Supporting Information Supporting Information Corrected July 15 , 2014 Yi et al. 10.1073/pnas.1404943111 SI Materials and Methods which no charge state could be determined were excluded MS/ Tumorsphere Growth Assay. Single-cell tumorsphere growth assays MS selection). Three independent experiments were performed. of mammary epithelial (HMLER) (CD44high/CD24low)SA and HMLER (CD44high/CD24low)FA population cells were per- Phosphorylation Site Localization. The probability of correct phos- formed with ultra-low attachment surface six-well plates (Corn- phorylation site localization for each phosphorylation site was ing) and the mammary epithelial cell basal medium (MEBM) measured using an Ascore algorithm (1). This algorithm con- medium (Lonza) was supplemented with B27 (Invitrogen), 20 ng/mL siders all phosphoforms of a peptide and uses the presence or EGF, and 20 ng/mL basic FGF (BD Biosciences), and 4 μg/mL absence of experimental fragment ions unique to each to create an ambiguity score (Ascore). Parameters included a window size of heparin (Sigma). Single cells were cultured for 8 d and images were ± taken with a Nikon camera. 100 m/z units and a fragment ion tolerance of 0.6 m/z units. Sites with an Ascore of ≥13 (P ≤ 0.05) were considered to be ≥ ≤ Soft Agar Colony Growth Assay. Single-cell soft agar colony for- confidently localized, and those with an Ascore of 19 (P 0.01) mation and growth assays were performed to identify the tumor- were considered to be localized with near certainty. More than ≥ igenesis capacity of HMLER (CD44high/CD24low)SA and HMLER a 2.5-fold ratio ( 60.02%) was considered as a significant (CD44high/CD24low)FA population cells in vitro. Briefly, base agar phosphorylation increase in phosphoproteome assays. As cellu- layer was 0.5% agar with MEBM, the top agar layer was 0.35% agar lar active are rapidly dephosphorylated by phospha- ∼ with mammary epithelial cell growth medium (MEGM) (500 mL tases in cells (2, 3), 24 kinases within 1.6- to 2-fold ratio (an ∼ MEBMwith2mLBPE,0.5mLinsulin,0.5mLrhEGF,0.5mL increase from 37.5% to 50%) were tracked and recovered as GA-1000, 0.5 mL hydrocortisone; Lonza). HMLER (CD44high/ phosphorylation increased kinases. A decrease of more than CD24low)SA and HMLER (CD44high/CD24low)FA population cells 60% was considered a significant phosphorylation decrease. (1 × 104) in single-cell suspensions were mixed with 2 mL 0.35% The postsearch data analyses were performed as previously de- agar with MEGM, and then the mixture was added onto the base scribed (4). agar layer of each well in the six-well plates. After about 2 wk, Database Searches, Data Filtering, Validation of Detection grossly visible colonies appeared. Images were taken with a Nikon Rate, and Phosphoproteome Analyses. The spectral data were camera. Colony diameters were analyzed and numbers counted searched with SEQUEST (1) against a database containing the with ImageJ software (http://imagej.nih.gov/ij/). human protein sequence database (www.ensembl.org/index.html) high low SA together with the reversed complement. The LC-MS/MS identi- Cell Viability Assay. HMLER (CD44 /CD24 ) and HMLER (CD44high/CD24low)FA population cells (1 × 104) were treated fications were filtered to 0.98% protein false discovery rate (FDR) and 0.1% peptide FDR. The peptide quantification and with indicated compounds with series of concentrations for 24 h. phosphorylation site localization were analyzed using in-house Cell viability assays were performed on the cells with CellTiter- software and Ascore as previously described (5). Glo luminescent cell viability assay kit (Promega) according to the manual. Three independent experiments were performed. Peptide Quantification. Peptide quantification was performed us- ing the Vista program. We required a signal-to-noise ratio (S/N) Strong Cation Exchange/Immobilized Metal Affinity Chromatography value >3 for both heavy and light species for quantification. For Phosphopeptide Enrichment and Liquid Chromatography Tandem peptides found exclusively as singlets (only a heavy or only a light Mass Spectrometry Analyses. In phosphopeptide enrichment by peak present), we reported the peak S/N ratio or its inverse, as strong cation exchange/immobilized metal affinity chromatog- a proxy for relative abundance measurement. For such peptides, raphy (SCX/IMAC), peptides were first separated at acidic pH on we required an S/N value >5 for the observed species. In addi- a polysulfethyl A semipreparative column (9.4 mm i.d. × 200 mm tion, if the S/N value of one member of a pair was <3, the length, 5-μm particle size, 200-Å pore size; PolyLC). Twelve partner value was required to be >5. Finally, to avoid quantifying fractions were collected and further enriched by IMAC, starting false positives, any identification from a singlet peak was re- with 30 μL of PHOS-Select beads (Sigma-Aldrich). Enriched quired to pass an identification threshold that was 10 times more phosphopeptides were eluted and desalted by STAGE-TIPS stringent (Q value of <0.001; precision >99.9%). Raw abun- (Thermo Scientific) as previously described (1). Phosphopep- dance ratios from each experiment were normalized based on tide-enriched fractions were analyzed by liquid chromatography the median distribution ratio. tandem mass spectrometry (LC-MS/MS) on an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) equipped with Accessibility of Raw Data and Bioinformatic Analysis. The five Excel a Thermo Fisher Scientific nanospray source, an Agilent 1100 files (Files S1–S5) and MS raw data are available at http:// Series binary HPLC pump, and a Famos autosampler. Phos- gwagner.med.harvard.edu/intranet/PNAS_Manuscript_2014/. phopeptides were separated on a 0.125 × 180 mm fused silica microcapillary column with an in needle tip (made in-house) Western Blot Assay. Total were extracted from breast with a ∼5-μm i.d. The silica microcapillary column was packed cancer stem cells (CSCs) with protease inhibitor (Roche) and with magicC18AQ C18 reverse-phase resin (5-μm particle size, phosphatase inhibitor (Sigma) for Western blot assays with 200-Å pore size; Michrom Bioresources). Separation was per- specific antibodies. Anti-p120 catenin and anti–p-p120 (Tyr280) formed by applying a 57-min gradient from 7% to 28% aceto- catenin antibodies were obtained from Syd Labs. Anti-GSK3β, nitrile in 0.125% formic acid. The mass spectrometer was anti-p(Ser9) GSK3β, anti-PKAC, anti-p(Thr197) PKAC, anti- operated with default settings: full MS [automatic gain control ERK1/2, anti-p(Thr202/Tyr204)-ERK1/2, anti-p(Ser217/221) (AGC), 1 × 106; resolution, 6 × 104; m/z range, 375–1,800; MEK1/2, anti-MEK1/2, anti-chemokine (C-X-C motif) re- maximum ion time, 1,000 ms]; MS/MS (AGC, 5 × 103; maximum ceptor 4 (CXCR4), anti–p-Ser/Thr, anti–β-actin, and anti-tubulin ion time, 120 ms; minimum signal threshold, 4 × 103; dynamic antibodies were ordered from Cell Signaling. Anti–p-Tyr (mono- exclusion time setting, 30 s; singly charged ions and ions for clonal) antibodies were ordered from Abcam. PKA inhibitor

Yi et al. www.pnas.org/cgi/content/short/1404943111 1of15 (14-22-Amide) was ordered from EMD. AMD3100 (A5602) was (http://networkin.info/search.php), and Kinasource (www.kinasource. ordered from Sigma. The short hairpin RNA (shRNA)-CXCR4 co.uk/Database/substrates.html) (9), phosphotase–substrates with plasmids and control shRNA plasmids were ordered from The PubMed, and pathways with the Kyoto Encyclopedia of RNAi Consortium (Broad Institute). Active SDF-1 protein was and Genomes (www.genome.jp/kegg/pathway.html). The – obtained from Abcam (ab78808). Each experiment was repeated phosphorylation site-specific substrate relationships were either at least three times. supplied in Kinasource or reported in the literature (Table S2, Cellular protein extraction and Western blot analysis was S3, and S7). All relationships of the kinase–substrate and performed with radioimmunoprecipitation assay buffer (50 mM phosphotase–substrate in this study are experimentally identified Tris·HCl pH 7.4, 150 mM NaCl, 1% Triton-X100, 0.1% SDS, × in human cells in previously published reports (refs. 5, 9, 10, and 0.25% Na-deoxycholate, 1 mM PMSF, 1 Roche complete mini 11 and Tables S3 and S7). The results were compared with 40 protease inhibitor mixture, 1× Pierce phosphatase inhibitor phosphoproteomic experiments of mammalian cells in the lit- mixture). The total Ser/Thr, or Tyr phosphorylation signal in- erature to validate the prediction for the biological function, tensities in Western blots were analyzed by Bio-Rad imaging system analysis software. The ratios of phosphorylation signal activation, and/or inhibition of phosphorylation sites. The net- intensities of samples compared with the control were expressed works were seeded on established SDF-1/CXCR4 signaling – as relative phosphorylation activities. pathways (12 14) with extended signal transduction based on the above-mentioned integrated bioinformatic analyses of phospho- Bioinformatic Analysis. Bioinformatic annotations were carried out proteins in the phosphoproteome. using Human Protein Reference Database (www.hprd.org)as previously described (4, 6). The phosphosites were identified Conserved Phosphorylation Site Analysis Across Species. Conserved using the Web-based program www.phosphosite.org and phosphorylation sites and motifs were analyzed with target PubMed database (www.ncbi.nlm.nih.gov/pubmed). The pre- protein sequences in Homo sapiens (PubMed protein database, viously detected phosphosites with unknown biological function www.ncbi.nlm.nih.gov/protein)andBLAST(http://blast.ncbi.nlm. are not labeled. LC-MS/MS data were analyzed for kinase– nih.gov/Blast.cgi) search of the following species: Homo sapiens, substrates using NetPhorest (7) (http://netphorest.info), for Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, kinase–substrate network reconstruction using NetworKIN (8) Arabidopsis thaliana, and Saccharomyces cerevisiae.

1. Villén J, Gygi SP (2008) The SCX/IMAC enrichment approach for global phosphorylation 9. Hernandez M, Lachmann A, Zhao S, Xiao K, Ma’ayan A (2010) Inferring the sign of analysis by mass spectrometry. Nat Protoc 3(10):1630–1638. kinase-substrate interactions by combining quantitative phosphoproteomics with 2. Alessi DR, et al. (1994) Identification of the sites in MAP kinase kinase-1 phosphorylated a literature-based mammalian kinome metwork. Proc IEEE Int Symp Bioinformatics by p74raf-1. EMBO J 13(7):1610–1619. Bioeng 2010:180–184. 3. Aoki K, Yamada M, Kunida K, Yasuda S, Matsuda M (2011) Processive phosphorylation 10. Wang X, et al. (2011) Characterization of the phosphoproteome in androgen- of ERK MAP kinase in mammalian cells. Proc Natl Acad Sci USA 108(31):12675–12680. repressed human prostate cancer cells by Fourier transform ion cyclotron resonance 4. Xiao K, et al. (2010) Global phosphorylation analysis of beta-arrestin-mediated mass spectrometry. J Proteome Res 10(9):3920–3928. signaling downstream of a seven transmembrane receptor (7TMR). Proc Natl Acad Sci 11. Christensen GL, et al. (2010) Quantitative phosphoproteomics dissection of seven- USA 107(34):15299–15304. transmembrane receptor signaling using full and biased agonists. Mol Cell Proteomics 5. Huttlin EL, et al. (2010) A tissue-specific atlas of mouse protein phosphorylation and 9(7):1540–1553. expression. Cell 143(7):1174–1189. 12. Teicher BA, Fricker SP (2010) CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer 6. Wu R, et al. (2011) Correct interpretation of comprehensive phosphorylation Res 16(11):2927–2931. dynamics requires normalization by protein expression changes. Mol Cell Proteomics 13. Busillo JM, Benovic JL (2007) Regulation of CXCR4 signaling. Biochim Biophys Acta 10(8):009654. 1768(4):952–963. 7. Miller ML, et al. (2008) Linear motif atlas for phosphorylation-dependent signaling. 14. Burger JA, Kipps TJ (2006) CXCR4: A key receptor in the crosstalk between tumor cells Sci Signal 1(35):ra2. and their microenvironment. Blood 107(5):1761–1767. 8. Linding R, et al. (2007) Systematic discovery of in vivo phosphorylation networks. Cell 129(7):1415–1426.

Yi et al. www.pnas.org/cgi/content/short/1404943111 2of15 Fig. S1. Tumorsphere growth in vitro. (A) Single HMLER (CD44high/CD24low)SA and HMLER (CD44high/CD24low)FA population cells were cultured for 8 d. Rep- resentative images are shown. (B) Soft agar colony assays of HMLER (CD44high/CD24low)SA and HMLER (CD44high/CD24low)FA population cells after culture for about 2 wk. Representative images (Left) and statistics results (Right) are shown. Error bars represent mean ± SD.

Yi et al. www.pnas.org/cgi/content/short/1404943111 3of15 Fig. S2. Total phosphorylation (p-Tyr, p-Ser/Thr) upon stromal cell-derived factor 1 (SDF-1) in breast CSCs over time, and CXCR4 knockdown. (A) Total p-Tyr phosphorylation change with 100 ng/mL SDF-1 on breast CSCs over time in Western blot assays. Relative total p-Tyr phosphorylation was expressed as thefold change relative to the control. (B) Total p-Ser/Thr phosphorylation change with 100 ng/mL SDF-1 on breast CSCs was monitored over time by Western blot assays. Relative total p-Ser/Thr phosphorylation was expressed as fold to control. Three independent experiments were performed. Error bars represent mean ± SD. Tubulin was used as a loading control. (C) CXCR4 was knocked down by shRNA-CXCR4 in breast CSCs.

Yi et al. www.pnas.org/cgi/content/short/1404943111 4of15 Fig. S3. Experimental workflow of the procedure for phosphopeptide enrichment and MS/MS analyses. The reductive dimethylated, mixed, alkylated, and digested peptides from Fig. 2A were desalted and separated by SCX chromatography. Twelve fractions with different phosphopeptide concentrations were collected, desalted, and enriched by IMAC. Each fraction was analyzed by LC-MS/MS. The mass spectra were analyzed by a sequence search, peptide and protein filtering, Ascore site assignment, and quantitative analysis.

Yi et al. www.pnas.org/cgi/content/short/1404943111 5of15 Fig. S4. Effects of SDF-1/CXCR4 on PLEC1 and ERK1/2 phosphorylation. (A) PLEC1 is a giant (500-KDa) and ubiquitous protein in mammalian cells, which acts as a link between the three main components of the cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. SDF-1/CXCR4 increases phosphorylation at 20 phosphosites in PLEC1. (B) Phosphorylation of ERK1/2 in SDF-1/CXCR4 signaling. SDF-1 (100 ng/mL for 10 min) and CXCR4 knockdown were used. SDF-1 elevates ERK1/2 phosphorylation in breast CSCs, whereas CXCR4 knockdown neutralizes the increase effects of SDF-1.

Yi et al. www.pnas.org/cgi/content/short/1404943111 6of15 Fig. S5. Reconstruction of SDF-1/CXCR4–induced phosphatase regulation loops. PPP1R2 (IPP-2) is an endogenous phosphatase inhibitor which inhibits myosin phosphatase (MP) activity. SDF-1/CXCR4 increases phosphorylation at Ser217 of PPP1R2. MP dephosphorylates both kinases [such as AMPK, mitogen-activated protein kinase (MAPK), PKC, and PAK] and phosphatases (such as PPP1R2 and PPP2R5D). The Thr320 residue of PPP1Cα and Thr316 of PPP1Cβ are conserved phosphosites. Ser311 of PPP1Cβ is the unique Ser in PPP1Cβ but not in other PPP1C isoforms. SDF-1/CXCR4 elevates phosphorylation of these Thr320, Thr316, and Ser311 residues. Tyrosine-protein phosphatase nonreceptor type 12/13/14 (PTPN12/13/14) are three members of the protein tyrosine phosphatase (PTP) family. SDF-1/CXCR4 increases phosphorylation of multiple phosphosites of PTPN12/13/14 members.

Yi et al. www.pnas.org/cgi/content/short/1404943111 7of15 Fig. S6. A global signaling network downstream of SDF-1/CXCR4 signaling in breast CSCs. Information flow in the predicted SDF-1/CXCR4 signaling network with feedback regulation systems [nested kinase negative feedback loops (such as PKA-Raf-1, PKA-GSK3β, Rps6ka1-SOS), and suppression by phosphatases (such as MP-ERK1, MP-AMPK), and antinegative regulation (nested phosphatase negative regulation loops (such as MP-PPP2R5D) and inhibition of phos- phatases by phosphatase inhibitor of PPP1R2]. For the proteins, blue indicates known proteins in SDF-1/CXCR4 signaling in both, black indicates those in the phosphoproteome, and gray represents the known signals in SDF-1/CXCR4 signaling not detected in phosphoproteome. Black arrows indicate the known phosphorylation relationship in SDF-1/CXCR4 signaling in both. Blue arrows indicate known direct interaction and phosphorylation relationship in both. Green arrows indicate the phosphorylation relationship in phosphoproteome. Pink arrows show the biological function regulation. Negative regulation by nested kinase feedback loops are indicated by green lines, dephosphorylation by phosphatases by purple lines, and inhibition of phosphatases by phosphatase in- hibitors by the cyan lines. Gray circles represent the known phosphoprotein complex in both.

Yi et al. www.pnas.org/cgi/content/short/1404943111 8of15 Table S1. Site pairs with equal or close phosphorylation increase fold in kinases Known dual Distance phosphorylation Kinases Site Increase fold Fold ratio (amino acids between) sites Biological functions

CamKKII S129 1.99 S129/S133 = 1 3 Cell migration, cell cycle, proliferation SS133 1.99 CDC2L2 SS191 3.15 S191/T197 = 1 5 Cell cycle, proliferation T197 3.15 CDK7 S164 2.1 S164/T170 = 1.1 5 + Cell cycle, proliferation, growth T170 1.9 CSNK2β S205 1.97 S205/S209 = 1.02 3 Unknown S209 1.93 CRKRS S323 1.99 S323/Y327 = 1 3 Cell migration, proliferation Y327 1.99 MAP4K4 S285 2.4 S285/S286 = 1 0 MAPK cascade, numerous cellular procedures S286 2.4 MAP3K11 S789 1.67 S789/S793 = 1 3 MAPK cascade, numerous cellular procedures S793 1.67 MLK7 S687 2.04 S687/S691 = 1 5 MAPK cascade, numerous cellular procedures S691 2.04 MAP2K2 S222 1.71 S226/S222 = 1.25 3 + MAPK cascade, numerous cellular procedures S226 2.15 ERK1 T202 1.6 T202/Y204 = 11 + MAPK cascade, numerous cellular procedures Y204 1.6 MARK3 S606 2.6 S606/S610 = 1.08 3 Gluconeogenesis, cellular polarity S610 2.4 MELK T460 3.79 T460/T466 = 1 3 Stem cell renewal, cell cycle, cell proliferation, radiation resistance T466 3.79 MRCKα S1638 4.27 S1638/S1641 = 1 2 Actin cytoskeletal reorganization, cell migration, invasion S1641 4.27 PI4K2α S462 2.67 S468/S462 = 1.18 4 Membrane trafficking, secretion regulation S468 3.17 PI4Kβ S258 2.07 S258/S266 = 1 7 Membrane trafficking S266 2.07 PKCδ S506 2.8 S506/T511 = 1 4 Cell proliferation, cell death, migration, pro- and antiapoptotic regulation T511 2.8 PKCe T309 4.11 T309/T314 = 1 4 Numerous cellular procedures, cancer growth and metastasis, a cancer marker T314 4.11 PKN1 S533 2.09 S533/S540 = 1 6 Cell migration, cell survival S540 2.09 SGK269 S568 2.02 S568/S572 = 1 3 Unknown S572 2.02 TLK1 S74 2.2 S74/S77 = 1 2 DNA repair S77 2.2

Yi et al. www.pnas.org/cgi/content/short/1404943111 9of15 Table S2. Phosphosite-specific kinase–substrate and phosphatase–substrate of SDF-1/CXCR4 regulated proteins in phosphoproteome ABCDEF

Known Detection of kinases Phosphatase Detection of phosphatases Phosphoproteins in Phosphosites in site-specific in column C in that dephosphorylates in column E in phosphoproteome phosphoproteome upstream kinase phosphoproteome the site in column B phosphoproteome

AMPKβ1 S182 CamKKII Yes PPP1C Yes CDK1 T14* MYT1 No CDC25 Yes Y15* No CDC25 Yes CDK7 S164 CDK1 Yes PPP1C Yes T170 CDK1 Yes FAK Y576 Src No GSK3β S9* PKA Yes PPP1C Yes MAP2K2 S222 Raf-1 Yes S226 Raf-1 Yes PPP1C Yes ERK1 T202 MEK2 Yes PPP1C Yes Y204 MEK2 Yes ERK3 S189 MEK2 Yes PAK4 S474 PAK4 Yes PPP1C Yes PDK1 S241 PKC Yes PKA T197 PDK-1, PKA Yes (both) PPP1C Yes PKCδ S506 PKC Yes PPP1C Yes Rps6ka1 S221 ERK1, GSK3 Yes (both) PPP1CA T320 CDK1 Yes PPP1C Yes MYPT1 S445 NAU.K.1 No S507 S910 NAU.K.1 No p120-catenin Y228 Src No PTPNs Yes S252 S320 Rb1 S780 PPP1C Yes Y790 CDK4 No S811 HNRNPK S284 ERK Yes MP Yes NFkB S903 ERK, GSK3β Yes (both) NFAT1 T326 ERK Yes S330 ERK Yes Histone H3.2 S28 ERK Yes VIM S51 PKA Yes S56 CDK1, ERK Yes (both) S66 PAK1 No MCM3 T767 CDK1 Yes MCM4 T110 CDK1 Yes NUCKS1 T179 CDK1 Yes Ets-1 S282 CamKKII Yes S285 CamKKII Yes RhoGEF11 T668 FAK Yes ZNF828 S627 CDK1 Yes

*Inhibitory phosphorylation sites.

Yi et al. www.pnas.org/cgi/content/short/1404943111 10 of 15 Table S3. Known activation promotion effector, kinase– substrate, and phosphatase–substrate detected in phosphoproteome Effector relationship Ref(s).

Activation promotion effector SOS1→Raf-1 (1, 2) Kinase–substrate Raf-1→MAP2K2 (3) MAP2K2→ERK1 (4) ERK1→Rps6ka1 (5, 6) ERK1→Ets-1 (7) ERK1→NFAT1 (8) ERK1→NFkB (9) ERK1→Histone H3.2 (10) ERK1→MARK3 (11) MAP4K4→MAP3K11 (12) MAP3K11→MAP2K2 (12) MAP4K4→MLK7 (13) MLK7→MAP2K2 (13) MAP2K2→ERK3 (14, 15) ERK3→MAPKAPK5 (16, 17) PKD1→PKA (18) PKA→MAP2K2 (19) FAK→PAK4 (20) PAK4→MAP2K2 (21) Phosphatase–substrate MP→ERK1 (22) MP→ERK3 (22) MP→MAP2K2 (22) PTPN12/13/14→ERK1 (23) PTPN12/13/14→FAK (24) PTPN12/13/14→p120-catenin (25)

1. Liu BX, Wei W, Broek D (1993) The catalytic domain of the mouse sos1 product activates Ras proteins in vivo and in vitro. Oncogene 8(11):3081–3084. 2. Galaktionov K, Jessus C, Beach D (1995) Raf1 interaction with Cdc25 phosphatase ties mitogenic signal transduction to cell cycle activation. Genes Dev 9(9):1046–1058. 3. Xuan YT, et al. (2005) Role of the -epsilon-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning. Circulation 112(13):1971–1978. 4. Khoo S, Cobb MH (1997) Activation of mitogen-activating protein kinase by glucose is not required for insulin secretion. Proc Natl Acad Sci USA 94(11):5599–5604. 5. Willard FS, Crouch MF (2001) MEK, ERK, and p90RSK are present on mitotic tubulin in Swiss 3T3 cells: A role for the MAP kinase pathway in regulating mitotic exit. Cell Signal 13(9): 653–664. 6. Hartkamp J, Troppmair J, Rapp UR (1999) The JNK/SAPK activator mixed lineage kinase 3 (MLK3) transforms NIH 3T3 cells in a MEK-dependent fashion. Cancer Res 59(9):2195–2202. 7. Yao R, Wang Y, Lubet RA, You M (2003) Differential in chemically induced mouse lung adenomas. Neoplasia 5(1):41–52. 8. Robbs BK, Lucena PI, Viola JP (2013) The transcription factor NFAT1 induces apoptosis through cooperation with Ras/Raf/MEK/ERK pathway and upregulation of TNF-α expression. Biochim Biophys Acta 1833(8):2016–2028. 9. Parthasarathy G, Philipp MT (2014) The MEK/ERK pathway is the primary conduit for Borrelia burgdorferi-induced inflammation and P53-mediated apoptosis in oligodendrocytes. Apoptosis 19(1):76–89. 10. Ding J, Swain JE, Smith GD (2011) -A regulates microtubule organizing center (MTOC) localization, dynamics, and histone-H3 phosphorylation in mouse oocytes. Mol Reprod Dev 78(2):80–90. 11. Müller J, Ory S, Copeland T, Piwnica-Worms H, Morrison DK (2001) C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1. Mol Cell 8(5):983–993. 12. Hagemann C, Blank JL (2001) The ups and downs of MEK kinase interactions. Cell Signal 13(12):863–875. 13. Handley ME, Rasaiyaah J, Chain BM, Katz DR (2007) Mixed lineage kinases (MLKs): A role in dendritic cells, inflammation and immunity? Int J Exp Pathol 88(2):111–126. 14. Hatzivassiliou G, et al. (2013) Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers. Nature 501(7466):232–236. 15. Raman M, Chen W, Cobb MH (2007) Differential regulation and properties of MAPKs. Oncogene 26(22):3100–3112. 16. Schumacher S, et al. (2004) Scaffolding by ERK3 regulates MK5 in development. EMBO J 23(24):4770–4779. 17. Seternes OM, et al. (2004) Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway. EMBO J 23(24):4780–4791. 18. Baines D (2013) Kinases as targets for ENaC regulation. Curr Mol Pharmacol 6(1):50–64. 19. Jiang Y, et al. (2001) Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase. Proc Natl Acad Sci USA 98(18):10102–10107. 20. Aksamitiene E, et al. (2011) Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells. Cell Signal 23(11):1794–1805. 21. Djeu JY, Jiang K, Wei S (2002) A view to a kill: Signals triggering cytotoxicity. Clin Cancer Res 8(3):636–640. 22. Grassie ME, Moffat LD, Walsh MP, MacDonald JA (2011) The myosin phosphatase targeting protein (MYPT) family: A regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ. Arch Biochem Biophys 510(2):147–159. 23. Freiss G, Chalbos D (2011) PTPN13/PTPL1: An important regulator of tumor aggressiveness. Anticancer Agents Med Chem 11(1):78–88. 24. Laczmanska I, Sasiadek MM (2011) Tyrosine phosphatases as a superfamily of tumor suppressors in colorectal cancer. Acta Biochim Pol 58(4):467–470. 25. Rhee I, Veillette A (2012) Protein tyrosine phosphatases in lymphocyte activation and autoimmunity. Nat Immunol 13(5):439–447.

Yi et al. www.pnas.org/cgi/content/short/1404943111 11 of 15 ie al. et Yi www.pnas.org/cgi/content/short/1404943111

Table S4. Evolutionary conservation of phosphorylation sites and motifs across species H. sapiens M. musculus Arabidopsis Phosphorylation sites Conserved motif Conserved motif No. Protein name in phosphoproteome Protein name and phosphorylation sites Protein name and phosphorylation sites

β LSSS * PPGP β LSSS * PPGP β MSSS PPGSP 1 AMPK 1*/2 • • AMPK 1*/2 • • SNF1 •• 2 CamKKII SSPQSSPR CamKKII SSPQSSPR 3 CDC2L1 YGSPLKAY CDC2L1 YGSPLKAY CDC2L YGSPIKPY 4 CDC2L2 SPLKAYTPVVVT CDK11 SPLKAYTPVVVT PKLP SPLKPYTHLVVT 5 CDC2L5 RPYTNKVITL CDK13 RPYTNKVITL CDKC1 TNRVITL 6CDK1GEGT*Y*GVVYK CDK1 GEGT*Y*GVVYK CDKA-1 GEGTYGVVYK 7CDK3RTYTHEVVT CDK3 RTYTHEVVT 8 CDK7 S*PNRAYT* CDK7 SPNRAYT CDKD3 SPNRKFT 9FAKDSTY*YKAS FAK DSTY*YKAS 10 GSK3β GRPRTTS*FAE GSK3β GRPRTTS*FAE 11 MAP2K2 SGQLIDS*MANS*FV MAP2K2 SGQLIDS*MANS*FV MKK2 TAGLANTFV 12 ERK1 DHTGFLT*EY*VATR ERK1 DHTGFLT*EY*VATR MPK4 ETDFMTEYVVTR 13 ERK3 GHLS*EGLVTK ERK3 GHLSEGLVTK MAPK11 TEYVVT 14 MAPKAPK5 KRKLLGTKPKD MAPKAPK5 KRKLLGTKPKD 15 PAK4 PRRKS*LVGTPY PAK4 PRRKSLVGTPY NP_564955 RNTFIGTP 16 PDK1 S*FVGTAQ PDK1 S*FVGTAQ PDK-1 TFVGTAA 17 PI4K2α RSSSESYTQS PI4K2α RSSSESYTQS 18 PKA VKGRTWT*LCGTPE;RVSINEK PKA VKGRTWT*LCGTPE;RVSINEK S6K2 RSNSMCGTTE 19 PKCδ FCGTPDYIAP PKCδ FCGTPDYIAP ATPK6 MCGTTEYMAP 20 Rps6ka1 YS*FCGTVE Rps6ka1 YSFCGTVE Atpk2 NSMCGTTE 21 PPP1Cα GRPITPPR PPP1Cα GRPITPPR PP1-6 RPGTPPH 22 MYPT1 GLRKTGS*YGALAE;LLGRSGS*YSYLEE MYPT1 GLRKTGS*YGALAE;LLGRSGS*YSYLEE Many of the SDF-1/CXCR4 regulated phosphosites in kinases and phosphatases are highly conserved across six eukaryotic species of H. sapiens, M. musculus, D. melanogaster, C. elegans, A. thaliana, and S. cerevisiae (see also Table S5). Red boldface and underlining represent phosphosites in phosphoproteome. Black boldface and underlining represent conserved phosphosites in other species. Double red dots represent the Ser/Thr-Pro(S/T-P) motif. *Characterized phosphosites in human and mouse (p-Ser site at AMPKβ2 is previously unknown). 2o 15 of 12 Table S5. Evolutionary conservation of phosphorylation sites and motifs across species

Drosophila C. elegans Yeast (S. cerevisiae) Conserved motif and Conserved motif and Conserved motif and No. Protein name Protein name Protein name phosphorylation sites phosphorylation sites phosphorylation sites PSSS P LPRPS P 1 AAKB-1 • • SIP2 • • GPFGPRSS SPNLSRP 2 GK12910 •••• Camkk1 3 GH14923 YGSPIKKY 4 GH14923P SPIKKYTSLVVT 5 CG7597 RPYTNKVITL CDTL7 ESRLYTNRVITL RNA polyII CDK1 YTNRVITL subunit 6 Cdk-1 GEGTYGVVYK Cdk-1 GEGTYGVVYK Cdc28p GEGTYGVVYK 7 CDC2 RIYTHEIVT P34CDC2 RVYTHEVVT 8 CDK7 SPNRIYT CDK7 SPNRNYT Kin28p APHEILT 9 RE69838p DQSYYHST Kin-32 DAVYTAS 10 Shaggy GRPRTSSFAE 11 MAPKK SGQLIDSMANSFV Mek-2 SGMLIDSMANSFV MEK SGNLVASLAKTNI 12 ROLLED DHTGFLTEYVATR MPK-1 DHTGFLTEYVATR Fus3p GMTEYVATR 13 ROLLED (b) GFLTEYVAT MPK-1(a) GFLTEYVAT Slt2p LTEYVAT 14 MAPK2 LLTKRRK 15 PAK-1 PKRKSLVGTPY PAK-2 PRRRSLVGTPY STE20 RTTMVGT 16 PK61C SFVGTAQ PDK-1 TFVGTAL Pkh2p SFVGTAE 17 PI4K2α RSSGSRFFSFTQR C56A3.8 QPTTASWDD 18 PKAC1 VKGRTWTLCGTPE;RISSTEK KIN-1 VKGRTWTLCGTPE;RISGTEK Tpk2p TWTLCGTP 19 PKCδ FCGTPDYMAP TPA-1 FCGTPDYISP Pkc1p FCGTPEFMAP 20 S6KII YSFCGTVE RSKN-1 YSFCGTVE Ypk1p DTFCGTPE 21 PP1α GRPLTPPR GSP-2 NRPVTPPR 22 Many of the SDF-1/CXCR4 regulated phosphosites in kinases and phosphatases are highly conserved across six eukaryotic species of H. sapiens, M. musculus, D. melanogaster, C. elegans, A. thaliana, and S. cerevisiae (see also Table S4). Red boldface and underlining represents phosphosites in phosphoproteome. Black boldface and underlining represents conserved phosphosites in other species. Double red dots represent the Ser/Thr-Pro(S/T-P) motif.

Yi et al. www.pnas.org/cgi/content/short/1404943111 13 of 15 Table S6. Conserved phosphorylation site in human PKC isozymes Human PKC isozymes Conserved motif

PKC-α RTFCGTPDYI PKC-β1 KTFCGTPDYI PKC-β2 KTFCGTPDYI PKC-γ RTFCGTPDYI PKC-δ STFCGTPDYI PKC-e TTFCGTPDYI PKC-η ATFCGTPDYI PKC-θ NTFCGTPDYI PKC-ι STFCGTPNYI PKC-ζ STFCGTPNYI PK-N1 STFCGTPEFL PK-N2 STFCGTPEFL PK-N3 STFCGTPEFL

Boldface represents Thr (T) sites detected in the phosphoproteome. The sequences of a highly conserved motif in all isozymes of PKC family are underlined.

Table S7. Other known promotion effector, kinase–substrate and phosphotase–substrate in phosphoproteome Effector relationship Ref(s).

Activation promotion factor β-arrestin→ERK1 (1) RhoGEF→GTP-RhoA (2) GTP-RhoA→ROCK (3) cAMP-PKA (4, 5) IFNAR2→STAT (6) Kinase–substrate PDK1→AMPK (7) PDK1→PKA (8) PKC→SDPR (9) PKC→Rictor (10) PKCδ→Raf-1 (11) ROCK1→MYPT1 (12) PKA→PPP1Cα (13) PKA→PPP2R5D (14) CDK1→CDK7 (15) CDK1→P53BP1 (16) CDK1→Dnmt1 (17) CDK1→CEP55 (18) CDK1→VIM (19) CDK1→MCM3 (20) CDK7→CDK1 (21) PDK1→NFkB (22) MARK2→MAP1A (23) GSK3β→PPP1R2 (24) PAK→actin (25) PAK→MLCK (25) FAK→PAK (26) FAK→RhoGEF (26) ROCK→MLCK (27) MLCK→MLC (28) MLC→actin (28) PKA→VIM (29) PI4K→PIP2 (30) Phosphatase–substrate MP→PPP1R2 (31) MP→PPP2R5D (31) MP→AMPK (31) MP→PKC (31) MP→PKA (31) PPPIR2→MP (31)

Yi et al. www.pnas.org/cgi/content/short/1404943111 14 of 15 1. Reiter E, Ahn S, Shukla AK, Lefkowitz RJ (2012) Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors. Annu Rev Pharmacol Toxicol 52:179–197. 2. Sah VP, Seasholtz TM, Sagi SA, Brown JH (2000) The role of Rho in G protein-coupled receptor signal transduction. Annu Rev Pharmacol Toxicol 40:459–489. 3. Schofield AV, Bernard O (2013) Rho-associated coiled-coil kinase (ROCK) signaling and disease. Crit Rev Biochem Mol Biol 48(4):301–316. 4. Kadamur G, Ross EM (2013) Mammalian phospholipase C. Annu Rev Physiol 75:127–154. 5. Taylor SS, Zhang P, Steichen JM, Keshwani MM, Kornev AP (2013) PKA: Lessons learned after twenty years. Biochim Biophys Acta 1834(7):1271–1278. 6. Su L, David M (2000) Distinct mechanisms of STAT phosphorylation via the interferon-alpha/beta receptor. Selective inhibition of STAT3 and STAT5 by piceatannol. J Biol Chem 275(17): 12661–12666. 7. Zou MH, et al. (2003) Activation of 5′-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endo- thelial cells. Role of peroxynitrite. J Biol Chem 278(36):34003–34010. 8. Romano RA, Kannan N, Kornev AP, Allison CJ, Taylor SS (2009) A chimeric mechanism for polyvalent trans-phosphorylation of PKA by PDK1. Protein Sci 18(7):1486–1497. 9. Baig A, Bao X, Wolf M, Haslam RJ (2009) The platelet protein kinase C substrate pleckstrin binds directly to SDPR protein. Platelets 20(7):446–457. 10. Dibble CC, Asara JM, Manning BD (2009) Characterization of Rictor phosphorylation sites reveals direct regulation of mTOR complex 2 by S6K1. Mol Cell Biol 29(21):5657–5670. 11. Kawauchi K, et al. (1996) Regulation of BCR- and PKC/Ca(2+)-mediated activation of the Raf1/MEK/MAPK pathway by protein-tyrosine kinase and -tyrosine phosphatase activities. Mol Immunol 33(3):287–296. 12. Hudson CA, Heesom KJ, López Bernal A (2012) Phasic contractions of isolated human myometrium are associated with Rho-kinase (ROCK)-dependent phosphorylation of myosin phosphatase-targeting subunit (MYPT1). Mol Hum Reprod 18(5):265–279. 13. Rotman T, Etkovitz N, Spiegel A, Rubinstein S, Breitbart H (2010) and protein kinase C(alpha)/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm. Reproduction 140(1):43–56. 14. Ahn JH, et al. (2007) Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit. Proc Natl Acad Sci USA 104(8):2979–2984. 15. Larochelle S, et al. (2007) Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. Mol Cell 25(6):839–850. 16. Castedo M, Perfettini JL, Roumier T, Kroemer G (2002) Cyclin-dependent kinase-1: Linking apoptosis to cell cycle and mitotic catastrophe. Cell Death Differ 9(12):1287–1293. 17. Lavoie G, St-Pierre Y (2011) Phosphorylation of human DNMT1: Implication of cyclin-dependent kinases. Biochem Biophys Res Commun 409(2):187–192. 18. Fabbro M, et al. (2005) Cdk1/Erk2- and -dependent phosphorylation of a centrosome protein, Cep55, is required for its recruitment to midbody and cytokinesis. Dev Cell 9(4): 477–488. 19. Yamaguchi T, et al. (2005) Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis. J Cell Biol 171(3):431–436. 20. Lin DI, Aggarwal P, Diehl JA (2008) Phosphorylation of MCM3 on Ser-112 regulates its incorporation into the MCM2-7 complex. Proc Natl Acad Sci USA 105(23):8079–8084. 21. Merrick KA, et al. (2008) Distinct activation pathways confer cyclin-binding specificity on Cdk1 and Cdk2 in human cells. Mol Cell 32(5):662–672. 22. Raimondi C, Falasca M (2011) Targeting PDK1 in cancer. Curr Med Chem 18(18):2763–2769. 23. Marx A, et al. (2006) Structural variations in the catalytic and ubiquitin-associated domains of microtubule-associated protein/microtubule affinity regulating kinase (MARK) 1 and MARK2. J Biol Chem 281(37):27586–27599. 24. Oliver CJ, Shenolikar S (1998) Physiologic importance of protein phosphatase inhibitors. Front Biosci 3:D961–972. 25. Chan PM, Manser E (2012) PAKs in human disease. Prog Mol Biol Transl Sci 106:171–187. 26. Lagares D, Kapoor M (2013) Targeting focal adhesion kinase in fibrotic diseases. BioDrugs 27(1):15–23. 27. McKenzie JA, Ridley AJ (2007) Roles of Rho/ROCK and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability. J Cell Physiol 213(1):221–228. 28. Shen Q, Rigor RR, Pivetti CD, Wu MH, Yuan SY (2010) Myosin light chain kinase in microvascular endothelial barrier function. Cardiovasc Res 87(2):272–280. 29. Eriksson JE, et al. (2004) Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments. J Cell Sci 117(Pt 6):919–932. 30. Balla A, et al. (2008) Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha. Mol Biol Cell 19(2):711–721. 31. Grassie ME, Moffat LD, Walsh MP, MacDonald JA (2011) The myosin phosphatase targeting protein (MYPT) family: A regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ. Arch Biochem Biophys 510(2):147–159.

Yi et al. www.pnas.org/cgi/content/short/1404943111 15 of 15