Supporting Information

Galan et al. 10.1073/pnas.1405601111 SI Experimental Procedures Sample Preparation and Trypsin Digestion. Cells were lysed in 1% GST-14-3-3 Fusion , Subtractive Fractionation, and Pull-Down sodium deoxycholate in 50 mM ammonium bicarbonate (pH 7.8) Assays. Fifty milliliters of overnight cultures of BL21 Escherichia and heated to 99 °C for 5 min. The mixture was then allowed to coli transformed with pGEX-4T-14-3-3e WT or K49E mutant cool down on ice and centrifuged at 13,000 × g for 10 min 5 mM were diluted into 500 mL and culture was induced with 1 mM DTT was added to the supernatant and incubated for 30 min at isopropyl β-D-1-thiogalactopyranoside overnight at 25 °C. Cells 56 °C, followed by alkylation with 15 mM iodoacetamide for 1 h were pelleted and resuspended in 40 mL of bacterial lysis buffer at 25 °C in the dark. Excess iodoacetamide was neutralized using (1× PBS/10 mM EDTA/0.1% Triton X, 1 mM PMSF, and 1× 5 mM DTT incubated for 15 min at 25 °C. Proteins were digested protease inhibitor mixture). Extracts were placed in a 50-mL overnight with sequencing-grade modified trypsin (enzyme: conical tube on ice and sonicated using a probe sonicator six ratio of 1:50) at 37 °C. The digested mixture was acid- times for 30 s with 30-s delays between blasts. After sonication, ified with 1% formic acid (FA) final concentration. The samples the extracts were centrifuged at 13,000 × g for 30 min and ali- were desalted using C18 (Waters) cartridge following the manufacturer’s instructions and then evaporated to dryness in a quoted into 1-mL tubes to be stored at −80 °C until further use. SpeedVac. For subtractive fractionation, we used a method previously de- scribed (1). Briefly, serum-starved A375 cells were lysed as de- Phosphopeptide Isolation, Offline Strong Cation Exchange Fractionation, scribed above. Cellular debris were removed by centrifugation for and Nanoflow Liquid Chromatography-MS/MS. The peptide samples 10 min at 13,000 × g. The supernatant, corresponding to 20 mg of were subjected to the TiO2 enrichment protocol as described in protein, was precleared by pouring it sequentially over glutathi- ref. 2. Briefly, sample loading, washing, and elution steps were one-Sepharose (GE/Amersham/Pharmacia), glutathione-Sephar- performed in homemade spin columns assembled following the ose bound to 500 μg of GST, and glutathione-Sepharose bound StageTip principle (3, 4) and composed of a 200-μL pipette tip with μ e to 250 g of GST-14-3-3 K49E. The flow-through was divided frit made of SDB-XC membrane (3M) and filled with TiO2 beads. equally and poured over GST-14-3-3e K49E (250 μg) or GST- SDB-XC material has similar hydrophobic properties to C18 and 14-3-3e WT (250 μg) (as shown in the schematic of Fig. 4A). The allows for combining phosphopeptide enrichment and desalting beads were then washed two times with 2 mL lysis buffer and steps. Centrifugation speed was set to 2,000 × g. Before peptide a final wash of 2 mL of lysis buffer lacking Nonidet P-40 and Brij loading, columns were equilibrated with 100 μL of loading buffer 35. Cellular proteins bound to each column were eluted stepwise [250 mM lactic acid in 70% acetonitrile (ACN) and 3% TFA]. μ with 400 μL of 400 mM MgCl2 in 50 mM Hepes, 400 μLof Peptides were solubilized in 100 L of loading buffer and applied μ 800 mM MgCl2 in 50 mM Hepes, and finally chased with 200 μL on a TiO2 column. Each column was washed with 100 Lof of 50 mM Hepes. Eluates were combined and precipitated with loading buffer followed by 2 × 100 μL of 125 mM asparagine and 15% trichloroacetic acid. Precipitated proteins were pelleted, glutamine in 70% ACN and 3% TFA and 100 μL of 70% ACN μ washed with acetone, resuspended in reducing sample buffer, and 3% TFA. Subsequent washing with 50 Lof1%FAwasused pH-adjusted with one-sixth volume of 1 M Tris base, boiled, and to equilibrate SDB-XC frit material. Phosphopeptides were × μ subjected to SDS/PAGE for Coomassie staining or immuno- eluted from TiO2 with 2 50- L portions of 500 mM Na2HPO4, blotting. For smaller-scale GST pull-downs, cell lysates were in- pH 7, and retained on SDB-XC frit. Peptides were desalted in 50 μ μ cubated with 10 μg of GST-14-3-3e WT or K49E for 2 h and L of 1% FA and subsequently eluted from SDB-XC in 50 Lof 50% ACN and 0.5% FA. Eluates were dried on a speedvac and washed four times with lysis buffer before elution with reducing − sample buffer, SDS/PAGE, and immunoblotting. stored at 80 °C. To increase phosphoproteome coverage before MS analysis, Stable Isotope Labeling by Amino Acids in Cell Culture. HEK293 and phosphopeptides were fractionated offline by strong cation ex- 12 14 12 14 change (SCX) chromatography. Peptides were solubilized in 100 A375 cells were grown in light ([ C6 N2]Lys, [ C6 N4]Arg) 13 15 13 15 μL of loading buffer (0.2% FA and 15% ACN) and loaded onto and heavy ([ C6 N2]Lys, [ C6, N4]Arg) DMEM (Cambridge Isotope Labs) where required. Both light and heavy cell types StageTips containing 6 mg of PolySULFOETHYL A SCX phase (5 μm, 300 angstrom). Then columns were washed with were supplemented with 10% dialyzed fetal bovine serum (In- μ vitrogen). For the use of pharmacological inhibitors, HEK293 an additional 50 L of the loading buffer and peptides were eluted in 100-μL salt steps with 40, 70, 100, 150, and 500 mM cells were serum-starved for 24 h and cells cultured in heavy NaCl dissolved in loading buffer. Flow-through and salt fractions media were treated with PD184352 (10 μM) or BI-D1870 were collected, dried on a speedvac, resuspended in 15 μLof4% (10 μM) for 30 min followed by phorbol-12-myristate-13-acetate FA, and analyzed by nano liquid chromatography-MS/MS. (PMA) (50 ng/mL) treatment for 30 min in both light and heavy SCX fractions obtained after offline fractionation were analyzed conditions. In A375 cells, cells were serum-starved for 24 h and by online reverse-phase chromatography coupled with an elec- μ μ treated with PD184352 (10 M) or BI-D1870 (10 M) for 2 h in trospray ionization interface to acquire MS (measuring intensity the heavy media. For the use of shRNA to target RSK1/2, and m/z ratio for peptides) and MS/MS (fragmentation spectra of HEK293 cells in heavy media were virally infected as described peptides) scans. A nanoflow HPLC system (Eksigent; Thermo above, serum-starved for 24 h, and stimulated with PMA (50 ng/ Fisher Scientific) was used for online reversed-phase chromato- mL) treatment for 30 min in both light and heavy conditions. graphic separation; peptides were loaded on a 5-mm-long trap A375 cells in heavy media were virally infected as described column (inner diameter 300 μm) in buffer A (0.2% FA) and above and serum-starved for 24 h. We performed two biological separated on 18-cm-long fused silica capillary analytical column replicates for each experimental design detailed above with (inner diameter 150 μm), both packed with 3 μm 200 Å Magic cross-labeling (changing the treatment condition on the light AQ C18 reverse-phase material (Michrom). Peptides were eluted cultured cells instead of heavy cultured cells) without significant by an increasing concentration of buffer B (0.2% FA in ACN) differences. from 5 to 40% in 100 min. Following the gradient elution, the

Galan et al. www.pnas.org/cgi/content/short/1405601111 1of10 column was washed with 80% buffer B and reequilibrated with also added to the database as well as reversed versions of all 5% buffer B. Peptides were eluted into the mass spectrometer at sequences. For searching, the enzyme specificity was set to a flow rate of 600 nL/min. The total run time was ∼125 min, trypsin with the maximum number of missed cleavages set to 2. including sample loading and column conditioning. Peptides were The precursor mass tolerance was set to 20 ppm for the first search analyzed using an automated data-dependent acquisition on a [used for nonlinear mass recalibration (6)] and then to 6 ppm LTQ-Orbitrap Elite mass spectrometer. Each MS scan was for the main search. Search criteria included a static modifica- acquired at a resolution of 240,000 FWHM (at 400 m/z)for + mass range 300–2,000 Th with the lock mass option enabled tion of cysteine residues of 57.0214 Da; a variable modification + (m/z: 445.120025) and was followed by up to 12 MS/MS data- of 15.9949 Da to include potential oxidation of methionines; and dependent scans on the most intense ions using collision- a modification of +79.966 on serine, threonine, or tyrosine for the induced activation (CID). AGC target values for MS and MS/ identification of phosphorylation. The false discovery rate for pep- MS scans were set to 1E6 (maximum fill time 500 ms) and 1E5 tide, protein, and site identification was set to 1%, the minimum (maximum fill time 50 ms), respectively. The precursor isolation peptide length was set to 6, and the “peptide requantification” window was set to 2 Th with CID normalized collision energy of function was enabled. To transfer identifications across different 35; the dynamic exclusion window was set to 60 s. runs, the “match between runs” option in MaxQuant was enabled with a retention time window of 1 min. Peptide distributions were Data Acquisition, Quantitation Analysis, and Bioinformatics. MS data were analyzed using MaxQuant (5, 6) software version 1.3.0.3 and analyzed with Excel and R. Bioinformatics analysis was done with searched against the UniProt subset of the human da- Ingenuity Pathway Analysis tools. To infer biological significance tabase (www.uniprot.org) containing 69,048 entries. A list of 248 all ratios showing a 1.5-fold change (ratio ≥1.5 or ratio ≤0.65) common laboratory contaminants included in MaxQuant was was required.

1. Ballif BA, Cao Z, Schwartz D, Carraway KL, 3rd, Gygi SP (2006) Identification of 14-3- 4. Ishihama Y, Rappsilber J, Mann M (2006) Modular stop and go extraction tips with 3epsilon substrates from embryonic murine brain. J Proteome Res 5(9):2372–2379. stacked disks for parallel and multidimensional Peptide fractionation in proteomics. J 2. Kanshin E, Michnick SW, Thibault P (2013) Displacement of N/Q-rich peptides on TiO2 Proteome Res 5(4):988–994. beads enhances the depth and coverage of yeast phosphoproteome analyses. J 5. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized Proteome Res 12(6):2905–2913. p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 3. Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted 26(12):1367–1372. laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in 6. Cox J, et al. (2011) Andromeda: A peptide search engine integrated into the MaxQuant proteomics. Anal Chem 75(3):663–670. environment. J Proteome Res 10(4):1794–1805.

Galan et al. www.pnas.org/cgi/content/short/1405601111 2of10 Fig. S1. Stable isotope labeling by amino acids in cell culture (SILAC) quantification of global protein abundance is not affected by RSK1/2 depletion and inhibitor treatments. (A and B) Proteomic scheme of sample conditions and peptide distribution of protein abundance in HEK293 and A375 cells treated with PD184352, BI-D1870, or RSK1/2 RNAi. Note that most peptide ratios are 1:1 between light and heavy counterparts, suggesting that cellular treatments did not significantly affect protein abundance.

Galan et al. www.pnas.org/cgi/content/short/1405601111 3of10 Fig. S2. Global phosphopeptide distribution and analysis of p90 ribosomal S6 kinase (RSK)-dependent phosphorylation events. (A) Distribution of phos- phopeptides identified and quantified from HEK293 treated with PD184352 or BI-D1870. (B) Distribution of phosphopeptides identified and quantified from A375 cells treated with PD184352 (10 μM) or BI-D1870 (10 μM). (C) Distribution of phosphopeptides identified and quantified from HEK293 and A375 cells treated with shRNA constructs targeting RSK1/2. (D) Bar graph representations of known and predicted RSK substrates identified in this study. (E) Ingenuity Pathway Analysis of Ontologies (GO) enriched from down-regulated phosphopeptides by MEK1/2 and RSK inhibition or RSK1/2 RNAi.

Galan et al. www.pnas.org/cgi/content/short/1405601111 4of10 Fig. S3. RSK regulates Ser457 phosphorylation and PDCD4 subcellular localization. (A) HEK293 cells expressing HA-tagged PDCD4 were imaged using im- munofluorescence microscopy. Following transfection, cells were serum-starved and treated with PD184352 (10 μM), BI-D1870 (10 μM), or SL0101 (50 μM) for 30 min before PMA (50 ng/mL) stimulation for 30 min. Cells were stained with phalloidin to visualize the actin cytoskeleton, anti-HA to monitor PDCD4 lo- calization, and DAPI to visualize nuclei. (B) HEK293 cells expressing different mutants of HA-tagged PDCD4 (S67A or S457A) were imaged using immuno- fluorescence microscopy. Transfected cells were serum-starved and stimulated with PMA (50 ng/mL) for 1 h. (C) HEK293 cells were cotransfected with PDCD4 and an empty vector or a constitutively activated form of MEK1 (MEK-DD) and treated with PD184352 (10 μM) or BI-D1870 (10 μM). Immunoprecipitated PDCD4 was then assayed for phosphorylation using the anti-RXXpS/T motif antibody. (D) HEK293 cells expressing HA-tagged PDCD4 and MEK-DD were imaged using immunofluorescence microscopy. Transfected cells were incubated with HA and Flag antibodies to monitor PDCD4 and MEK expression, respectively. DAPI was used to visualize nuclei.

Galan et al. www.pnas.org/cgi/content/short/1405601111 5of10 Fig. S4. Analysis of the 14-3-3 interactome in melanoma cells. (A and B) Ingenuity Pathway Analysis of GO terms enrichments for molecular function and canonical pathways. (C) HEK293 cells were transfected with WT PDCD4, serum-starved, and stimulated with PMA (50 ng/mL) for 30 min before being harvested. Lysates were used in a GST pull-down assay for 14-3-3 (WT) binding to PDCD4 with increasing concentrations (micromolar) of the inhibitory R18 peptide.

Galan et al. www.pnas.org/cgi/content/short/1405601111 6of10 Fig. S5. RSK regulates 14-3-3 binding and degradation of PDCD4. (A) Sequence alignment showing conservation of Ser76 and Ser457 among different vertebrate species. (B) HEK293 cells were transfected with WT PDCD4 or the phosphorylation mutant S67A, serum-starved, and stimulated with PMA for 30 min. Binding to WT 14-3-3 was analyzed by GST pull-down assay. (C) HEK293 cells were transfected with WT PDCD4 or the phosphorylation double mutant S76/ 457A, serum-starved, and stimulated with PMA for 30 min. The 14-3-3 binding was analyzed as described in B.(D) HEK293 cells were transfected with PDCD4 and an empty vector or difopein and treated with PMA (50 ng/mL) during a cycloheximide (CHX) (100 μg/mL) time course. Extracts were prepared at each time point and analyzed by immunoblotting. (E) Densitometric analysis of PDCD4 was performed on the CHX time course shown in D and normalized to ERK1/2 band intensities. The data were then expressed relative to respective controls (t = 0).

Galan et al. www.pnas.org/cgi/content/short/1405601111 7of10 Table S1. Predicted RSK substrates ranked based on PSPL and SILAC scores Rank Accession no. Gene name Protein name PSPL score SILAC score Final score

1 Q9UPN9 TRIM33 E3 ubiquitin-protein ligase TRIM33 0.560 0.618 1.178 2 Q14157 UBAP2L Ubiquitin-associated protein 2-like 0.163 0.993 1.156 3 P62753 RPS6 40S ribosomal protein S6 0.682 0.252 0.934 4 O94763 URI1 19 ORF 2 0.801 0.072 0.873 5 Q9UGU5 HMGXB4 HMG box domain containing 4 0.567 0.250 0.817 6 O60343 TBC1D4 TBC1 domain family, member 4 0.683 0.108 0.791 7 Q92625 ANKS1A Ankyrin repeat and SAM-containing protein 1A 0.747 0.034 0.781 8 Q8IYH5 ZZZ3 ZZ-type zinc fi nger-containing protein 3 0.533 0.234 0.767 9 Q8TD19 NEK9 Serine/threonine-protein kinase Nek9 0.528 0.238 0.766 10 Q9NTI5 PDS5B Sister chromatid cohesion protein PDS5B 0.667 0.087 0.754 11 Q53EL6 PDCD4 Programmed cell death protein 4 0.645 0.102 0.747 12 Q8TEU7 RAPGEF6 Rap guanine nucleotide exchange factor (GEF) 6 0.473 0.274 0.747 13 Q92615 LARP4B La ribonucleoprotein domain family, member 4B 0.707 0.039 0.746 14 Q13535 ATR Serine/threonine-protein kinase ATR 0.663 0.072 0.735 15 Q14678 KANK1 KN motif and ankyrin repeat domains 1 0.679 0.051 0.730 16 Q7Z4V5 HDGFRP2 Hepatoma-derived growth factor-related protein 2 0.618 0.109 0.727 17 Q96ST2 IWS1 Protein IWS1 homolog 0.620 0.105 0.725 18 Q9HC44 GPBP1L1 GC-rich promoter binding protein 1-like 1 0.687 0.035 0.722 19 Q99759 MAP3K3 Mitogen-activated protein kinase kinase kinase 3 0.580 0.138 0.718 20 Q8N3D4 EHBP1L1 EH domain binding protein 1-like 1 0.685 0.031 0.716 21 Q96D71 REPS1 RALBP1 associated Eps domain containing 1 0.652 0.063 0.715 22 Q96F86 EDC3 Enhancer of mRNA decapping 3 0.645 0.066 0.711 23 Q16566 CAMK4 Calcium/calmodulin-dependent protein kinase IV 0.399 0.295 0.694 24 Q9NZN5 ARHGEF12 Rho guanine nucleotide exchange factor (GEF) 12 0.526 0.159 0.685 25 Q01831 XPC DNA repair protein complementing XP-C cells 0.637 0.046 0.683 26 Q09161 NCBP1 Nuclear cap-binding protein subunit 1 0.399 0.279 0.678 27 Q5SW79 CEP170 Centrosomal protein of 170 kDa 0.576 0.094 0.670 28 P55196 MLLT4 Afadin 0.604 0.064 0.668 29 Q8NDI1 EHBP1 EH domain binding protein 1 0.450 0.214 0.664 30 Q9BWT3 PAPOLG Poly(A) polymerase gamma 0.432 0.226 0.658 31 O43379 WDR62 WD repeat domain 62 0.554 0.101 0.655 32 O43765 SGTA Small glutamine-rich TPR-containing protein α 0.549 0.102 0.651 33 Q07889 SOS1 Son of sevenless homolog 1 0.417 0.234 0.651 34 O60716 CTNND1 Catenin delta-1 0.611 0.039 0.650 35 Q6ZU35 KIAA1211 KIAA1211 0.604 0.044 0.648 36 P21333 FLNA Filamin-A 0.585 0.049 0.634 37 O14974 PPP1R12A Protein phosphatase 1 regulatory subunit 12A 0.451 0.182 0.633 38 P23588 EIF4B Eukaryotic translation initiation factor 4B 0.514 0.118 0.632 Proteins highlighted in green are known RSK substrate. PDCD4 is shown in blue. The remaining potential RSK substrates can be found in Dataset S3.

Galan et al. www.pnas.org/cgi/content/short/1405601111 8of10 Table S2. Predicted RSK substrates that were also found to interact with WT 14-3-3 in melanoma cells Accession no. Gene name Protein name No. of peptides Source (PMID)

A1A5G0 CLASP1 CLIP-associated protein 1 22

Q9Y4H2 IRS2 Insulin receptor substrate 2 13 9312143 O75122 CLASP2 CLIP-associated protein 2 8 Q86W92 PPFIBP1 Liprin-beta-1 8 Q6Y7W6 GIGYF2 PERQ amino acid-rich-GYF domain- 8 contain protein 2 Q9Y2H2 INPP5F Phosphatidylinositide phosphatase SAC2 7 Echinoderm microtubule associated Q32P44 EML3 6 protein-like 3 O75592 MYCBP2 E3 ubiquitin-protein ligase MYCBP2 6 Q69PK0 LARP1 La-related protein 1 5 17353931 P04049 RAF1 RAF proto-oncogene serine/threonine-protein 5 8085158 kinase O60825 PFKFB2 6-phosphofructo-2-kinase/fructose- 5 2,6-bisphosphatase 2 Q86SQ0 PHLDB2 Pleckstrin homology-like family B member 2 5 P23588 EIF4B Eukaryotic translation initiation factor 4B 4 17361185 Q14C86 GAPVD1 (GAP) and VPS9 domain-containing protein 1 4 Q13625 TP53BP2 Apoptosis stimulating of p53 proteins 2 3 Q9ULL8 SHROOM4 Protein Shroom4 3 O95425 SVIL Supervillin 3 Q9NSV6 CDKN2AIP CDKN2 interacting protein 2 Q96F86 EDC3 Enhancer of mRNA-decapping proteins 3 2 20051463 Q16875 PFKFB3 6-phosphofructo-2-kinase/fructose- 2 2,6-bisphosphatase Q5SW79 CEP170 Centrosomal proteins of 170 kDa 2 Q99759 MAP3K3 Mitogen-activated protein kinase kinase 2 16407301 kinase 3 Q9HAU0 PLEKHA5 Pleckstrin homology domain-containing 2 family A/5 Q6WKZ4 RAB11FIP1 Rab11 family-interacting protein 1 2 O96013 PAK4 Serine/threonine-protein kinase PAK 4 2 Q96N67 DOCK7 Dedicator of cytokinesis protein 7 2 Q9UBF8 PI4KB Phosphatidylinositol 4-kinase beta 2 Q53EL6 PDCD4 Programmed cell death protein 4 2 This study O43166 SIPA1 Signal-induced proliferation-associated 2 1-like protein P40818 USP8 Ubiquitin carboxyl-terminal hydrolase 8 2 17720156 Q2M218 AAK1 Adapter associated kinase 1 1 Q7Z401 DENND4A C-myc promoter binding proteins 1 P22681 CBL Casitas B lymphoma proto-oncogene 1 8663231 Q9BW34 EEF1D EEF1D protein 1 Q14814 MEF2D Myocite specific enhancer factor 2D 1 11433030 Q14678 KANK1 KN motif/ankyrin repeat domain-containing 1 18458160 proteins Q09666 AHNAK Neuroblast differentiation-associated protein 1 Q96RK0 CIC Proteins Capicua homolog 1 21087211

Proteins highlighted in green are known 14-3-3 substrates. Proteins highlighted in blue are both RSK substrates and 14-3-3–interacting proteins. The remaining protein substrates can be found in Dataset S5. PMID, PubMed ID.

Galan et al. www.pnas.org/cgi/content/short/1405601111 9of10 Other Supporting Information Files

Dataset S1 (XLSX) Dataset S2 (XLSX) Dataset S3 (XLSX) Dataset S4 (XLSX) Dataset S5 (XLSX)

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