Comparative receptor tyrosine kinase profiling identifies a novel role for AXL in human stem cell pluripotency.

Human Molecular Genetics, 2014, Vol. 23, No. 7 doi:10.1093/hmg/ddt571 Advance Access published on November 11, 2013

1802–1816

Comparative receptor tyrosine kinase profiling identifies a novel role for AXL in human stem cell pluripotency Mi-Young Son1,2,{, Binna Seol1,{, Yong-Mahn Han2 and Yee Sook Cho1,3,∗ 1

Received July 25, 2013; Revised October 25, 2013; Accepted November 6, 2013

The extensive molecular characterization of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) is required before they can be applied in the future for personalized medicine and drug discovery. Despite the efforts that have been made with kinome analyses, we still lack in-depth insights into the molecular signatures of receptor tyrosine kinases (RTKs) that are related to pluripotency. Here, we present the first detailed and distinct repertoire of RTK characteristic for hPSC pluripotency by determining both the expression and phosphorylation profiles of RTKs in hESCs and hiPSCs using reverse transcriptase – polymerase chain reaction with degenerate primers that target conserved tyrosine kinase domains and phospho-RTK array, respectively. Among the RTKs tested, the up-regulation of EPHA1, ERBB2, FGFR4 and VEGFR2 and the down-regulation of AXL, EPHA4, PDGFRB and TYRO3 in terms of both their expression and phosphorylation levels were predominantly related to the maintenance of hPSC pluripotency. Notably, the specific inhibition of AXL was significantly advantageous in maintaining undifferentiated hESCs and hiPSCs and for the overall efficiency and kinetics of hiPSC generation. Additionally, a global phosphoproteomic analysis showed that ∼30% of the proteins (293 of 970 phosphoproteins) showed differential phosphorylation upon AXL inhibition in undifferentiated hPSCs, revealing the potential contribution of AXL-mediated phosphorylation dynamics to pluripotency-related signaling networks. Our findings provide a novel molecular signature of AXL in pluripotency control that will complement existing pluripotency-kinome networks.

INTRODUCTION Protein tyrosine kinases (PTKs) are central components of signaling networks that orchestrate various physiological functions, such as cell growth, survival, metabolism and differentiation, in numerous cell types, including human embryonic stem cells (hESCs) (1 – 3) and human-induced pluripotent stem cells (hiPSCs) (4). Currently, 91 PTKs have been identified in the human genome; these PTKs fall within two major groups: 59 receptor tyrosine kinases (RTKs) and 32 non-receptor, cytoplasmic PTKs (5). RTKs comprise an extracellular ligand binding domain, a transmembrane domain and an intracellular kinase domain and transduce extracellular signals upon ligand binding to intracellular downstream signaling cascades that

evoke various cellular processes (6,7). RTK ligands include various growth factors, cytokines and hormones (8). Considerable effort has been made to determine the distinct roles of RTKs and their specific ligands in hPSCs using various genetic and biochemical methods. High-throughput approaches, including microarrays (9 – 11), serial analysis of gene expression (12) and expressed sequence tags (13), have identified distinct gene expression patterns of RTKs in hPSCs, including three fibroblast growth factor receptors (FGFR1, FGFR2 and FGFR4), human epidermal growth factor receptor 2 (ERBB2) and insulin-like growth factor 1 receptor (IGF1R). As a result, it has become possible to decipher their molecular mechanisms and the pathways involved in hPSC pluripotency

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To whom correspondence should be addressed. Tel: +82 428604479; Fax: +82 428604608; Email: [email protected] These authors contributed equally to this work.

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# The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Downloaded from http://hmg.oxfordjournals.org/ at Uniwersytet Warszawski Biblioteka Uniwersytecka on April 13, 2015

Stem Cell Research Center, KRIBB, 125 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea 2Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea 3 Department of Functional Genomics, University of Science & Technology, 217 Gajungro, Yuseong-gu, Daejeon 305-333, Republic of Korea

Human Molecular Genetics, 2014, Vol. 23, No. 7

RESULTS Differential expression of RTK genes in hPSCs As representative hPSCs, we tested H9 hESCs and human foreskin fibroblast (hFF)-derived iPSCs induced by the four Yamanaka factors [OCT4 (O), SOX2 (S), KLF4 (K) and c-MYC (M); OSKM]. The expression profiles of the RTKs observed in undifferentiated hPSCs were compared with those of differentiated hPSCs. Both differentiating embryoid bodies (EBs) derived from hPSCs and retinoic acid (RA)-differentiated hPSCs were used as representative differentiated cells. The undifferentiated state of hPSCs was confirmed by compact and round colony morphology, a high level of alkaline phosphatase (AP) activity and high expression of the pluripotency markers OCT4, NANOG, SSEA4, tumor-related antigen (TRA)-1-60 and REX1 (Supplementary Material, Fig. S1). Mixtures of PTK genes were selectively amplified by RT– PCR using degenerate primer sets spanning the PTK subdomain VII and IX regions of undifferentiated and differentiated hPSCs (Fig. 1A). As shown in Figure 1, the amplified PCR product was a single band of 150 –170 bp in length that was cloned and directly sequenced. Of the 1681 positive clones that were identified, 91.4% (748 of 818 clones) and 87.5% (631 of 721 clones) of the clones encoded known human PTKs from hESCs and reprogrammed hiPSCs, respectively (Fig. 1B). In addition, 80.8%

(256 of 317 clones) of the clones contained fragments of known human PTKs from the terminally differentiated hFFs that were used for reprogramming. In total, 40 human PTKs, including RTKs and NRTKs, were expressed in hPSCs (Fig. 1A and Supplementary Material, Fig. S2). Minor non-PTKs, including serine/threonine kinases, were also detected among the nonspecific PCR products due to mispriming. Putative novel genes containing the catalytic subunit of PTKs were also identified, and their identities are under examination. Among the PTK genes that were examined, 75.2% (615 of 818 clones) and 71.2% (513 of 721 clones) of the clones originated from RTK genes from hESCs and hiPSCs, respectively (Fig. 1A). To identify new candidate RTKs associated with hPSC pluripotency control, the differences in gene frequency between the undifferentiated and differentiated hPSCs were analyzed and are listed in Table 1. Overall, 213 clones from hESCs, 200 clones from hESC-EBs and 202 clones from RA-differentiated hESCs were assessed. Of the 22 RTKs (a gene frequency cut-off .0.5%), eight RTKs (EPHA1, EPHA7, ERBB1, ERBB2, ERBB3, FGFR2, FGFR4 and VEGFR2) were transcriptionally up-regulated in undifferentiated hESCs, while six RTKs (AXL, EPHA2, EPHA4, EPHB4, PDGFRB and TYRO3) were downregulated compared with the differentiated hESCs. In parallel, 205 clones from hiPSCs, 142 clones from EBs and 166 clones from RA-differentiated hiPSCs were analyzed. Of the 17 RTKs (a gene frequency cut-off .0.5%), seven RTKs (EPHA1, EPHB4, ERBB2, FGFR1, FGFR4, IGF1R and VEGFR2) were transcriptionally up-regulated in undifferentiated hiPSCs, while seven RTKs (AXL, DDR1, EPHA4, EPHB2, HGFR, PDGFRB and TYRO3) were down-regulated compared with the differentiated hiPSCs. Relatively fewer RTKs were detected in hiPSCs compared with hESCs, but both cell types appeared to share common pluripotency-related RTK gene targets, such as the FGFRs (Table 1). The RTKs detected in hPSCs included Axl (a Tyro3 PTK), DDR (discoidin domain receptor), EphR (ephrin receptor), EGFR (epidermal growth factor receptor), FGFR, InsR (insulin receptor), HGFR (hepatocyte growth factor receptor), TrkR (tropomyosin receptor), PDGFR (PDGF receptor) and VEGFR (vascular endothelial growth factor receptor; Table 1). DDR2 was detected in primary hFFs but not in hPSCs. Four RTKs (EPHA1, ERBB2, FGFR4 and VEGFR2) were identified as being commonly up-regulated in both undifferentiated hESCs and hiPSCs, while four RTKs (AXL, EPHA4, PDGFRB and TYRO3) were down-regulated compared with differentiated hESCs and hiPSCs. In agreement, real-time quantitative polymerase chain reaction (qPCR) analysis also confirmed the up-regulation in four RTK genes (EPHA1, ERBB2, FGFR4 and VEGFR2) and down-regulation in four RTK genes (AXL, EPHA4, PDGFRB and TYRO3) in both undifferentiated hESCs and hiPSCs compared with the differentiated hESCs and hiPSCs (Fig. 2A and B). We also verified this observation in two independently derived hESC lines (HUES7 and H1) and human lung fibroblast-derived iPSCs (Supplementary Material, Fig. S3). Therefore, the expression patterns of these selected RTKs do not appear to be cell line-specific. The cellular functions and pathways of the RTK genes identified in this study are presented in Supplementary Material, Table S1. These RTK profiles obtained using gene frequency analyses may provide valuable information on the dynamic behavior of these kinases, which control the coordinated balance between undifferentiated growth and differentiation in hPSCs.


control (9,11,14–16). Detailed functional assays have also confirmed that RTK-dependent signaling plays key roles in regulating hPSC pluripotency. The activation of the IGFII/ IGF1R pathway helps to maintain hESCs in an undifferentiated state (1). The inactivation of the ERBB2 pathway disturbs the maintenance of undifferentiated hESCs (15,16). Several RTK ligands, such as basic fibroblast growth factor (bFGF) (17), IGF2 (1), platelet-derived growth factor (PDGF) (18), heregulin-1b (16) and neurotrophins (3) to hPSC cultures, can support the undifferentiated growth of hPSCs in vitro. bFGF potentially perpetuates hPSC self-renewal and pluripotency via the activation of multiple FGFR-dependent intracellular signaling pathways, such as phosphatidylinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) (2,19). Currently, bFGF is the most commonly used growth supplement in routine culture of hPSCs (20). However, further work is required to fully understand the significant roles of RTK-dependent signaling networks in influencing the self-renewal capacity and pluripotency of hPSCs. Here, we provide for the first time the hPSC-specific repertoire of 23 RTK transcripts and 42 phospho-RTK proteins, which reflect both the undifferentiated and differentiated states of hPSCs. These proteins were identified using parallel degenerative reverse transcriptase– polymerase chain reaction (RT – PCR) and phospho-RTK array analyses. Based on the profiling data, AXL was identified as a key factor for hPSC pluripotency control. Importantly, AXL antagonism positively contributes to the undifferentiated growth of hPSCs and the reprogramming of human somatic cells to hiPSCs. Further, AXL-modulated protein phosphorylation dynamics were also analyzed in-depth using standard phosphoproteomic techniques followed by liquid chromatography/mass spectrometry with tandem mass spectrometry (LC-MS/MS). Our results will help bridge the gap between RTK-mediated protein phosphorylation dynamics and the pluripotency regulatory network.

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Differential phosphorylation/activation of RTKs in hPSCs We next examined the phosphorylation status of key regulatory RTKs using a human phospho-RTK array containing 42 different RTKs in hPSCs. Consistent with our gene frequency analysis, four RTKs (EPHA1, 1.9-fold; ERBB2, 1.8-fold; FGFR4, 2.3-fold; VEGFR2, 2.6-fold) were highly phosphorylated (P-values ranging from ,0.05 to ,0.01) and thus active in undifferentiated hESCs compared with the RA-differentiated hESCs (Fig. 3A). In parallel, another four RTKs (AXL, 2.2-fold; EPHA4, 2.1-fold; PDGFRb, 1.6-fold; TYRO3, 2.0-fold) were highly phosphorylated and thus active in differentiated hESCs, compared with undifferentiated hESCs (Fig. 3B). These results demonstrate the existence of variable and distinctive RTK expression profiles at the transcriptional and post-translational levels, which are characteristic of undifferentiated and differentiated hPSCs.

Inhibition of AXL improves the maintenance of hPSC pluripotency We tested small molecule inhibitors that are specific for the identified RTKs in hPSCs to investigate whether they can modulate the state of pluripotency. In general, hESCs were maintained in an undifferentiated state using feeder-free conditions on Matrigel with daily changes of fresh mouse embryonic fibroblasts-conditioned medium (MEF-CM), whereas they underwent differentiation when cultured with UM without feeder factors (Fig. 4A). Among the tested compounds, the daily addition of either warfarin (0.1–1 mM) or R428 (1 nM), which are selective inhibitors of AXL, to UM significantly blocked differentiation and instead promoted

the maintenance of undifferentiated hESCs, as confirmed by their typical hESC-like morphology and enriched AP activity (Fig. 4A). The mRNA expression of the hESC markers OCT4, NANOG and REX1 was significantly restored by treatment with AXLspecific inhibitors (warfarin and R428) in hESCs cultured with UM (Fig. 4B). Consistently, immunocytochemical analysis also showed an enriched expression of hESC-specific OCT4, SSEA-4 and TRA-1-60 in hESCs cultured with UM containing either warfarin or R428 (Fig. 4C). In addition to promoting selfrenewal in hESCs, warfarin and R428 also significantly increased the growth of undifferentiated hESCs maintained in CM (Fig. 4D). Most importantly, we demonstrated that warfarin and R428 did not adversely influence the differentiation potential of hESCs. qPCR and immunocytochemical analyses of EBs derived from warfarinor R428-maintained hESCs showed the expression of tri-lineage germ layer markers, indicating that they retained their pluripotent differentiation potential (Fig. 4E and F). The positive effects of AXL inhibitors on the growth and maintenance of undifferentiated hPSCs were also confirmed under feeder-containing and chemically defined feeder-free culture systems (Supplementary Material, Fig. S4). These results indicate that blocking the AXLactivated signaling pathway, which we expected to provide pro-differentiation signaling, is significantly advantageous for maintaining the undifferentiated state of hPSCs. Inhibition of AXL enhances reprogramming of human somatic cells into hiPSCs Next, we further examined whether the AXL inhibitors can enhance the reprogramming of human somatic cells to a


Figure 1. Differential display analysis of the PTK profile in hPSCs. (A) Experimental scheme of the PTK profile analysis by degenerate RT– PCR. (a) Design of degenerate primers. The degenerate PCR primer sequences were derived from the conserved DFG and DVW motifs of PTK catalytic domains VII and IX, respectively. (b) PTK amplicon amplification by PCR. PCR was performed using a combination of degenerate primers, F1, F2, F3 and R, as described in the Materials and methods. PCR products (150– 170 bp) were eluted from gels and purified. (c) Cloning and sequencing. PCR-amplified products were subcloned into T-vectors. The recombinant plasmids were sequenced and then compared with GenBank sequences from the National Center for Biotechnology Information using the BLAST algorithm. (B) Identification of PTKs expressed in hPSCs by RT –PCR using degenerate primers. Known, genes of known function that are not human PTK or serine/threonine kinase genes; no hits, no significant similarity to any known gene by BLASTN.

Table 1. Expression profiles of RTKs in hPSCs Gene

AXL DDR DDR EPHR EPHR EPHR EPHR EPHR EPHR EGFR EGFR EGFR FGFR FGFR FGFR InsR AXL HGFR HGFR TrkR PDGFR PDGFR AXL VEGFR

Amino acid sequence DFGLSKKIYNGDYY – – RQGRIAKMPVKWIAIESLADRVYTSKSDVW DFGMSRNLYAGDYY – – RVQGRAVLPIRWMAWECILMGKFTTASDVW DFGMSRNLYSGDYY – – RIQGRAVLPIRWMSWESILLGKFTTASDVW DFGLTRLL –DDFDGTYETQ-G-GKIPIRWTAPEAIAHRIFTTASDVW DFGLSRVLEDDP-EATYTTS-G-GKIPIRWTAPEAISYRKFTSASDVW DFGMSRVLEDDP-EAAYTTR-G-GKIPIRWTAPEAIAYRKFTSASDVW DFGLSRVIEDDP-EAVYTTT-G-GKIPVRWTAPEAIQYRKFTSASDVW DFGLSRFLEDDTSDPTYTSALG-GKIPIRWTAPEAIQYRKFTSASDVW DFGLSRFLEENSSDPTYTSSLG-GKIPIRWTAPEAIAFRKFTSASDAW DFGLAKLLGAEEKE – -YHAEG-GKVPIKWMALESILHRIYTHQSDVW DFGLARLLDIDETE – -YHADG-GKVPIKWMALESILRRRFTHQSDVW DFGVADLLPPDDKQ – -LLYSE-AKTPIKWMALESIHFGKYTHQSDVW DFGLARDIHHIDYY – – KKTTNGRLPVKWMAPEALFDRIYTHQSDVW DFGLARDINNIDYY – – KKTTNGRLPVKWMAPEALFDRVYTHQSDVW DFGLARGVHHIDYY – – KKTSNGRLPVKWMAPEALFDRVYTHQSDVW DFGMTRDIYETDYY – – RKGGKGLLPVRWMSPESLKDGVFTTYSDVW DFGLSKKIYSGDYY – – RQGRIAKMPVKWIAIESLADRVYTSKSDVW DFGLARDMYDKEYY – SVHNKTGAKLPVKWMALESLQTQKFTTKSDVW DFGLARDILDREYYS– VQQHRHARLPVKWMALESLQTYRFTTKSDVW DFGMSRDVYSTDYY – – RVGGHTMLPIRWMPPESIMYRKFTTESDVW DFGLARDIMHDSNY – – VSKGSTFLPVKWMAPESIFDNLYTTLSDVW DFGLARDIMRDSNY – – ISKGSTFLPLKWMAPESIFNSLYTTLSDVW DFGLSRKIYSGDYY – – RQGCASKLPVKWLALESLADNLYTVQSDVW DFGLARDIYKDPDY – – VRKGDARLPLKWMAPETIFDRVYTIQSDVW

Frequency (%)∗ hES hES-EB

hES-RA

0.9 7.5 0.0 6.1 0.0 2.8 1.4 19.7 0.5 0.5 11.3 1.4 37.6 0.9 5.6 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.5 2.8 100

12.9 37.1 0.0 3.0 0.5 4.0 0.0 21.3 1.5 0.0 1.0 0.0 6.9 0.0 0.5 0.0 0.5 0.5 0.0 2.0 0.0 5.0 3.5 0.0 100

4.5 4.0 0.0 1.5 0.5 15.5 0.5 19.5 1.0 0.0 6.5 1.0 40.0 0.5 0.0 0.0 0.0 0.0 1.0 0.0 0.5 1.5 2.0 0.0 100

PE† ¼ NF ¼ ¼ NF ¼ ¼ ¼ ¼ ¼

Frequency (%)∗ hFF hiPS

hiPS-EB

hiPS-RA

17.9 3.8 1.4 0.0 0.9 11.3 0.0 3.8 0.0 0.9 3.3 0.0 38.2 1.4 0.0 0.0 1.4 3.8 0.0 0.0 0.5 2.8 8.5 0.0 100

7.7 22.5 0.0 3.5 0.0 6.3 0.7 22.5 0.7 0.0 4.9 0.0 16.2 0.0 2.1 0.0 0.7 0.7 0.0 0.0 0.0 4.9 6.3 0.0 100

15.7 45.2 0.0 2.4 0.6 1.8 0.0 19.9 0.6 0.0 0.6 0.0 1.8 0.0 0.6 0.0 0.0 0.6 0.0 0.0 0.0 6.0 4.2 0.0 100

1.5 13.2 0.0 7.8 0.0 0.0 0.5 17.6 1.0 0.0 26.8 0.0 23.4 0.0 5.4 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 2.0 100

∗ The †

frequency of each kinase was evaluated by dividing the number of clones of a kinase gene by the number of total clones from each sample. PE indicates the patterns of expression of a gene in undifferentiated hPSC as follows; , up-regulated; , down-regulated; ¼, not differentially expressed; NF, not found.

PE† NF ¼ ¼ NF NF NF ¼ NF NF NF


AXL DDR1 DDR2 EPHA1 EPHA2 EPHA4 EPHA7 EPHB2 EPHB4 ERBB1 ERBB2 ERBB3 FGFR1 FGFR2 FGFR4 IGF1R MERTK HGFR MST1R NTRK3 PDGFRA PDGFRB TYRO3 VEGFR2

RTK family

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pluripotent state. To generate hiPSCs, hFFs were virally infected with the four canonical reprogramming factors, OSKM, in either the presence or the absence of an AXL inhibitor under the feederfree conditions depicted in Figure 5A. The reprogramming efficiency was assessed by the numbers of AP+ hESC-like colonies at day 18 after re-plating on Matrigel (Fig. 5A). Warfarin significantly promoted the reprogramming efficiency up to 17.7-fold, with the most effective concentration being 0.5 mM (Fig. 5B); 1 nM of R428 also allowed up to a 10.7-fold increase in reprogramming efficiency compared with the untreated controls (Fig. 5B). We also found that tightly packed colonies with well-defined borders appeared 6 or 4 days earlier in warfarin- or R428-treated cells than in untreated controls, respectively. hESClike iPSC colonies were selected at approximately day 13 or 16 after transduction in reprogramming conditions with warfarin or R428, respectively, while iPSC colonies were picked at day 23 after transduction in the untreated control group, indicating

improved reprogramming kinetics by treatment with the AXL inhibitors (Fig. 5C). Warfarin-derived hiPSCs (W-iPSC) and R428-derived hiPSCs (R-iPSC) were highly expressed pluripotency markers, including OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81 (Fig. 5D). Genomic DNA PCR revealed that all four transgenes (OSKM) were integrated in the W-iPSC and R-iPSC lines tested (Fig. 5E). W-iPSCs and R-iPSCs displayed a normal karyotype after prolonged culturing (Fig. 5F). To confirm the pluripotency of the W-iPSCs and R-iPSCs, differentiation potentials were verified in vitro and in vivo. Immunocytochemical analyses for endoderm (SOX17 and a-FP), mesoderm [DESMIN and a-smooth muscle actin (a-SMA)] and ectoderm (TUJ1 and NESTIN) markers confirmed successful in vitro differentiation of W-iPSCs and R-iPSCs (Fig. 5G). After transplantation into nude mice, W-iPSCs and R-iPSCs formed teratomas consisting of representative derivatives of all three germ layers, including gut like epithelium


Figure 2. Expression analysis of selected RTK genes in hPSCs. The expression patterns of RTK genes detected by gene frequency analysis were analyzed by real-time quantitative polymerase chain reaction (qPCR) using gene-specific primers. Four RTK genes, EPHA1, ERBB2, FGFR4 and VEGFR2, were consistently up-regulated in undifferentiated hPSCs, such as hESCs and hiPSCs (A), whereas four RTK genes, AXL, EPHA4, PDGFRb and TYRO3, were consistently down-regulated in undifferentiated hPSCs (B). Undifferentiated hESCs and hiPSCs were used as a control. hPSCs, such as hESCs and hiPSCs, were induced to differentiate using EB formation or RA treatment. Fold changes of expression levels are relative to hESCs or hiPSCs. ∗ P , 0.05 and ∗∗ P , 0.01, by the t-test.


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(endoderm), myxoid tissue and adipocytes (mesoderm) and neural rosettes (ectoderm) (Fig. 5H). These results demonstrated that W-iPSCs and R-iPSCs are able to differentiate into three germ layers in vitro and in vivo. Collectively, our results suggest that the inhibition of AXL increased both the efficiency and kinetics of human somatic cell reprogramming. AXL-modulated phosphoprotein dynamics are closely linked to the pluripotent signaling networks For a broader understanding of how AXL inhibition is related to the signaling pathways that govern self-renewal capacities and pluripotency, we attempted to map the signaling networks that respond to AXL inhibition using a quantitative phosphoproteomic approach to the hESCs. Our quantitative phosphoproteomics experimental strategy using a TiO2 phosphopeptide enrichment method is shown in Figure 6A. The levels of AXL phosphorylation in hESCs treated with an AXL inhibitor (1 nM of R428) rapidly decreased (within 30 min) to 6.0% of the untreated control levels (Fig. 6B). In control hESCs, 1858 phosphopeptides were found, which corresponded to 665 phosphoproteins (Fig. 6C and Supplementary Material, Table S2). In R428-treated hESCs, 2813 phosphopeptides were found, which corresponded to 826 phosphoproteins (Fig. 6C and Supplementary Material, Table S2). Among all the identified phosphoproteins, we found that 30.21% (293 of 970 phosphoproteins) of the phosphoproteins in the R428-treated hESCs was significantly altered based on a 2-fold up- or down-regulation cut-off level. While 76 proteins showed a significant increase (.2-fold) in phosphorylation, 217 proteins showed a significant decrease (,0.5-fold). The 293 identified phosphoproteins that were up- or downregulated by the AXL inhibitor R428 were classified according to their Gene Ontology descriptions using information from the Gene Ontology and PANTHER classification systems. The phosphoproteins identified in our study are related to a variety of biological processes, including the metabolic processes, cellular processes, cell communication, developmental processes, cell

cycle, cellular component organization, system process, apoptosis, cell adhesion and others (Fig. 6D). A significant number of these proteins were attributed with biological functions that are involved in self-renewal and pluripotency, such as cellular processes (18.1%), developmental processes (7.7%), cell cycle (7.3%) and apoptosis (3.6%). To further understand the relationship between the 293 identified proteins and the possible roles of inhibition of AXL, we performed pathway and network analysis using both the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Ingenuity Pathway Analysis (IPA) databases. Supplementary Material, Table S3, lists the seven most significantly enriched KEGG pathways upon AXL inhibition. We found that several signaling pathways [adherens junction (hsa04520), nucleotide-binding oligomerization domain-like receptor signaling pathway (hsa04621), spliceosome (hsa03040), DNA replication (hsa03030), insulin signaling pathway (hsa04910), ubiquitinmediated proteolysis (hsa04120) and progesterone-mediated oocyte maturation (hsa04914)] were significantly altered. The IPA program identified networks that differed slightly from the pathways that were identified in the KEGG analysis. The five canonical pathways of differentially phosphorylated proteins after AXL inhibition that were found with IPA were the protein ubiquitination pathway, EIF2 signaling, semaphorin signaling in neurons, PPAR signaling and DNA double-strand break repair by non-homologous end joining (Supplementary Material, Table S4). Next, we analyzed whether inhibiting AXL could activate the intracellular signaling networks that are important for maintaining hPSCs. Recent works have established that a fine-balanced signaling network involving cross-talk between the PI3K/AKT, ERK, Wnt and TGFb pathways is critical for sustaining hPSC selfrenewal and pluripotency (7,21). In relation, the downstream signaling proteins of those signaling pathways were differentially phosphorylated in response to AXL inhibitor treatment (Fig. 6E). When compared with the untreated cells, ERK was increased in phosphorylation (2.25-fold), whereas GSK3b and TAK1 (MAP3K7) were decreased in phosphorylation


Figure 3. Differential expression of phosphorylated RTKs in hPSCs. Human phospho-RTK arrays were used to examine RTK phosphorylation levels in lysates from undifferentiated hESCs (UNDIFF) as a control and from differentiated hESCs (DIFF). Each array was identically exposed to detection reagents and film. EPHA1, ERBB2, FGFR4 and VEGFR2 showed increased phosphorylation levels in undifferentiated hESCs (A), whereas the induction in hESC differentiation was accompanied by the increased phosphorylation of AXL, EPHA4, PDGFRb and TYRO3 (B). A representative array from two independent experiments (top) was quantified using densitometry (bottom). Mean pixel density + SE of duplicate spots, normalized against duplicate phosphotyrosine-positive control spots ¼ 100. ∗ P , 0.05 and ∗∗ P , 0.01, by the t-test.

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Figure 4. Effect of AXL inhibition on the maintenance of pluripotency in hPSCs. hESCs cultured with MEF-conditioned medium (CM) or unconditioned medium (UM) containing the indicated chemicals in feeder-free condition for 6 days. (A) Representative scanned images of alkaline phosphatase (AP)-stained 35 mm culture dishes at day 6 (top). Relative AP expression measured by scanning densitometry of AP-stained hESCs, normalized to CM control (bottom). (B) Real-time qPCR analysis for the expression of OCT4, NANOG and REX1. The results are displayed as the mRNA level relative to the CM control. The data are presented as the means + SE (n ¼ 3). ∗ P , 0.05 and ∗∗ P , 0.01 compared with UM. (C) Immunocytochemical analysis of OCT4, SSEA-4 and TRA-1-60. hESCs were cultured in the presence of 0.5 mM warfarin or 1 nM R428. Nuclei were visualized with DAPI. Bar ¼ 500 mm. (D) Effect of AXL inhibition on the growth of hESCs. The growth curve was obtained by trypan-blue staining and counting cells. The data are presented as the mean + SE (n ¼ 3). ∗∗ P , 0.01, by the t-test. RT– PCR (E) and immunocytochemical analysis (F) of the expression of markers of the three germ layers in EBs induced in the presence of 0.5 mM warfarin or 1 nM R428. Bar ¼ 50 mm.


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Figure 5. Inhibiting AXL promotes the reprogramming of human somatic cells into iPSCs. (A) Schematic diagram of the reprogramming protocol used. (B) The AXL inhibitors enhance reprogramming efficiency. hFFs were reprogrammed with retroviruses encoding the transcription factors OCT4, SOX2, KLF4 and c-MYC in the presence of the indicated chemicals under feeder-free conditions. Representative images of AP+ iPSC colonies (top); the number of AP+ colonies was counted at day 23 (bottom). (C) The AXL inhibitors promote reprogramming kinetics. The reprogramming kinetics was represented as the number of days required for colony selection. The data are presented as the mean + SE (n ¼ 3). ∗∗ P , 0.01 compared with untreated control. (D) Immunocytochemical staining for pluripotency markers. Nuclei were stained with DAPI. Bar ¼ 500 mm. (E) RT –PCR analysis showing the genomic integration of the exogenous factors. (F) Karyotype analysis of W-hiPSC and R-hiPSCs. (G) In vitro differentiation of W-hiPSC and R-hiPSCs. Immunocytochemical analyses of various differentiation markers for the three germ layers: with endoderm (SOX17 and a-FP), mesoderm [DESMIN and a-smooth muscle actin (a-SMA)] and ectoderm (TUJ1 and NESTIN). Nuclei were stained with DAPI (blue). Scale bar, 100 mm. (H) In vivo differentiation assay using a teratoma formation. The histological analysis of teratoma derived from W-hiPSC and R-hiPSCs by hematoxylin and eosin staining. Teratomas of W-hiPSC and R-hiPSCs differentiated into all three germ layers: endoderm (gut-like epithelium), mesoderm (myxoid tissue and adipocytes) and ectoderm (neural rosettes).

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(0.50-fold and 0.46-fold, respectively). Additionally, RIF1, a telomere-binding protein, was increased in phosphorylation (2.72-fold) (Fig. 6E), suggesting that telomere length is also influenced by AXL inhibition. Notably, many of the ubiquitinproteasome system (UPS) members were also differentially

phosphorylated upon R428 treatment, suggesting possible links to pluripotency regulators, including OCT4, NANOG and SOX2, which are modified by ubiquitination (22). In addition, we found that the structural components of 26S proteasome (PSMD1 and PSMD2, 0.37-fold and 0.46-fold, respectively),


Figure 6. AXL inhibition plays a role in the maintenance of pluripotency based on the phosphoproteome analysis. (A) Flow diagram of the quantitative phosphoproteomics experiment. (B) Western blot analysis of AXL phosphorylation levels after R428 treatment. Protein bands were quantified via densitometry (right). (C) A Venn diagram of control and R428-stimulated phosphorylated proteins in hESCs. (D) A pie chart of the functional distribution of R428-stimulated phosphoproteins based on biological processes (Gene Ontology Consortium). Proteins involved in cellular process and developmental process were further classified. (E) The observed differential phosphorylation upon R428 treatment in multiple signaling pathways plays an important role in maintaining pluripotency. The phosphoproteins that were identified in this study are indicated in bold. (F) Downstream targets of the core transcriptional regulatory circuitry regulated by phosphorylation. A Venn diagram showing the overlap of OCT4, NANO and SOX2 targets that were differentially phosphorylated upon R428 treatment. Increased phosphorylation is shown in red (.2-fold), whereas decreased phosphorylation is shown in blue (,0.5-fold) (E and F).


DISCUSSION In this study, we identified 23 RTKs that were differentially expressed in undifferentiated hESCs or hiPSCs and differentiated cells derived from those cells. We also simultaneously assessed the phosphorylation status of 42 different RTKs using human phospho-RTK array analysis. To our knowledge, this study is the first time that the differential expression and phosphorylation patterns of RTKs have been characterized in hPSCs, such as hESCs and hiPSCs. Previous studies have emphasized the important roles of RTKs and their specific ligands on the modulation of hPSC selfrenewal and pluripotency. Xu et al. (20) determined that bFGF/ FGFR signaling is critical for hESC self-renewal. IGF1R, which is activated by IGF2, appears to play a role in maintaining pluripotency in hESCs (1). PDGF promotes intracellular S1P signaling by activating sphingosine kinase (SPK), which in turn converts sphingosine to S1P. The combination of exogenous PDGF and S1P maintains hESCs in the undifferentiated state through the activation of ERK and S1P/SPK (18). Neurotrophins are a family of peptide growth factors that includes nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5, which bind to their respective TrkRs (24). The addition of neurotrophins to hESC cultures can increase the clonal survival of hESCs and improve their growth (3). However, relatively little is known regarding which RTKs and downstream pathways are activated by corresponding ligands and whether the activation of RTKs by ligands governs the cell fate choices of hPSCs. It is expected that global gene expression profiling studies of hESCs may identify critical genes involved in the regulation of pluripotency and the differentiation of hPSCs. Data obtained

during the past few years have shown that several genes and mechanisms are recognized as key participants in pluripotency and differentiation of hPSC through the transcriptional profiling of various stem cell populations using microarrays (9,11,25– 27). Based on degenerate PCR and phosphoprotein array analyses in this study, we determined that the differential distinctive expression of RTKs in hESCs and hiPSCs is dependent on pluripotency. EPHA1, ERBB2, FGFR4 and VEGFR2 transcripts and phosphoproteins were commonly enriched in undifferentiated hESCs and hiPSCs, whereas AXL, EPHA4, PDGFRB and TYRO3 were commonly enriched in differentiated cells. These results are in agreement with those of previous studies (11,16), indicating the high expression of ERBB2 and FGFR4 in undifferentiated hESCs. To more comprehensively analyze the expression of RTKs across all human cell types, we compared our data with the existing transcriptome data in undifferentiated hPSCs and in various human tissues using the Amazonia expression atlas (http://amazonia.montp.inserm.fr) (25,28) (Supplementary Material, Table S5). We found that the up-regulated RTKs, such as EPHA1, ERBB2, FGFR4 and VEGFR2, are indeed predominantly expressed in undifferentiated hPSCs compared with normal adult tissues, whereas AXL, EPHA4 and PDGFRB, which are down-regulated RTKs, are relatively highly expressed in differentiated tissues. However, TYRO3 is expressed in both undifferentiated hESCs and hiPSCs as well as in neural tissue. These differential expression patterns of RTKs likely reflect the developmental stage-specific and tissue-specific regulation of the corresponding genes. Although transcriptome data are useful, it has been shown that mRNA levels partially reflect the state of the cells, such as the specific point in the cell cycle, and mRNA levels do not always correlate with protein abundance and activity (26,29,30). Therefore, a combined transcriptome and proteome analysis is a powerful approach in studying the key factors and mechanisms underlying hPSC pluripotency and differentiation. In particular, our study highlights AXL as a new potential target of RTKs that contributes to hESC self-renewal and pluripotency. AXL is an RTK member of the Axl family, which includes AXL, TYRO3 (or SKY) and MER, and was originally discovered in patients with chronic myelogenous leukemia (31). AXL exhibits transforming potential and is overexpressed in a variety of human cancers (32,33). AXL and Gas6 (growth arrest-specific protein 6), the biological ligand of AXL, are involved in various cellular responses, including proliferation, cell survival, migration and angiogenesis; these activities require the concurrent activation of the RAS/RAF/MAPK and PI3K/AKT/S6K signaling cascades (34–38). A recent report showed that the AXL-Gas6 pathway functions in lineage-specific differentiation programs of many tissue and cell types, such as adipogenesis, differentiation into glial progenitor cells, maturation of natural killer cells and osteogenic differentiation (39–42). However, to date, the role of AXL has not been investigated in hPSCs. We are the first group to show that AXL is expressed at high levels in differentiated hESCs and hiPSCs and that the activity of AXL, as measured by its phosphorylation, also increases upon differentiation, suggesting a role in actively promoting the differentiation of hPSCs. Thus, our results support the new hypothesis that inhibiting AXL helps maintain and regain hPSC pluripotency by inhibiting the AXL-activated, differentiation-inducing signaling pathway. We provide evidence that the use of either warfarin or R428, which are specific AXL inhibitors, to hPSC cultures is greatly


ubiquitin C-terminal hydrolases (USP14 and USP42, 0.46-fold and 0.35-fold, respectively), a ubiquitin-specific peptidase (USP10, 0.37-fold), a ubiquitin-conjugating enzyme (UBE2J1, 0.19-fold) and an E3 ubiquitin-protein ligase (NEDD4L, 0.45-fold) showed decreased levels of phosphorylation, whereas a ubiquitin conjugation factor (UBE4B, 3.84-fold) and a ubiquitin C-terminal hydrolase (USP8, 7.03-fold) showed increased levels (Fig. 6E). These results suggest that multiple signaling pathways may cooperate with the AXL pathway to contribute to pluripotency control. To further investigate whether inhibiting AXL affected any downstream targets of the core transcription factors, OCT4, NANOG and SOX2, we mapped our phosphoproteome data to the chromatin immunoprecipitation data sets reported by Boyer et al. (23). Of the 293 differentially regulated phosphoproteins, 30 proteins were encoded by target genes of at least one of these transcription factors (Fig. 6F). Interestingly, these phosphorylated proteins are predominantly transcription factors (e.g. SALL2, PRDM14, HDGF and SLC4A1AP), other DNA-binding proteins (e.g. EIF2A, UBP10, ACIN1, RIF1 and TOP2A), signaling molecules (e.g. SEMA6A and TRIP10), cell cycle regulators (e.g. BUB1B and WEE1) and cytoskeletal remodeling proteins (e.g. ADD3, DNM2, MAGED2 and PARD3). Therefore, the observed changes in phosphorylation of the 30 proteins that are controlled by OCT4, NANOG and SOX2 reflect the importance of AXL signaling in pluripotency.

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self-renewal network are controlled by the interplay of kinases and phosphatases (38). The activities of the core transcription factors, including OCT4, SOX2 and NANOG, and their downstream targets were found to be regulated by phosphorylation upon differentiation signals (e.g. BMP4) or the addition of pluripotency-inducing signals (e.g. FGF-2) (27,38). These reports imply that changing the state of hESCs is accompanied by alterations in the phosphorylation levels of many components of the core transcriptional circuitry in hESCs. Interestingly, by mapping the AXL-regulated phosphoproteins to chromatin immunoprecipitation data that determined the downstream targets of OCT4, SOX2 and NANOG, we found that 10.24% (30 proteins) of the 293 regulated phosphoproteins were targets of at least one of those core transcription factors (23). This result strongly suggests that the AXL-dependent signaling pathway may directly or indirectly link the core transcriptional regulatory circuitry in hPSCs to maintain the undifferentiated state of hESCs, while AXL may negatively regulate the OCT4/ SOX2/NANOG complex to maintain undifferentiated hPSCs. This hypothesis was supported by the observation that previously reported stem cell-associated factors, including SALL2, RIF1, SPAG9, PRDM14, TOP2A, WEE1, RNF31 and SEMA6A, were also identified among the phosphoproteins in our study. SALL2, SPAG9 and SEMA6A are used as markers of pluripotency (54–56). The knockdown of RIF1 leads to the differentiation of ESCs (52), whereas the loss of RNF31 induces pluripotency-associated gene expression (51). PRDM14 expression is important for maintaining naive pluripotency by regulating DNA methylation (57,58). The appropriate cell cycle progression by cell cycle regulators, such as TOP2A and WEE1, is essential for maintaining pluripotency (59,60). Although the mechanism behind the differential phosphorylation status of various residues in the 30 proteins is not well understood in hESCs, the observed overall alterations in the phosphorylation status of these proteins, regardless of whether phosphorylation is increased or decreased, may underlie the change in hESC state. Therefore, establishing the connection between quantitative differences in the phosphorylation of specific sites of downstream targets upon R428 treatment and the regulation of pluripotency is the next critical step in elucidating the AXL-mediated core transcriptional regulatory networks involved in the control of hESC identity and reprogramming. The further identification of this novel pathway will greatly benefit our understanding of hPSCs at the molecular level and potentially lead to the discovery of novel targets for pluripotency and reprogramming.

CONCLUSION Our study defines the distinctive RTK profiles and signatures of hPSCs and provides valuable insights into pluripotency-related signaling networks. Importantly, we propose the pharmacological antagonism of AXL receptors as a potentially novel strategy for improving the maintenance of hPSC self-renewal and pluripotency and enhancing the reprogramming process of human somatic cells into hiPSCs under feeder-free, chemically defined conditions. We also determined that blocking the AXLdependent pathway mediated key changes in the components of multiple pathways related to hPSC pluripotency, such as ERK, WNT, BMP, RIF1, components of the UPS and downstream


beneficial for blocking differentiation, improving the undifferentiated growth of hPSCs, eliminating the use of feeder cells and animal serum and enabling large-scale propagation. hESCs cultured in warfarin- and R428-containing medium successfully retained their pluripotent differentiation potential and self-renewal after long-term cultures. Consistent with our results on AXL expression, a high expression level of Gas6, the endogenous ligand for AXL, was observed in various differentiated cell types compared with undifferentiated hESCs and hiPSCs in a meta-analysis of 38 different array experiments (http://amazonia.montp.inserm.fr/) (28). However, no studies have reported that Gas6 could induce hESCs or hiPSCs to differentiate into specific cell types. Therefore, further investigations that determine whether the differentiation of hPSCs can be thus driven and which differentiated cell types could be generated by AXL activation through its ligand Gas6 are required to clarify these issues. Apart from promoting the undifferentiated growth of hPSCs in vitro, the AXL inhibitors warfarin and R428 are also enhancers of human somatic cell reprogramming to hiPSCs. The addition of AXL inhibitors enhances both the overall efficiency of iPSC generation and the kinetics of reprogramming. During the reprogramming of fibroblasts to iPSCs, fibroblasts must be converted into an epithelial phenotype via mesenchymal-to-epithelial transition (MET) (43,44). Several studies have shown that inhibiting the epithelial-mesenchymal transition (EMT), i.e. reversing it to MET, greatly increases reprogramming efficiency (4,45,46), suggesting that MET is an important cellular event during reprogramming. Similarly, a recent study demonstrated that AXL is a unique EMT effector that is essential for breast cancer progression (47). In addition, AXL functions as a downstream effector of TGFb1 signaling, which inhibits iPSC generation by inducing EMT (48). Thus, our results suggest that inhibiting AXL signaling facilitates hiPSC generation, possibly through suppressing EMT. The improved maintenance and re-acquisition of pluripotency acquired by inhibiting AXL signaling are certainly the results of altered intracellular phosphorylation signaling linked to pluripotency and differentiation. Here, we used LC-MS/MS-based quantitative proteomics to monitor dynamic alterations in the phosphoproteome of hESCs upon AXL inhibition. Changing the status of hESCs by R428 treatment, a specific AXL inhibitor, was immediately followed by a dynamic rearrangement of the phospho-signaling pathways. Within 30 min of R428 treatment, approximately one-third of the phosphoproteome patterns, including major components of the canonical pathways that support self-renewal and pluripotency, such as ERK, GSK3 and TAK1, were changed in hESCs. We also found that AXLmediated changes in pluripotency-related factors, such as RIF1, regulated telomere length (49) and UPS machinery, restricting protein abundance (50,51). These results are in agreement with previous studies that have shown that telomere length is associated with developmental pluripotency (52,53). UPS is important in pluripotency and reprogramming in stem cells by controlling levels of key pluripotency factors (50,51). The top-ranked differentially phosphorylated proteins affected by R428 (Supplementary Material, Table S6) were assumed to be alternate biologic targets for AXL-mediated functions in hPSCs. In hESCs, the phosphorylation status of pluripotency-associated proteins dramatically changes during differentiation, suggesting that the activities of many proteins involved in the pluripotency and


components of core transcription factors (Oct4, Nanog and Sox2). We demonstrate that the RTKs presented here are promising candidates that pave the way to understanding the molecular circuitry of pluripotency and reprogramming and advancing technologies for hPSC applications in regenerative medicine and drug discovery.

MATERIALS AND METHODS Reagents

hPSC culture hESC line H9 (NIH code, WA09; WiCell Research Institute, Madison, WI, USA), H1 (NIH code WA01), HUES7 (Harvard University, Cambridge, MA, USA) and the hiPSC lines were routinely maintained as described previously (37). For feeder-free cultures, hPSCs were grown on plates coated with MatrigelTM (BD Biosciences) in MEF-CM, UM or mTeSR1 medium (StemCell Technologies, Vancouver, Canada). MEF-CM was prepared using g-irradiated MEFs as previously described (61) and was supplemented with 8 ng/ml bFGF before use. The cultured hPSCs were passaged with 1 mg/ml collagenase IV (Invitrogen, Carlsbad, CA, USA) treatment every 6–7 days. All cultures were routinely screened for mycoplasma, and a normal karyotype was monitored by chromosomal G-band analysis using ChIPSKaryo (Chromosome Image Processing System, GenDix, Inc.). Differentiation of hESCs hPSCs were differentiated by direct treatment with 1 mM RA (Sigma) for 5 days. The culture medium was changed daily. RA was added every day to fresh culture medium. EBs were generated as described previously (62). Briefly, 7-day-old hPSCs were dissociated into small clumps with dispase (Invitrogen), which allowed cells to aggregate on the non-tissue culture-treated plastic Petri dish in human embryoid body medium consisting of knockout Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen), 20% fetal bovine serum (FBS, Invitrogen), 1% nonessential amino acids (NEAA), 1 mM L-glutamine, 0.1 mM b-mercaptoethanol and 1% penicillin/streptomycin.

were used in previous studies at an annealing temperature of 428C for five cycles and then at 508C for 25 cycles (15). The 150–170 bp PCR products were purified with QIAquick Gel Extraction Kit (Qiagen) and subcloned into a T-vector (Promega, Madison, WI, USA). Randomly selected positive clones were further purified with the Plasmid High Throughput DNA Prep Kit (Core Bio System, Seoul, Korea) and used for auto-sequencing analysis using an ABI Prism 3700 DNA analyzer (PE Applied Biosystems, Foster City, CA, USA). The individual sequences were analyzed using BLASTN with default parameters against the GenBank database from the National Center for Biotechnology Information. The frequency for each kinase was analyzed by dividing the number of clones for the kinase gene by the number of total clones of human origin from each sample. Human phospho-RTK arrays The activation of RTKs and their downstream signaling pathways during differentiation were analyzed using the Proteome Profiler Array Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. Briefly, RTK array membranes were blocked, incubated with 200 mg of total protein from hPSCs overnight at 48C, washed and incubated with horseradish peroxidaseconjugated anti-phosphotyrosine for 2 h at room temperature. The membranes were washed again before development with ECL western blotting detection reagent (GE Healthcare), and RTK spots were visualized using Kodak BioMax film (Kodak, Rochester, NY, USA). The level of phosphorylated RTK was densitometrically quantified with Image Gauge software (Fuji Photo Film GMBH) and normalized to the internal phosphotyrosine-positive control spots. hiPSC generation Human newborn foreskin fibroblasts (hFFs, catalog number CRL-2097; ATCC, Manassas, VA, USA) were maintained in DMEM containing 10% FBS (Invitrogen), 1% NEAA, 1 mM L-glutamine (Invitrogen) and 0.1 mM b-mercaptoethanol (Sigma). To generate iPSCs, hFFs (1 × 105 cells/well) were transduced with pMX-based retroviruses encoding human OCT4, SOX2, KLF4 and c-MYC (Addgene Inc., Cambridge, MA, USA). Retrovirus production was performed as described in the supplementary materials and methods. Four days after transduction, hFFs were replated in Matrigel-coated 6-well dishes (5–6 × 104 cells/well). The following day, the medium was replaced with MEF-CM supplemented with 10 ng/ml bFGF. The medium was changed every other day. In-gel digestion and phosphopeptide enrichment

Differential display analysis for evaluating the RTK profile in hPSCs Total RNA was isolated from cells with an RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse-transcribed with a SuperScript First-strand Synthesis System Kit (Invitrogen) according to the manufacturer’s instructions. The degenerate PCR primer sequences were based on the conserved DFG and DVW motifs of the tyrosine kinase catalytic domains VII and IX. The primers are listed in Supplementary Material, Table S7. The PCR was performed using the degenerate PCR primer sets that

Each 250 mg hESC lysate was digested using a trypsin in-gel digestion procedure shortly following separation by sodium dodecyl sulphate– polyacrylamide gel electrophoresis. The bands were manually excised, and the gel pieces were destained and washed prior to in-gel digestion. In-gel digestion was performed with 250 ng/ml sequencing grade modified trypsin (Promega) dissolved in 50 mM NH4HCO4 buffer (pH 7.8) at 378C overnight. The digested peptides were extracted using 5% formic acid in acetonitrile, and the supernatant was dried in a Speed-Vac and stored at 2208C.


Selective AXL inhibitors, warfarin (Sigma, St Louis, MO, USA) and R428 (Synkinase, Shanghai, China) were dissolved in dimethyl sulfoxide (Sigma) to a final concentration of 10 mM and stored as aliquot in the deep-freezer (2708C). Prior to use, they were diluted with culture medium to yield the desired concentrations.

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Analysis by nano-LC-ESI-MS/MS The proteolytic peptides were loaded onto a fused silica microcapillary column (50 cm × 75 mm, PepMapw) packed with ˚ ). LC separation was conC18 reversed phase resin (2 mm, 100 A ducted under a linear gradient of 3 – 50% solvent B (ACN containing 0.1% formic acid) combined with solvent A (DW containing 0.1% formic acid) at a flow rate of 250 nl/min for 60 min. The column was directly connected to a Q Exactive mass spectrometer (Finnigan, CA) equipped with a nanoflow high-performance liquid chromatography instrument (EasynLC, Thermo Fisher Scientific). Each full MS data set was acquired using a data-dependent top eight method that dynamically selected the most abundant precursor ions from the survey scan (300 – 2000 Da) for higher-energy collisional dissociation fragmentation. The dynamic exclusion duration was 15 s, and the isolation window of the precursor was performed with two. The survey scans were acquired at a resolution of 70 000 at m/z 200, and the resolution for HCD spectra was set to 17 500 at m/z 200. Data analysis The acquired LC-electrospray ionization (ESI)-MS/MS fragment spectra were searched in the Proteome Discoverer (version 1.3) software against the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) nonredundant human database. The searching conditions were trypsin enzyme specificity; a permissible level for two missed cleavages; respective precursor and fragment mass tolerances of 10 ppm and 0.8 Da; a 1% false discovery rate; and variable modifications of carbamidomethylation of cysteine (+57 Da), oxidation of methionine (+16 Da) residues and phosphorylation of serine, threonine or tyrosine (+80 Da).

Bioinformatic analysis Gene Ontology (http://www.geneontology.org) and KEGG analyses were performed using David (http://david.abcc.ncifcrf. gov/content.jsp?file=about_us.html), STRING (http://string-db. org/) and ClueGO. The associations of regulated genes with specific biological processes were assessed using PANTHER (Protein ANalysis THrough Evolutionary Relationships, http:// www.pantherdb.org/). To perform functional enrichment tests of the candidate genes, we used the Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood, CA) system for both canonical pathways and molecular networks. Statistical analysis All data are expressed as the mean + SEM. Differences between groups were analyzed using Student’s t-test. A P-value of ≤0.05 was considered significant. All data are representative of at least three independent experiments.

SUPPLEMENTARY MATERIAL Supplementary Material is available at HMG online. Conflict of Interest statement. None declared.

FUNDING This work was supported by the grant from the Korean Ministry of Health and Welfare (A084697). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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After digestion, the digested peptides were acidified with trifluoroacetic acid and subjected to desalting using C18 tips (catalog no. 87784, Pierce). After elution from the C18 resin, the sample was dried and then re-dissolved in B buffer (57% acetonitrile (ACN)/26% lactic acid), as required for the TiO2 phosphopeptide enrichment kit (catalog no. 88301, Pierce). The TiO2 phosphopeptide enrichment procedure, which enriches the proteolytic digests for phosphopeptides, was performed with the Phosphopeptide Enrichment Kit (catalog no. 88301, Pierce). Each group of peptide digests was applied to the column, and the phosphopeptides were bound by incubation at room temperature for 10 min four times. The column was washed five times with each of the following solutions: 20 ml of buffer B (57% ACN/26% lactic acid) and 20 ml of buffer A (80% ACN). The phosphopeptides were eluted from the TiO2 column with 50 ml of 5% NH4OH, followed by elution with 50 ml of 50% ACN. The eluates were combined, and the resulting sample was acidified and dried. Prior to LC-MS/MS analysis, the TiO2-enriched phosphopeptides were desalted with PepClean C18 spin columns (Thermo Scientific, Rockford, IL, USA) using the manufacturer’s protocol. The phosphopeptides that bound to the PepClean C18 spin columns were eluted with 20 ml of 70% ACN/0.1% formic acid, dried in a Speed-Vac and stored at 2208C until they were injected into the LC-MS/MS instrument.


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Kinome-wide shRNA screen identifies the receptor tyrosine kinase AXL as a key regulator for mesenchymal glioblastoma stem-like cells.

AXL receptor tyrosine kinase as a therapeutic target in NSCLC.

Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase.

Axl receptor tyrosine kinase expression in breast cancer.

Axl receptor tyrosine kinase is a potential therapeutic target in renal cell carcinoma.

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

AXL receptor tyrosine kinase is required for T cell priming and antiviral immunity.

Comparative proteomic profiling identifies potential prognostic factors for human clear cell renal cell carcinoma.

The Axl receptor tyrosine kinase is a discriminator of macrophage function in the inflamed lung.

Deep transcriptome profiling of mammalian stem cells supports a regulatory role for retrotransposons in pluripotency maintenance.

The role of AXL and the in vitro activity of the receptor tyrosine kinase inhibitor BGB324 in Ewing sarcoma.

AXL kinase as a novel target for cancer therapy.

The receptor tyrosine kinase AXL promotes migration and invasion in colorectal cancer.

Targeting the degradation of AXL receptor tyrosine kinase to overcome resistance in gefitinib-resistant non-small cell lung cancer.

The AXL receptor tyrosine kinase is associated with adverse prognosis and distant metastasis in esophageal squamous cell carcinoma.

Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene.

A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor.

β signaling by Axl receptor tyrosine kinase modulates HBV immunity.

A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations.

Potassium as a pluripotency-associated element identified through inorganic element profiling in human pluripotent stem cells.

Human-Mouse Chimerism Validates Human Stem Cell Pluripotency.

Receptor tyrosine kinase Axl is required for resistance of leukemic cells to FLT3-targeted therapy in acute myeloid leukemia.

A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells.

Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis.