m6A RNA methylation regulates the transcription factors JUN and JUNB in TGF-ß-induced epithelial-mesenchymal transition of lung cancer cells.

N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3'UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta-induced EMT of lung cancer cells.

ASH2L, a COMPASS core subunit, is involved in the cell invasion and migration of triple-negative breast cancer cells through the epigenetic control of histone H3 lysine 4 methylation.

ASH2L (Absent-Small-Homeotic-2-Like protein) is a core subunit of the COMPASS (COMplex of Proteins ASsociated with Set1) complex, the most notable writer of the methylation of histone H3 lysine 4 (H3K4). The COMPASS complex regulates active promoters or enhancers for gene expression, and its dysfunction is associated with aberrant development and disease. Here, we demonstrated that ASH2L mediated the cell invasion and migration activity of triple-negative breast cancer cells through the interaction with the COMPASS components and the target genomic regions. Transcriptome analysis indicated a potential correlation between ASH2L and the genes involved in inflammatory/immune responses. Among them, we found that the intrinsic expression of IL1B (interleukin 1 beta), an essential proinflammatory gene, was directly regulated by ASH2L. These results revealed a novel role of ASH2L on the maintenance of breast cancer malignancy possibly through H3K4 methylation of the target inflammatory/immune responsive genes.

KDM2B is involved in the epigenetic regulation of TGF-ß-induced epithelial-mesenchymal transition in lung and pancreatic cancer cell lines.

Polycomb repressive complex-1 (PRC1) induces transcriptional repression by regulating monoubiquitination of lysine 119 of histone H2A (H2AK119) and as such is involved in a number of biological and pathological processes including cancer development. Previously we demonstrated that PRC2, which catalyzes the methylation of histone H3K27, has an essential function in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung and pancreatic cancer cell lines. Since the cooperative activities of PRC1 and PRC2 are thought to be important for transcriptional repression in EMT program, we investigated the role of KDM2B, a member of PRC1 complex, on TGF-ß-induced EMT in this study. Knockdown of KDM2B inhibited TGF-ß-induced morphological conversion of the cells and enhanced cell migration and invasion potentials as well as the expression changes of EMT-related marker genes. Overexpression of KDM2B influenced the expression of several epithelial marker genes such as CDH1, miR200a, and CGN and enhanced the effects of TGF-ß. Mechanistic investigations revealed that KDM2B specifically recognized the regulatory regions of CDH1, miR200a, and CGN genes and induced histone H2AK119 monoubiquitination as a component of PRC1 complex, thereby mediating the subsequent EZH2 recruitment and histone H3K27 methylation process required for gene repression. Studies using KDM2B mutants confirmed that its DNA recognition property but not its histone H3 demethylase activity was indispensable for its function during EMT. This study demonstrated the significance of the regulation of histone H2A ubiquitination in EMT process and provided the possibility to develop novel therapeutic strategies for the treatment of cancer metastasis.

The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB.

N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-ß-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-ß-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-ß-induced EMT of lung cancer cells through the regulation of JUNB.

MEG8 long noncoding RNA contributes to epigenetic progression of the epithelial-mesenchymal transition of lung and pancreatic cancer cells.

Long noncoding RNAs (lncRNAs) are important regulatory molecules in various biological and pathological processes, including cancer development. We have previously shown that the MEG3 lncRNA plays an essential role in transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of human lung cancer cells. In this study, we investigated the function of another lncRNA, MEG8, which shares the DLK1-DIO3 locus with MEG3, in the regulation of EMT. MEG8 lncRNA expression was immediately induced during TGF-ß-mediated EMT of A549 and LC2/ad lung cancer and Panc1 pancreatic cancer cell lines. MEG8 overexpression specifically suppressed the expression of microRNA-34a and microRNA-203 genes, resulting in up-regulation of SNAIL family transcriptional repressor 1 (SNAI1) and SNAI2 transcription factors, which repressed expression of cadherin 1 (CDH1)/E-cadherin. Mechanistic investigations revealed that MEG8 associates with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) protein and induces its recruitment to the regulatory regions of the two microRNA genes for histone H3 methylation and transcriptional repression. Interestingly, expression of both MEG8 and MEG3, but not each individually, could induce EMT-related cell morphological changes and increased cell motility in the absence of TGF-ß by activating the gene expression program required for EMT. MEG8 knockdown indicated that endogenous MEG8 lncRNA is indispensable for TGF-ß-induced EMT in A549 lung cancer and Panc1 pancreatic cancer cells. Our findings indicate that MEG8 lncRNA significantly contributes to epigenetic EMT induction and increase our understanding of the lncRNA-mediated regulatory mechanisms involved in malignant progression of cancer.

Sox13 is a novel early marker for hair follicle development.

Sox13, a group D member of the Sry-related high-mobility group box (Sox) transcription factor family, is expressed in various tissues including the hair follicle. However, its spatiotemporal expression patterns in the hair follicle and its role in hair development remain to be elucidated. To address these questions, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the LacZ reporter gene was inserted in-frame into exon 2, which contains the translation initiation codon. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode, and subsequently in the hair germ and the hair peg during early hair follicle development. In postnatal catagen and anagen, Sox13 was detected in the epithelial sheath, whereas in telogen, Sox13 was localized in the bulge region, where hair follicle stem cells reside. Immunohistochemistry with an anti-ß-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that Sox13 was predominantly expressed in the outer root sheath in anagen. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically indistinguishable from those of wild type mice. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a useful marker for early hair follicle development.

MEG3 Long Noncoding RNA Contributes to the Epigenetic Regulation of Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines.

Histone methylation is implicated in a number of biological and pathological processes, including cancer development. In this study, we investigated the molecular mechanism for the recruitment of Polycomb repressive complex-2 (PRC2) and its accessory component, JARID2, to chromatin, which regulates methylation of lysine 27 of histone H3 (H3K27), during epithelial-mesenchymal transition (EMT) of cancer cells. The expression of MEG3 long noncoding RNA (lncRNA), which could interact with JARID2, was clearly increased during transforming growth factor-ß (TGF-ß)-induced EMT of human lung cancer cell lines. Knockdown of MEG3 inhibited TGF-ß-mediated changes in cell morphology and cell motility characteristic of EMT and counteracted TGF-ß-dependent changes in the expression of EMT-related genes such as CDH1, ZEB family, and the microRNA-200 family. Overexpression of MEG3 influenced the expression of these genes and enhanced the effects of TGF-ß in their expressions. Chromatin immunoprecipitation (ChIP) revealed that MEG3 regulated the recruitment of JARID2 and EZH2 and histone H3 methylation on the regulatory regions of CDH1 and microRNA-200 family genes for transcriptional repression. RNA immunoprecipitation and chromatin isolation by RNA purification assays indicated that MEG3 could associate with JARID2 and the regulatory regions of target genes to recruit the complex. This study demonstrated a crucial role of MEG3 lncRNA in the epigenetic regulation of the EMT process in lung cancer cells.

Epigenetic regulation of epithelial-mesenchymal transition by KDM6A histone demethylase in lung cancer cells.

Histone methylation is associated with various biological and pathological processes including cancer development. KDM6A is a candidate tumor suppressor gene that encodes a histone H3 lysine 27 (H3K27) demethylase. In this study, we discovered that ectopic expression of KDM6A antagonized TGF-ß-induced epithelial-mesenchymal transition (EMT) and cell migration of lung cancer cell lines through its demethylase activity. KDM6A counteracted TGF-ß-dependent changes in the expression of EMT-related genes such as CDH1/E-cadherin, FN1/Fibronectin, ZEB family and microRNA-200 family. Mechanistic investigations revealed that KDM6A inhibited the recruitment of EZH2 histone H3K27 methyltransferase and H3K27 methylation on the regulatory regions of the target genes such as CDH1 and microRNA-200 family. Knockdown of KDM6A did not proceed EMT by itself, but influenced the expression of specific target genes critical for EMT, suggesting that endogenous KDM6A was involved in EMT-inducing transcriptional program. This study demonstrated a novel regulatory role of KDM6A histone demethylase in the epigenetic control of EMT process in lung cancer cells.

Jmjd5 functions as a regulator of p53 signaling during mouse embryogenesis.

Genetic studies have shown that aberrant activation of p53 signaling leads to embryonic lethality. Maintenance of a fine balance of the p53 protein level is critical for normal development. Previously, we have reported that Jmjd5, a member of the Jumonji C (JmjC) family, regulates embryonic cell proliferation through the control of Cdkn1a expression. Since Cdkn1a is the representative p53-regulated gene, we have examined whether the expression of other p53 target genes is coincidentally upregulated with Cdkn1a in Jmjd5-deficient embryos. The expression of a subset of p53-regulated genes was increased in both Jmjd5 hypomorphic mouse embryonic fibroblasts (MEFs) and Jmjd5-deficient embryos at embryonic day 8.25 without the induced expression of Trp53. Intercrossing of Jmjd5-deficient mice with Trp53 knockout mice showed that the growth defect of Jmjd5 mutant cells was significantly recovered under a Trp53 null genetic background. Chromatin immunoprecipitation analysis in Jmjd5 hypomorphic MEFs indicated the increased recruitment of p53 at several p53 target gene loci, such as Cdkn1a, Pmaip1, and Mdm2. These results suggest that Jmjd5 is involved in the transcriptional regulation of a subset of p53-regulated genes, possibly through the control of p53 recruitment at the gene loci. In Jmjd5-deficient embryos, the enhanced recruitment of p53 might result in the abnormal activation of p53 signaling leading to embryonic lethality.

Jmjd5, an H3K36me2 histone demethylase, modulates embryonic cell proliferation through the regulation of Cdkn1a expression.

Covalent modifications of histones play an important role in chromatin architecture and dynamics. In particular, histone lysine methylation is important for transcriptional control during diverse biological processes. The nuclear protein Jmjd5 (also called Kdm8) is a histone lysine demethylase that contains a JmjC domain in the C-terminal region. In this study, we have generated Jmjd5-deficient mice (Jmjd5(Δ)(/)(Δ)) to investigate the in vivo function of Jmjd5. Jmjd5(Δ)(/)(Δ) embryos showed severe growth retardation, resulting in embryonic lethality at the mid-gestation stage. Mouse embryonic fibroblasts (MEFs) derived from Jmjd5 hypomorphic embryos (Jmjd5(neo/neo)) also showed the growth defect. Quantitative PCR analysis of various cell cycle regulators indicated that only Cdkn1a expression was upregulated in Jmjd5(neo/neo) MEFs and Jmjd5(Δ)(/)(Δ) embryos. A knockdown assay with Cdkn1a-specific small interfering RNAs revealed that the growth defect of Jmjd5(neo/neo) MEFs was significantly rescued. In addition, a genetic study using Jmjd5(Δ)(/)(Δ); Cdkn1a(Δ)(/)(Δ) double-knockout mice showed that the growth retardation of Jmjd5(Δ)(/)(Δ) embryos was partially rescued by Cdkn1a deficiency. Chromatin immunoprecipitation analysis showed that increased di-methylated lysine 36 of histone H3 (H3K36me2) and reduced recruitment of endogenous Jmjd5 were detected in the transcribed regions of Cdkn1a in Jmjd5(neo/neo) MEFs. Taken together, these results suggest that Jmjd5 physiologically moderates embryonic cell proliferation through the epigenetic control of Cdkn1a expression.

EED regulates epithelial-mesenchymal transition of cancer cells induced by TGF-ß.

Histone methylation is involved in various biological and pathological processes including cancer development. In this study, we found that EED, a component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in epithelial-mesenchymal transition (EMT) of cancer cells induced by Transforming Growth Factor-beta (TGF-ß). The expression of EED was increased during TGF-ß-induced EMT and knockdown of EED inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. EED knockdown antagonized TGF-ß-dependent expression changes of EMT-related genes such as CDH1, ZEB1, ZEB2 and microRNA-200 (miR-200) family. Chromatin immunoprecipitation assays showed that EED was implicated in TGF-ß-induced transcriptional repression of CDH1 and miR-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of EED, which regulates PRC2 activity and histone methylation during TGF-ß-induced EMT of cancer cells.

Roles of histone methyl-modifying enzymes in development and progression of cancer.

Retroviral insertional mutagenesis in mice is considered a powerful forward genetic strategy to identify disease genes involved in cancer. Our high-throughput screens led to frequent identification of the genes encoding the enzymes engaged in histone lysine methylation. Histone methylation can positively or negatively impact on gene transcription, and then fulfill important roles in developmental control and cell-fate decisions. A tremendous amount of progress has accelerated the characterization of histone methylations and the enzymes that regulate them. Deregulation of these histone methyl-modifying enzymes has been increasingly recognized as a hallmark of cancer in the last few years. However, in most cases, we have only limited understanding for the molecular mechanisms by which these enzymes contribute to cancer development and progression. In this review, we summarize the current knowledge regarding some of the best-validated examples of histone lysine methyltransferases and demethylases associated with oncogenesis and discuss their potential mechanisms of action.

KDM8 epigenetically controls cardiac metabolism to prevent initiation of dilated cardiomyopathy.

Cardiac metabolism is deranged in heart failure, but underlying mechanisms remain unclear. Here, we show that lysine demethylase 8 (Kdm8) maintains an active mitochondrial gene network by repressing Tbx15, thus preventing dilated cardiomyopathy leading to lethal heart failure. Deletion of Kdm8 in mouse cardiomyocytes increased H3K36me2 with activation of Tbx15 and repression of target genes in the NAD+ pathway before dilated cardiomyopathy initiated. NAD+ supplementation prevented dilated cardiomyopathy in Kdm8 mutant mice, and TBX15 overexpression blunted NAD+-activated cardiomyocyte respiration. Furthermore, KDM8 was downregulated in human hearts affected by dilated cardiomyopathy, and higher TBX15 expression defines a subgroup of affected hearts with the strongest downregulation of genes encoding mitochondrial proteins. Thus, KDM8 represses TBX15 to maintain cardiac metabolism. Our results suggest that epigenetic dysregulation of metabolic gene networks initiates myocardium deterioration toward heart failure and could underlie heterogeneity of dilated cardiomyopathy.

PLU1 histone demethylase decreases the expression of KAT5 and enhances the invasive activity of the cells.

PLU1 is a candidate oncogene that encodes H3K4 (Lys(4) of histone H3) demethylase. In the present study, we found that ectopic expression of PLU1 enhanced the invasive potential of the weakly invasive cells dependent on its demethylase activity. PLU1 was shown to repress the expression of the KAT5 gene through its H3K4 demethylation on the promoter. The regulation of KAT5 by PLU1 was suggested to be responsible for PLU1-induced cell invasion. First, knockdown of KAT5 similarly increased the invasive potential of the cells. Secondly, knockdown of PLU1 in the highly invasive cancer cells increased KAT5 expression and reduced the invasive activity. Thirdly, simultaneous knockdown of KAT5 partially relieved the suppression of cell invasion imposed by PLU1 knockdown. Finally, we found that CD82, which was transcriptionally regulated by KAT5, might be a candidate effector of cell invasion promoted by PLU1. The present study demonstrated a functional contribution of PLU1 overexpression with concomitant epigenetic dysregulation in cancer progression.

The tumor suppressor Rb and its related Rbl2 genes are regulated by Utx histone demethylase.

Utx is a candidate tumor suppressor gene that encodes histone H3 lysine 27 (H3K27) demethylase. In this study, we found that ectopic expression of Utx enhanced the expression of retinoblastoma tumor suppressor gene Rb and its related gene Rbl2. This activation was dependent on the demethylase activity of Utx, and was suggested to contribute to the decreased cell proliferation induced by Utx. A chromatin immunoprecipitation assay showed that over-expressed Utx was associated with the promoter regions of Rb and Rbl2 resulting in the removal of repressive H3K27 tri-methylation and the increase in active H3K4 tri-methylation. Furthermore, siRNA-mediated knockdown of Utx revealed the recruitment of endogenous Utx protein on the promoters of Rb and Rbl2 genes. These results indicate that Rb and Rbl2 are downstream target genes of Utx and may play important roles in Utx-mediated cell growth control.

Transient IGF-1R inhibition combined with osimertinib eradicates AXL-low expressing EGFR mutated lung cancer.

Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC.

Jmjd2c histone demethylase enhances the expression of Mdm2 oncogene.

Jmjd2c is a candidate oncogene that encodes histone lysine demethylase. In this study, we discovered that over-expression of Jmjd2c increased the expression of Mdm2 oncogene dependent on its demethylase activity, which led to the reduction of p53 tumor suppressor gene product in the cells. A chromatin immunoprecipitation assay showed that Jmjd2c was recruited to the P2 promoter region of Mdm2 gene resulting in demethylation of histone H3 lysine 9, as typically found in actively transcribed genes. Furthermore, siRNA-mediated knockdown of Jmjd2c caused the reduction of Mdm2 expression in the cells. These results indicate that Mdm2 oncogene is a downstream target of Jmjd2c and may play an important role in Jmjd2c-mediated oncogenesis.

DOT1L histone methyltransferase regulates the expression of BCAT1 and is involved in sphere formation and cell migration of breast cancer cell lines.

DOT1L is a histone H3 lysine 79 (H3K79) methyltransferase mainly implicated in leukemia. Here we analyzed the function of DOT1L in breast cancer cells. The expression of DOT1L was up-regulated in malignant breast cancer tissues. Over-expression of DOT1L significantly increased the sphere formation and the cell migration activities of MCF7 breast cancer cell line. In contrast, knockdown of DOT1L reduced the cell migration activity of MDA-MB-231 breast cancer cell line. BCAT1, which encodes a branched-chain amino acid transaminase, was identified as one of the target genes controlled by DOT1L through the regulation of H3K79 methylation. Mechanistic investigation revealed that BCAT1 might be an important regulator responsible for DOT1L-mediated sphere formation and cell migration in breast cancer cells.

Contribution of JNK, Mek, Mos and PI-3K signaling to GVBD in Xenopus oocytes.

In Xenopus oocytes, induction of the G2/M transition by progesterone is a complex process that is promoted by a network of signaling molecules whose cumulative effect results in the activation of maturation promoting factor (MPF) and germinal vesicle breakdown (GVBD). We examined the role of Mos, Mek, PI-3 kinase and c-Jun N-terminal kinase (JNK) in progesterone stimulation of GVBD. Expression of an activated form of JNK neither induced nor enhanced progesterone-mediated GVBD in oocytes, suggesting a limited role in cell-cycle progression. We blocked Mek, Mos and PI-3 kinase activities by a variety of means that included expression of dominant-negative kinase suppressor of Ras (DnKSR), expression of a dominant-negative PI-3 kinase (DnPI3K), treatment of oocytes with a Mek inhibitor (U1026) or PI-3 kinase (LY294002) inhibitor, and introduction of Mos antisense morpholinos. Inhibition of any one pathway alone failed to block GVBD induced by either high or low concentrations of progesterone. In contrast, inhibiting Mos or Mek function in addition to abrogating PI-3 kinase activity effectively blocked oocyte maturation. Furthermore, by expressing suboptimal amounts of Mos in conjunction with an activated form of Mek and an activated form of the p110 catalytic subunit of PI-3 kinase, we show cooperation among these signaling molecules toward the induction of GVBD. Moreover, expression of optimal amounts of these three proteins in conjunction with inhibitors of Mos, Mek or PI-3 kinase demonstrated that activated Mek-induced GVBD is independent of Mos or PI-3 kinase activity. In addition, Mos-induced GVBD is dependent upon Mek activity, but does not require PI-3 kinase activity. Finally, Mos appears to be a major contributor to GVBD induced by activated PI-3 kinase, while Mek is a minor contributor to this process.

Tyr-298 in ephrinB1 is critical for an interaction with the Grb4 adaptor protein.

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are thought to play a role in the regulation of cell adhesion and migration during development by mediating cell-to-cell signalling events. The transmembrane ephrinB protein is a bidirectional signalling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase residing on another cell. The reverse signal is transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Previous work from our laboratory has implicated the activated FGFR1 (fibroblast growth factor receptor 1) as a regulator of a de-adhesion signal that results from overexpression of ephrinB1. In the present study, we report the isolation of Xenopus Grb4 (growth-factor-receptor-bound protein 4), an ephrinB1-interacting protein, and we show that when expressed in Xenopus oocytes, ephrinB1 interacts with Grb4 in the presence of an activated FGFR1. Amino acid substitutions were generated in Grb4, and the resulting mutants were expressed along with ephrinB1 and an activated FGFR in Xenopus oocytes. Co-immunoprecipitation analysis shows that the FLVR motif within the Src homology 2 domain of Xenopus Grb4 is vital for this phosphorylation-dependent interaction with ephrinB1. More importantly, using deletion and substitution analysis we identify the tyrosine residue at position 298 of ephrinB1 as being required for the physical interaction with Grb4, whereas Tyr-305 and Tyr-310 are dispensable. Moreover, we show that the region between amino acids 301 and 304 of ephrinB1 is also required for this critical tyrosine-phosphorylation-dependent event.