Dimerization-deficient 14-3-3 forms present reduced phosphoproteins binding. (A) Schematic representation of 14-3-3 ζ GST-fusion variants used in the study. (B) GST-fusion proteins were purified from ³⁵S-Met-labeled COS-7 cells coexpressing the indicated GST-fusion proteins and either myc-Raf (lanes 1-4) or myc-14-3-3 (lanes 5-9) and resolved using 10% SDS-PAGE. An autoradiogram showing the ³⁵S-labeled proteins is presented. Indicated are the migrations of the GST-fusion proteins, endogenous 14-3-3 and the myc-epitope tagged Raf and 14-3-3. (C) GST-fused wild-type 14-3-3 (wt), dimerization-deficient 14-3-3 (dm) or amino-terminally truncated 14-3-3 (ct) were purified from ³²P-labeled COS-7 cells treated as indicated with 100 nM EGF or 150 nM calyculin A (cal A) for 15 min. The recovered proteins were separated using a continuous 6-15% gradient (left) or a 6.5% (right) SDS-PAGE and analyzed by autoradiography. Indicated are the positions of the molecular weight markers and of the GST-14-3-3 fusion protein (left only).

Raf phospho-serine 259 antibody and a pan phospho-specific 14-3-3 binding site antibody display high specificity for their phosphorylated targets. (A) Myc-epitope-tagged c-Raf-1 variants were immunoprecipitated from COS-7 cells expressing wild-type (lanes 1-7), S259A (lanes 8-14), or S259/621A (lanes 15-17) myc-Raf alone, or coexpressing constitutive (ca, lanes 4, 5, 11, and 12) or kinase-dead (KD, lanes 6, 7, 13, and 14) AMP kinase, treated with 100 ng/ml EGF or 100 nM calyculin A for 15 min as indicated. The resulting Raf proteins were separated with 10% SDS-PAGE and immunoblotted with either anti-myc antibody (clone 9E10, bottom), phospho-Raf S259 (top; Cell Signaling Technology) or with a pan phospho-specific 14-3-3 binding site antibody (middle; Cell Signaling Technology). The position of myc-Raf in the various blots is indicated. (B and C) Serum-deprived COS-7 cells expressing wild-type (B) or S259A (C) myc-Raf variants were metabolically labeled with ³²P and stimulated with 100 ng/ml EGF for 15 min as indicated. myc-Raf proteins were immunoprecipitated with a myc-epitope tag antibody and subjected to phosphopeptide mapping as described in MATERIALS AND METHODS. Representative autoradiograms of the Raf phosphopeptide maps and a schematic representation of the phosphopeptide spots are presented. The locations of phospho-S43, -S621, and -S259 peptides are indicated. Note that the phospho-S259 spot is missing in the map of the Raf S259A mutant. The identity of the other phosphopeptide spots is unknown. (D) myc-Immunoprecipitates from vector control (lane 1), wild-type myc-Raf (lanes 2 and 4-6) or S259/621A myc Raf (lane 3)-transfected COS cells were treated with vehicle (lanes 1-3), alkaline phosphatase (CIAP, 20 U, lanes 4 and 5; Promega), or protein phosphatase 2A (UBI, PP2A, 0.5 U, lanes 6 and 7) for 30 min at 30°C. Lane 5 also included the alkaline phosphatase inhibitor PNPP (50 mM) and lane 7 the protein phosphatase inhibitor calyculin A (cal A, 10 nM). After dephosphorylation, the myc-immunoprecipitates were washed twice and analyzed as in A. Immunoblotting results with the pan phospho-specific 14-3-3 binding site antibody (top) and myc (bottom) are presented. (E) Twenty nanomoles of each synthetic peptide: Raf 613-627 (LPKINRSASEPSLHR, 613-627, lane 1), Raf 613-627 phosphopeptide phosphorylated on a site corresponding to Raf S621 (LPKINRSApSEPSLHR, pS621, lane 2), and Raf 461-486 phosphopeptide phosphorylated on two sites, corresponding to Raf S471 and T481 (AKNIIHRDMKpSNNIFLHEGLpTVKIGD, pS471/pT481, lane 3) were separated on a 17.5% SDS-PAGE and immunoblotted with the pan phospho-specific 14-3-3 binding site antibody.

Dimerization-deficient 14-3-3-associated proteins are not recognized by the pan phospho-specific 14-3-3 binding site antibody. (A and B) Serum-deprived COS-7 cells transiently expressing wild-type GST-14-3-3 (50 ng of DNA/10-cm plate, lanes 1-5 or 250 ng/plate, lanes 6-11) or dimerization-deficient GST 14-3-3 (250 ng/plate, lanes 12-14), were ³⁵S-Met labeled and treated with either carrier (lanes 1, 6, 11, and 12) for 60 min or calyculin A (100 nM, lanes 2-5, 7-10, and 14) for the indicated times. Cells were extracted in the absence (lanes 1-10, 12, and 14) or presence (lanes 11 and 13) of 300 μM synthetic Raf competitor phosphopeptide, and the recovered GST-fusion proteins were analyzed as described in Figure 2. The migration position of the molecular weight protein markers, GST-14-3-3 and coassociated cellular vimentin are indicated. To stress the difference between wild-type and dimerization-deficient 14-3-3-associated proteins and to demonstrate the ability of the competitor phosphopeptide to completely dissociate all associated proteins from wild-type 14-3-3 provided are two exposure levels (A and B).

Dimerization diminishes 14-3-3 susceptibility to phosphorylation. (A) Serum-deprived COS-7 cells expressing either wild-type GST-14-3-3 (lanes 1-5) or dimerization-deficient GST-14-3-3 (lanes 6-10) were metabolically labeled with ³²P for 2 h and stimulated with 100 ng/ml EGF and/or 300 nM calyculin A for 15 min as indicated. GSH-bead-pulled-down proteins were resolved using 8.5% SDS-PAGE and analyzed using autoradiography (bottom) and phosphorimaging (PI units, top). The migration positions of GST-14-3-3 and coassociated cellular vimentin are indicated. (B) COS-7 cells expressing dimerization-deficient GST-14-3-3 (left) or wild-type GST-14-3-3 (right) were deprived of serum for 18 h and were either left untreated (lane 1) or treated with 100 ng/ml EGF (lanes 2-4), 100 nM rapamycin (lanes 5-8), 200 nM phorbol 12-myristate 13-acetate (lanes 8 and 9, left; lanes 8-10, right) or 10% fetal calf serum (lanes 10-12, left; lanes 11-13, right) for the indicated periods. GSH-bead-pulled-down proteins were resolved using 8.5% SDS-PAGE and immunoblotted with the pan phospho-specific 14-3-3 binding site antibody. Indicated are the migration positions of the molecular weight protein markers and the GST-14-3-3 protein.

Binding of dimerization-deficient 14-3-3 to DAF-16 is independent of DAF-16 phosphorylation. (A) Schematic representation of DAF-16 mutants used in the study. (B) HEK-293 cells were transfected with GST-14-3-3 (lanes 1-12) or dimerization-deficient GST-14-3-3 (lanes 13-24) together with m2-FLAG-epitope-tagged DAF-16 (wt, lanes 1-3 and 13-15), T54A DAF-16 (lanes 4-6 and 16-18), S240A/T242A DAF-16 (2A, lanes 7-9 and 19-21), or T54A/S240A/T242A DAF-16 (3A, lanes 10-12 and 22-24). The transfected cells were either left untreated or were serum deprived for 18 h in the absence or presence of the PI-3 kinase inhibitor LY294002 (LY, 10 μM) as indicated. GSH-bead-pulled-down proteins were resolved using 10% SDS-PAGE and immunoblotted for m2-DAF-16 (Sigma-Aldrich). The migration position of m2-DAF-16 is indicated. (C) Nuclear/cytoplasmic extracts of HEK-293 cells expressing control vector (lanes 1 and 2), m2-FLAG-DAF-16 (lanes 3-8), T54A DAF-16 (lanes 9 and 10), or T54A/S240A/T242A DAF-16 (3A, lanes 11 and 12) grown in serum (lanes 1-4 and 9-12) or deprived of serum in the presence of vehicle (lanes 5 and 6) or LY294002 (10 μM, lanes 7 and 8) were separated on 8.5% SDS-PAGE and were immunoblotted with the pan phospho-specific 14-3-3 binding site antibody (top) or with m2-FLAG antibody (bottom). The location of DAF-16 is indicated.

14-3-3 binds all of its target proteins in COS-7 cells via the phosphopeptide binding pocket, and most of the associated proteins contain a phosphopeptide recognized by a pan phospho-specific 14-3-3 binding site antibody. Serum-deprived COS-7 cells transiently expressing GST (50 ng of DNA/10-cm plate, lane 1) or GST-fused 14-3-3 ζ (10 ng/plate, lanes 2-6 or 50 ng/plate lanes 7-14) were ³⁵S-Met labeled and treated with either carrier (lanes 2, 7, and 12) for 60 min or calyculin A (100 nM, lanes 3-6, 8-11, 13, and 14) for the indicated times. Cells were extracted in the absence (lanes 1-11 and 14) or presence (lanes 12 and 13) of 300 μM synthetic competitor phosphopeptide corresponding to the Raf S621 14-3-3 binding site. GST-fusion proteins were precipitated using GSH-Sepharose beads, and the resulting proteins were separated using a continuous 6-15% gradient SDS-PAGE and visualized using either autoradiography (top) or by immunoblotting with the pan phospho-specific 14-3-3 binding site antibody (bottom). The migration positions of the molecular weight protein markers, GST-fusion proteins, and coassociated cellular vimentin are indicated. It is important to note that presented are exposure levels that most optimally represent the general result; however, our experiments used multiple exposures of both the ³⁵S-Labeling and immunoblotting experiments to make a comprehensive comparison of the two.

Binding of dimerization-deficient 14-3-3 to Raf is independent of Raf phosphorylation. (A and B) GST-fusion proteins purified from ³²P-labeled COS-7 cells coexpressing myc-Raf and either wild-type GST-14-3-3 (wt) or dimerization-deficient GST-14-3-3 (dm) (A) or wild-type GST-14-3-3 or amino-terminally truncated GST-14-3-3 (ct) (B) were resolved using SDS-PAGE. The GST-14-3-3-associated myc-Raf was analyzed by myc immunoblotting (A and B, bottom) to quantify Raf recovery and by phosphorimaging to quantify ³²P incorporation in Raf (A, middle; B, top). Indicated are the migration positions of myc Raf and the ³²P-myc-Raf. Top, A, graphic representation of the phosphorimaging quantification of ³²P in myc-Raf presented as PhosphorImager (PI) units. (C) COS-7 cells were transfected with myc-epitope tagged wild-type Raf (lanes 1-4), S259A Raf (lanes 5-7), or S259/621A Raf (lanes 8-10) together with either GST (lane 1), GST-14-3-3 (wt, lanes 2, 5, and 8), dimerization-deficient GST-14-3-3 (dm, lanes 3, 6, and 9), or carboxy-terminal GST-14-3-3 (ct, lanes 4, 7, and 10). Total cell extracts and GSH-bead-pulled-down proteins were resolved using 8.5% SDS-PAGE and immunoblotted for myc-Raf (bottom) or phospho-S259 Raf (top). The migration position of myc-Raf and phospho-S259-myc-Raf are indicated. The lower double band in the phospho-S259 Raf blots corresponds probably to the endogenous phospho-S259 Raf protein.