Phosphorylation Regulates Human OCT4 INAUGURAL ARTICLE

Phosphorylation regulates human OCT4 INAUGURAL ARTICLE

Justin Brumbaugha,b, Zhonggang Houb, Jason D. Russellc, Sara E. Howdenb, Pengzhi Yub, Aaron R. Ledvinac, Joshua J. Coona,c, and James A. Thomsonb,d,e,1

aDepartment of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706-1532; bMorgridge Institute for Research, Madison, WI 53715; cDepartment of Chemistry, University of Wisconsin, Madison, WI 53706-1322; dDepartment of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53706; and eDepartment of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2008.

Contributed by James A. Thomson, March 7, 2012 (sent for review December 17, 2011) The transcription factor OCT4 is fundamental to maintaining ulatory mechanism were not determined. Several large-scale pro- pluripotency and self-renewal. To better understand protein-level teomic studies have identified protein phosphorylation events in regulation of OCT4, we applied liquid chromatography–MS to pluripotent cells, but only three phosphorylation sites on OCT4 identify 14 localized sites of phosphorylation, 11 of which were were previously described (29, 30). To this end, our current un- previously unknown. Functional analysis of two sites, T234 and derstanding of the extent and function of OCT4 phosphorylation S235, suggested that phosphorylation within the homeobox re- has been very limited. gion of OCT4 negatively regulates its activity by interrupting se- In this study, we combined high mass accuracy MS with mul- quence-specific DNA binding. Mutating T234 and S235 to mimic tiple dissociation techniques to identify 14 phosphorylation sites constitutive phosphorylation at these sites reduces transcriptional on OCT4, 11 of which are newly described. Our mutational activation from an OCT4-responsive reporter and decreases analysis of the two phosphorylation sites located within the reprogramming efficiency. We also cataloged 144 unique phos- OCT4 homeobox domain (T234 and S235) suggests that phosphopeptides on known OCT4 interacting partners, including phorylation negatively regulates OCT4 by disrupting sequence- SOX2 and SALL4, that copurified during immunoprecipitation. specific DNA binding. We also identified 144 phosphorylation These proteins were enriched for phosphorylation at motifs asso- sites in OCT4 binding partners and found that ERK phosphor-

ciated with ERK signaling. Likewise, OCT4 harbored several puta- ylation motifs are highly enriched both in the binding partners BIOLOGY

tive ERK phosphorylation sites. Kinase assays confirmed that and OCT4 itself. Finally, we found that purified OCT4 is phos- DEVELOPMENTAL ERK2 phosphorylated these sites in vitro, providing a direct link phorylated at sites outside of the homeobox domain by ERK, between ERK signaling and the transcriptional machinery that identifying OCT4 as a possible direct downstream target of FGF governs pluripotency. signaling, one of the key pathways that promotes human ES cell self-renewal. proteomics | posttranslational regulation Results CT4 is a homeobox transcription factor that was first Phosphoproteomic Analysis. Endogenous OCT4 protein was af- Oidentified for its essential role in early mammalian de- finity-purified from human ES cells using five lysis methods velopment (1–3). It is expressed in totipotent and pluripotent (Materials and Methods and Fig. S1A). We included a range of cells and down-regulated on differentiation (4–6). OCT4 is re- detergents and salt concentrations to optimize conditions for quired to maintain pluripotency both in vivo and in cell culture either sequence coverage or identification of binding partners. fl – (2, 7, 8), and it is indispensable for transcription factor-mediated Applying nano ow liquid chromatography MS, we sequenced fi reprogramming (9–12). Together with NANOG and SOX2, over 75% of the OCT4 protein and identi ed 24 phosphopep- OCT4 carries out these functions by activating transcription of tides that corresponded to 14 unique, localized phosphorylation < A genes that support pluripotency and repressing genes involved in sites [ 1% false discovery rate] (Fig. 1 and Table S1). Tandem fi development (13–17). mass spectra were manually validated to con rm all 14 OCT4 fi B A variety of proteins, including NANOG, SOX2, and OCT4 modi cations. Fig. 1 shows a representative spectrum; all ions fi itself, form an intricate regulatory loop that balances OCT4 ex- from the c- and z-ion series were identi ed in this example, fi pression (15, 18–20). Genomic and epigenetic studies have con rming the localization of the phosphorylation site. Note identified additional mechanisms that directly or indirectly in- that digestion with GluC combined with RIPA lysis dramatically fi fluence OCT4 expression, providing a detailed view of its tran- increased phosphorylation identi cations, although trypsin ac- scriptional regulation (21–26). OCT4 function is closely tied to counted for greater overall sequence coverage for OCT4 (Fig. C fi its regulation, because decreasing OCT4 mRNA levels cause 1 ). Although it is dif cult to compare directly between prep- differentiation into trophoblast, and increasing OCT4 expression aration techniques because of sample and instrument variability, by as little as 1.5-fold causes differentiation to primitive endo- it is clear that RIPA buffer provided the best phosphoproteomic derm (7). The relative stoichiometry of OCT4 and SOX2 is also coverage, averaging more than 12 phosphopeptide spectral fi matches and 3 unique phosphopeptides per run for OCT4 alone important for establishing pluripotency, because the ef ciency of B reprogramming is dependent on the proportion of OCT4 and (Fig. S1 ). SOX2 In pluripotent cells, OCT4 forms a heterodimer with SOX2 transcripts (9). Thus, precise regulation of OCT4 is es- – sential for pluripotency. to regulate transcription (13 17). In analyzing OCT4 samples, Although OCT4 transcriptional regulation has been exten- sively studied, far less is known about its posttranslational regulation. Previous studies have speculated that phosphorylation Author contributions: J.B., Z.H., J.D.R., S.E.H., J.J.C., and J.A.T. designed research; J.B., Z.H., J.D.R., S.E.H., P.Y., and A.R.L. performed research; J.B., Z.H., J.D.R., and P.Y. analyzed data; controls OCT4 activity (27, 28). For example, differences in and J.B. and J.A.T. wrote the paper. electrophoretic mobility suggested that the homeobox domain of The authors declare no conflict of interest. OCT4 is differentially phosphorylated when expressed in 293 1To whom correspondence should be addressed. E-mail: jthomson@morgridgeinstitute. cells compared with HeLa cells (27). Intriguingly, these different org. ’ states correlated with OCT4 s ability to activate transcription This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. from a reporter; however, specific phosphorylation sites and reg- 1073/pnas.1203874109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1203874109 PNAS Early Edition | 1of7 Downloaded by guest on September 29, 2021 A GluC Trypsin Both enzymes P PO4 site

Previously IDed PO site Putative ERK PO site * 4 4 OCT4 * PP P PPP P P*P *PP P P P SP TP SSP 1 POU-domain Homeobox 360 SOX2 * PP P P**P P SPS 1 HMG-box 317 B P C K R T S I E N R +2 MH-NH3 Total OCT4 sequence Phosphorylation coverage +3 +2 MH MH c c6 c7 5 coverage for OCT4 z 1 z c2 z 2 4 MH-H O 7.1% 2 12.1% z c z 3 3 6 21.4% c4 20.3% z5

c1 72.6% 71.4% z7

100 200 300 400 500 600 700 800 900 1000 1100 m/z

Fig. 1. OCT4 is phosphorylated at 14 unique localized sites. (A) A schematic showing sequence coverage and phosphorylation sites identiﬁed for OCT4 and SOX2. (B) A representative mass spectrum showing sequence coverage and phosphorylation at S235 on OCT4. The c- and z-ions are annotated and labeled on the spectrum. (C) Pie charts that show the percent of sequenced peptides or phosphorylation sites that were identiﬁed by trypsin (blue), GluC (green), or both enzymes (gray).

we identified one phosphorylation site from SOX2 that constitutively phosphorylated proteins, respectively. Based on copurified with OCT4. To extend this dataset, we then directly EMSA, the T234E_S235E mutant exhibited greatly decreased purified endogenous SOX2 for analysis by MS (Fig. 1A). DNA binding capacity compared with the WT protein and the In total, four phosphopeptides were observed from SOX2, T234A_S235A mutant (Fig. 2A). We estimate that the binding from which six unique phosphorylation sites were localized to constants for the WT protein and T234A_S235A mutant are aspecificresidue(Table S1). As before, all phosphorylation both ∼100 nM, whereas the T234E_S235E mutant had a bind- sites were manually verified. This work identified three pre- ingconstantthatexceededtherangesthatwetested(>400 viously identified sites and three unknown phosphorylation nM). To ensure that DNA binding was specific in our system, sites (29, 30, 31). we also performed EMSAs with a mutant probe, in which 3 nt within each octamer binding motif were changed (Materials and Phosphorylation of T234 and S235 on OCT4 Regulates DNA Binding. Methods). In contrast to the WT probe, OCT4 protein did not Phosphorylation sites on OCT4 were scattered throughout the produce a distinct shift with the mutant probe, indicating that protein (Fig. 1A); however, two sites (T234 and S235) were reduced electrophoretic mobility observed with the WT probe present near the N terminus of the OCT4 homeobox domain. is representative of sequence-specificDNA–protein inter- Based on structural modeling of the OCT4–DNA complex, actions (Fig. 2B). To determine whether individual phosphor- previous work suggested that this region is located adjacent to ylation of T234 and S235 impacts DNA binding, we mutated DNA (Fig. S2A) (28, 32). Both phosphorylation sites are highly each residue to glutamic acid. Unlike the T234E_S235E double conserved in POU5F1 across species and also in the POU family mutant, single mutation did not entirely disrupt DNA binding of proteins (Fig. S2 B and C). By searching the human protein (Fig. 2C). Together, these results suggest that phosphorylation reference database (33), we identified several candidate kinases within the OCT4 homeodomain disrupts sequence-specific for phosphorylation of T234 and S235 (Table S2). However, DNA binding, and the effects of multiple phosphorylation in vitro phosphorylation with purified OCT4 and commercially events are additive. available kinases (PKA, PKC, PIM1, PAK2, and AKT2) was unsuccessful. Phosphorylation of T234 and S235 on OCT4 Modulates Transcriptional To determine the effect of phosphorylation at these positions, Activation. To explore whether phosphorylation of OCT4 and the we recombinantly expressed two OCT4 mutants (T234A_S235A subsequent reduction in affinity for its DNA targets altered its and T234E_S235E) that mimic the nonphosphorylated and ability to activate transcription, we transfected 293FT cells with

2of7 | www.pnas.org/cgi/doi/10.1073/pnas.1203874109 Brumbaugh et al. Downloaded by guest on September 29, 2021 A B INAUGURAL ARTICLE WT 00AA EE 0WT WT Wildtype probe Mutant probe Wildtype probe

OCT4/DNA complex

Free probe

C ES 00TE WT 0EE 0 Wildtype probe

OCT4/DNA complex

Free probe

fi fi A Fig. 2. A construct that mimics the constitutively phosphorylated OCT4 protein exhibits greatly decreased sequence-speci c DNA binding af nity. ( ) BIOLOGY Recombinantly expressed WT OCT4 and the T234A_S235A (AA) mutant bind DNA, causing a distinct shift by EMSA. The T234E_S235E (EE) mutant exhibits a far DEVELOPMENTAL lower capacity for DNA binding. Protein concentrations used for each sample were 400, 200, 100, and 0 nM. (B) Shifts observed by EMSA are sequence-specific, because switching only 2 nt (mutant probe) eliminates a distinct gel shift. Protein concentrations used for each sample were ∼800, 400, 200, 100, 50, and 0 nM. (C) Single mutation of T234 (ES) or S235E (TE) to glutamic acid does not disrupt DNA binding to the same extent as the double mutant T234E_S235E (EE). Protein concentrations used for each sample were 400, 200, 100, and 0 nM.

an OCT4-responsive luciferase reporter and WT OCT4, the replicates varied. Regardless of efficiency, these experiments T234A_S235A mutant, or the T234E_S235E mutant. Consistent show that each of the OCT4 constructs is functional and capable with the EMSA results, T234E_S235E showed a significant of activating transcription from OCT4 target genes to initiate decrease in transcriptional activation compared with both the pluripotency. WT and T234A_S235A mutant (P < 0.0005). Meanwhile, the T234A_S235A mutant activated transcription at a level com- OCT4 Interacting Proteins. In addition to OCT4 phosphorylation, mensurate with the WT protein (Fig. 3A). The differences ob- we identified 46 known OCT4 binding partners, including SOX2, served were not the result of inefficient transfection between SALL3, SALL4, and LIN28A (34–36); 32 binding partners har- constructs, because flow cytometry indicated that an equivalent bored at least one phosphorylation site, and many were multi- number of cells was positive for each construct (Fig. 3B). phosphorylated, accounting for 144 phosphopeptides, of which Likewise, the lower transcriptional activation exhibited by the 65 phosphorylation isoforms were localized with greater than T234E_S235E mutant was not caused by inefficient translation 95% certainty. We also discovered methylation on two OCT4 or rapid protein degradation, because similar amounts of pro- binding partners (ILF2 and EWSR1) and acetylation on DHX9. tein were present in cells transfected with each construct (Fig. 3 Fig. 4 represents an OCT4-centered protein interaction network C D and and Fig. S3). To ensure that the protein was properly overlaid with posttranslational modification data for selected fl localized, we performed immuno uorescence and found that proteins (Dataset S1 has a full listing). We applied the Motif-X all three OCT4 constructs were present exclusively in the nu- program to determine whether localized phosphorylation sites cleus of 293FT cells (Fig. S4). We conclude that phosphory- on OCT4 and its binding partners share common motifs (37). lation of OCT4 at T234 and S235 reduces its capacity to Two motifs were identified as significantly enriched in this dataset activate transcription and that this effect is likely the result of (P < 0.000001) (Fig. 4B). We then queried the human protein reduced affinity for DNA rather than variation in protein levels reference database and found that one motif, RX[S/T], has been or localization. linked to PKC and cAMP-activated PKA. The other motif satis- fi Phosphorylation of T234 and S235 on OCT4 Decreases Reprogramming ed the minimal requirements needed to direct ERK1 and ERK2 Efficiency. To determine the role that these phosphorylation sites phosphorylation (38). Together, these results suggest that proteins fi play in reprogramming, we moved the T234A_S235A and within the OCT4 complex are modi ed by the same kinases. It T234E_S235E mutants into a lentiviral vector and initiated should be noted that ERK shares a preferred phosphorylation reprogramming as previously described (9). Interestingly, both motif with cyclin-dependent kinase 5 (CDK5). Although we can- mutants were capable of reprogramming somatic cells to induced not rule out phosphorylation by other proline-directed kinases pluripotent stem (iPS) cells; however, the T234E_S235E mutant such as CDKs, the putative ERK motifs are particularly intriguing, produced far fewer iPS colonies than both the T234A_S235A because FGF/ERK signaling plays a central role in promoting mutant and WT construct (n = 15) (Fig. 3E). The median self-renewal of human ES cells. However, direct downstream number of colonies generated by the T234A_S235A mutant was transcription factor targets of FGF/ERK signaling have not been lower than WT OCT4; however, the efficiency for individual previously identified (39).

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1 30

20 0.5 POU5F1 positive cells (%) 10 Mean normalized luciferase activity 0 0 WT AA EE CNTL pGL3-basic WT AA EE CNTL C D E 4 WT AA EE CNTL Outlier 200 Mean 1.5 140 100 3 80 60 50 1 2 40 POU5F1 30

efficiency 1 20 0.5 Normalized protein level

WT-Normalized reprogramming 40 0 30

GAPDH WT AA EE CNTL AA EE

Fig. 3. The impact of OCT4 T234_S235 mutation on transcription activation and reprogramming efficiency. (A) Luciferase assays normalized to a Renilla control. Error bars represent SD. *P < 0.0005. AA, OCT4 T234A_S235A; CNTL, pCEP4 control (without OCT4 expression); EE, OCT4 S234E_T235E; pGL3-basic, basal luciferase reporter without an OCT4 promoter. (B) Transfection efficiency as assessed by flow cytometry for each of the OCT4 constructs. (C) A Western blot showing protein for each construct. Analysis was performed in triplicate. Fig. S3 shows whole-membrane images. (D) Quantitative data for the Western blots described in C.(E) Reprogramming efficiency for T234A_S235A and T234E_S235E mutants. The number of colonies produced for each mutant was normalized to the number of colonies produced using WT protein under the same conditions. The dashed line represents WT reprogramming efficiency for comparison (n = 15).

ERK2 Phosphorylates OCT4 in Vitro. Given the enrichment for pu- homeobox portion of OCT4 for additional analysis. Structural tative ERK phosphorylation sites among OCT4 binding partners, modeling based on Oct1 suggested that these sites may be po- we searched OCT4 for phosphorylation sites that match the sitioned in close proximity to the phosphate backbone of DNA minimal ERK motif; 3 of 14 phosphorylation sites on OCT4, (Fig. S2A) (32). We, therefore, postulated that the addition of S111, T118, and S355 are putative ERK sites (Fig. 1A). To a negatively charged phosphoryl moiety on T234 and S235 causes confirm that ERK2 phosphorylates these sites in vitro, we per- electrostatic repulsion and diminishes the DNA binding capacity formed kinase assays using recombinant OCT4 protein and ac- of OCT4. EMSA results support this hypothesis, because the tive ERK2. As a control, we conducted concomitant experiments T234E_S235E mutant that mimics the constitutively phosphor- using PAK2, PIM1, and PKA. We then labeled each sample ylated protein showed greatly reduced affinity for DNA har- A using isobaric tags and performed quantitative MS. Fig. 5 boring the octamer binding motif. The effect of phosphorylation shows a representative spectrum and reporter region for a pep- at these sites is likely additive, because single mutants (T234E or tide that was phosphorylated at S111. A peak at a mass to charge S235E) do not reduce the DNA binding capacity of OCT4 to the ratio of 126 is representative of ERK phosphorylation, whereas same extent as the double mutant T234E_S235E. Recent work peaks at mass to charge ratios of 127, 130, or 131 represent suggested that a similar site in the homeobox domain of mouse phosphorylation by PAK2, PIM1, or PKA, respectively. All of Oct4 is phosphorylated based on a predictive algorithm (28). the putative ERK phosphorylation sites identified in our dis- Although the modification was never directly identified in plu- covery experiment were specifically phosphorylated by ERK2 (Fig. 5B). In contrast, phosphorylation at S289 and S290 was ripotent cells, the work provided evidence that mutation of this present in PIM1- and PKA-treated samples but was not detected site disrupts transcriptional activation from an OCT4 reporter in the ERK2-treated sample (Fig. 5C). We conclude that ERK2 (28). Our results are consistent with this work, because the is capable of specifically phosphorylating S111, T118, and S355 T234E_S235E mutant decreases transcriptional activation ca- fl in vitro. pacity of OCT4 to background levels. Based on immuno uorescence and quantitative protein measurements, reduced activation Discussion is not the result of improper localization or rapid degradation This research extends the current knowledge regarding post- of the T234E_S235E mutant. We conclude that phosphorylation translational modification of OCT4. A notable aspect of this of T234 and S235 regulates OCT4 transcription primarily by al- work is that we purified OCT4 directly from human ES cells tering its DNA binding capacity. without overexpression to map physiologically relevant post- Reprogramming experiments using the T234E_S235E and translational modifications. In total, we cataloged 11 previously T234A_S235A mutants provided a perspective on the biological unknown phosphorylation sites on OCT4 and confirmed all 3 consequence of OCT4 phosphorylation during reprogramming. previously reported sites. To explore the functional relevance of The mutant mimicking the constitutively phosphorylated protein OCT4 modifications, we chose two phosphorylation sites in the exhibited greatly reduced reprogramming efficiency compared

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P P P P P P P P P P ARID3B P MITF LIG3 MSH6

P P P P P P P P P P P P P P RIF1 LIN28A SOX2 MBD3 MTA3 P P

MTA1 P P P P P P P P P P P P P P P P P P P P P SALL4 OCT4 P P P P CHD4 GATAD2A

P P P P P P P P PML ZMYM2 ILF2 P WWP2 EWSR1 P SMARCA4 P Mee P Mee P Me ACTL6A

P P P DHX9 P P TRIM24 P P RBM14 P SMARCA5 Ac P P P P P P P P P P P P P P P P P B ERK1/ERK2 kinase substrate motif PKC/PKA kinase substrate motif BIOLOGY D A DEVELOPMENTAL P RG PPC G ARSA G S R F D REP KRG F A DD L KQL L HR G S D T P EA P GT P AKQ I HE I LG A D GL LR D R P I KQ E F L Q R NV H YF T S P A VS H N CCVK MGK EF E NN VK QC E H T SS F M N L I HT NRS S I V TPTY VSW YP V YTYS -4 -3 -2 -1X 0P +1 +2 +3 +4 -4 -3 R-2 -1X 0 +1 +2 +3 +4

Fig. 4. An OCT4-centered phosphorylation network. (A) Selected OCT4 binding partners were processed through the STRING database and overlaid with posttranslational modifications identified in proteomic experiments. Thick lines represent direct association with OCT4, whereas thin lines indicate association with periphery components in the complex. Dataset S1 has a full listing of OCT4 binding partners and their corresponding modifications. (B) Sequence logos representing phosphorylation motifs that were enriched in OCT4 and associated proteins (P < 0.000001). X, the site of phosphorylation (either phosphoserine or phosphothreonine.

with WT OCT4. Still, the mutant was capable of producing iPS data suggest that ERK2 directly phosphorylates OCT4 at mul- colonies, indicating that (i) the mutant protein was functional at tiple sites. All of these sites are located outside of annotated some level and (ii) other forms of regulation are required to DNA binding domains, which may imply positive regulation either completely inactivate or degrade OCT4 protein. The through ERK phosphorylation. In support of this hypothesis, T234A_S235A mutant, which mimics a version of OCT4 that recent work showed that activating ERK2 in differentiating cells cannot be phosphorylated at these sites, generated colonies with maintains NANOG, another transcription factor that is key in a mean efficiency commensurate to the WT protein. These data maintaining pluripotency (39). Although still somewhat specu- point to a negative regulatory role for phosphorylation of T234 lative, our results suggest that FGF signaling through ERK and S235 on OCT4. phosphorylation directly regulates OCT4 and associated proteins By applying a variety of lysis conditions, we also copurified that establish and maintain pluripotency. known OCT4 binding partners. Included in this analysis was phosphorylation of S251 on SOX2, indicating that SOX2 is Materials and Methods modified as a component of the OCT4 complex. Almost all of Cell Lysis. Human ES cells (line H1) were harvested using TrypLE (Invitrogen) the proteins in this dataset were posttranslationally modified and washed three times in PBS (Invitrogen). Roughly 108 cells were used for through phosphorylation, methylation, or acetylation. Remarkably, each experiment. The cells were lysed using RIPA buffer, nuclear isolation we found that OCT4 binding partners share highly significant buffer, urea buffer, nonidet P-40 buffer, or sonication buffer (SI Materials phosphorylation motifs (P < 0.000001), suggesting coregulation and Methods). of these physically associated proteins through a common upstream kinase. One of the motifs satisfies the minimal require- MS. Tandem MS was performed using a NanoAcquity UPLC system (Waters) coupled to a QLT-Orbitrap with electron transfer dissociation (Thermo Fisher ments for phosphorylation by ERK1/2, which functions in the Scientific). Mass spectrometer instrument methods consisted of one MS1 (re- FGF pathway. solving power = 30,000–60,000) scan followed by data-dependent MS2 scans For years, it has been known that FGF signaling promotes the of the 10 most intense precursors. In the case of high-resolution MS2, nominal self-renewal of human pluripotent stem cells (40, 41); however, resolution was set to 7,500. After peptide selection, dynamic exclusion was the mechanism by which FGF signaling interacts with the tran- used. Precursors with unassigned charges states or charge states of one [and scriptional machinery of pluripotency has remained elusive. Our two for electron transfer dissociation (ETD) scans] were also excluded.

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y Reporter ion region 7

126.1275

y8 +2 y7 125 126 127 128 129 130 131 132 y3 +2 y12 +2 b y 6 8 b y y6 b3 5 4 y b b12 b b 9 10 y 4 7 10 b13-H2PO4

200 400 600 800 1000 1200 1400 1600 1800 m/z B C 4 4 3.5 3.5 3 3 2.5 2.5 2 2 1.5 1.5 1 1 0.5 0.5

Mean normalized relative abundance 0 Mean normalized relative abundance 0 ERK2 PAK2 PIM1 PKA ERK2 PAK2 PIM1 PKA ERK2 PAK2 PIM1 PKA ERK2 PAK2 PIM1 PKA S111 T118 S355 S289, S290

Fig. 5. ERK2 specifically phosphorylates OCT4 in vitro. (A) A representative spectrum showing sequence coverage for an OCT4 peptide with phosphorylation at S111. The lift-out section shows the reporter region for isobaric tags. Peaks corresponding to each tag are proportional to the relative abundance of the phosphorylated peptide in the corresponding sample: 126, ERK2; 127, PAK2; 130, PIM1; 131, PKA. (B) Bar graphs showing the relative abundance of phosphorylated OCT4 peptides from the indicated kinase assays. Included are all three putative ERK phosphorylation sites identified from endogenous OCT4 in the discovery experiments. Error bars represent SD for all peptide spectral matches quantified in this experiment. (C) Bar graphs showing the relative abundance of a phosphorylated OCT4 peptide that does not contain a putative ERK2 phosphorylation site.

Electrophoretic Mobility Shift Assays. The sequences for the DNA probes used T234E_S235E-IRES2-Sox2 were used in place of pSin-EF2-Oct4-IRES2-Sox2 in this experiment were previously published (42) (SI Materials and Methods). where indicated. Protein samples were handled at 4 °C at all times. DNA probes were diluted Kinase Assays. All in vitro kinase assays were carried out in 40 mM Tris·HCl (pH to 5 nM in 20 mM Tris·HCl (pH 8), 150 mM NaCl, 2 mM MgCl2,1 mM DTT, 0.025 U didc, and 4% Ficoll and added to the indicated protein amounts. 7.5), 20 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, and 0.1 mM ATP (Promega). Approximately 1 μg recombinant OCT4 was added for each reaction. Then, Samples were incubated for 30 min at 4 °C in the dark and then loaded onto 0.25 μg ERK2 (Promega), 0.25 μg PAK2 (Millipore), 0.25 μg PIM1 (Millipore), 5% TBE gels (Biorad). The gels were run for 1.5 h at 100 V. The results were or 2.5 units PKA (Sigma) were added for 30 min at 23 °C. The final reaction fi imaged using an LAS-3000 Imaging System (Fuji lm). All quantitation was volume was 25 μL. The samples were then subjected to digestion as de- ’ determined according to manufacturer s instructions with MultiGauge scribed above and labeled with TMT isobaric tags (Thermo-Fisher) according software, version 2.0 (Fujifilm). to the manufacturer’s recommendations. MS was carried out as above. Quantitation was processed using the COMPASS software suite (43). All Transfection and Luciferase Reporter Assays. 293FT cells were transfected by quantitative values were mean-normalized, and SD was calculated based on electroporation (SI Materials and Methods). Luciferase assays were carried values associated with peptide spectral matches. out per the manufacturer’s recommendations, and all values were normalized to the Renilla control. Signal intensities were then mean-normalized for ACKNOWLEDGMENTS. We thank Kelly Hoadley and James Keck for help fi each experiment. with recombinant protein expression and kindly providing the modi ed pGEX4T-1 plasmid. We also thank Krista Eastman, Ron Stewart, and Brian McIntosh for critical reading of this manuscript and A. J. Bureta for help with Reprogramming. Reprogramming was carried out as previously described (10), graphics. We are grateful to Alex Hebert for help with quantitative except that pSin-EF2-Oct4_T234A_S235A-IRES2-Sox2 and pSin-EF2-Oct4_ data analysis.

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