.

Model for the assembly of 14-3-3:Shc scaffolds. Stimulation with 50 ng/ml GM-CSF (GM) results in the activation of tyrosine kinases including JAK2 and Src family of kinases. Although we have shown that the activated receptor constitutes a high order dodecamer (33), only two subunits of the receptor are illustrated. JAK2 can phosphorylate Tyr⁵⁷⁷ of βc, whereas Src family kinases can phosphorylate Tyr¹⁷⁹ of 14-3-3ζ. The phosphorylation of 14-3-3ζ on Tyr¹⁷⁹ provides a docking site for the direct binding of the SH2 domain of Shc. The 14-3-3:Shc scaffold bound to Tyr⁵⁷⁷ of the βc subunit of the GM-CSF receptor is specifically required for the activation of the PI 3-kinase/Akt pathway and the regulation of cell survival.