Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation

Interactions between receptor tyrosine kinases of the Eph family and their ligands, ephrins, are implicated in establishment of organ boundaries and repulsive guidance of cell migration during development, but the mechanisms by which this is achieved are unclear. Here we show that activation of endogenous EphA2 kinase induces an inactive conformation of integrins and inhibits cell spreading, migration and integrin-mediated adhesion. Moreover, EphA2 is constitutively associated with focal-adhesion kinase (FAK) in resting cells. Within one minute after stimulation of EphA2 with its ligand, ephrin-A1, the protein tyrosine phosphatase SHP2 is recruited to EphA2; this is followed by dephosphorylation of FAK and paxillin, and dissociation of the FAK-EphA2 complex. We conclude that Eph kinases mediate some of their functions by negatively regulating integrins and FAK.     

EphrinA1 activates a Src/focal adhesion kinase-mediated motility response leading to rho-dependent actino/myosin contractility

Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell repulsive motility through heterotypic cell-cell interactions. Several Ephs, including EphA2, are greatly overexpressed in certain tumors, in correlation with poor prognosis and high vascularity in cancer tissues. The ability of several Eph receptors to regulate cell migration and invasion likely contribute to tumor progression and metastasis. We report here that in prostatic carcinoma cells ephrinA1 elicits a repulsive response that is executed through a Rho-dependent actino/myosin contractility activation, ultimately leading to retraction of the cell body. This appears to occur through assembly of an EphA2-associated complex involving the two kinases Src and focal adhesion kinase (FAK). EphrinA1-mediated repulsion leads to the selective phosphorylation of Tyr-576/577 of FAK, enhancing FAK kinase activity. The repulsive response elicited by ephrinA1 in prostatic carcinoma cells is mainly driven by a Rho-mediated phosphorylation of myosin light chain II, in which Src and FAK activation are required steps. Consequently, Src and FAK are upstream regulators of the overall response induced by ephrinA1/EphA2, instructing cells to retract the cell body and to move away, probably facilitating dissemination and tissue invasion of ephrin-sensitive carcinomas.     

Novel protein kinases and molecular mechanisms of autoinhibition

During the past year, crystal structures of the PDK-1, ITK, Aurora-A, c-KIT and FLT-3 protein kinases in complex with several ATP-competitive inhibitors have been determined. Some structures have crystallized in catalytically active conformations, whereas others appear to be in inactive or native conformations. The differences between these two classes of structures provide further understanding of how kinase activity may be self-regulated in the cellular environment and how phosphorylation can modulate signalling at a molecular level. All of these structures provide a basis for designing selective protein kinase inhibitors of use in the treatment of cancer and autoimmune disease.     

Activation of erythropoietin-producing hepatocellular receptor A2 attenuates cell adhesion of human fallopian tube epithelial cells via focal adhesion kinase dephosphorylation

Tyrosine kinase receptor erythropoietin-producing hepatocellular receptor A2 (EphA2) and its predominant ligand EphrinA1 have been studied extensively for their roles of mediating cell adhesion in epithelial cells. However, EphA2 signaling in human fallopian tube epithelial cells is poorly understood. In this study, primary cultured fallopian tube epithelial cells were used as a model treated with EphrinA1-Fc or IgG-Fc (control), to explore the role of EphA2 signal and its network involved in the regulation of cell adhesion of tubal epithelia cells. The activation of EphA2 and focal adhesion kinase (FAK) was evaluated by western blotting assay in the cultured fallopian tube epithelia cells, of which the cell adhesion activity was determined by MTT assay. A significantly negative correlation was found between phosphorylated-EphA2 (Pho-EphA2) and phosphorylated-FAK (Pho-FAK) after exposure to EphrinA1-Fc (P = 0.000; r = -0.848). EphrinA1-Fc increased Pho-EphA2 and reduced Pho-FAK in seconds, with the apex level of Pho-EphA2 and the nadir level of Pho-FAK detected at the same time (10 min). Cell adhesion of the cultured cells supplemented with EphrinA1-Fc appeared to be weaker than that of the controls at the later time points of the treatment (from 30 to 120 min) (P < 0.05). Taken together, the EphrinA1 addition directly induces an elevated Pho-EphA2 accompanied by a decreased Pho-FAK in human fallopian tube epithelia cells. Furthermore, activation of EphA2 participates in the regulation of fallopian tube cell adhesion via FAK dephosphorylation.     

7-[1H-Indol-2-yl]-2,3-dihydro-isoindol-1-ones as dual Aurora-A/VEGF-R2 kinase inhibitors: design, synthesis, and biological activity

A novel series of 7-[1H-indol-2-yl]-2,3-dihydro-isoindol-1-ones designed to be inhibitors of VEGF-R2 kinase was synthesized and found to potently inhibit VEGF-R2 and Aurora-A kinases. The structure-based design, synthesis, and initial SAR of the series are discussed.     

Irradiation affects cellular properties and Eph receptor expression in human melanoma cells

X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated.     

Irradiation affects cellular properties and Eph receptor expression in human melanoma cells

X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated.     

Searching for biomarkers of Aurora-A kinase activity: identification of in vitro substrates through a modified KESTREL approach

Aurora-A, -B, and -C are members of a small family of mitotic serine/threonine kinases that regulate centrosome maturation, chromosome segregation, and cytokinesis. They are often overexpressed in different human tumor types and have been identified as attractive targets for anticancer drug development. As specific inhibitors of the Aurora kinases are entering phase I clinical trials, there is a high need for appropriate Aurora-A biomarkers to follow mechanism of action or response. To identify novel Aurora-A substrates potentially useful as specific biomarkers we applied several modifications to the original KESTREL (Kinase Substrate Tracking and Elucidation) method in conjunction with gel electrophoresis and MALDI-MS and LC-MS/MS. The major modifications to the method included the introduction of a heating step to inactivate endogenous kinases after cell lysis and the execution of the in vitro kinase reaction in the presence of 5 mM Mg(2+) and at high (1 mM) ATP concentration. Total and fractionated extracts from nocodazole-treated HeLa cells were used as a source of Aurora-A substrates. Using this approach, we were able to detect a number of Aurora-A specific phospholabeled signals and to identify vimentin as a putative Aurora-A substrate. Vimentin was then confirmed as an in vitro substrate of Aurora-A by the phosphorylation of the recombinant protein followed by MS and antibody detection.     

Overexpression of Aurora-A enhances invasion and matrix metalloproteinase-2 expression in esophageal squamous cell carcinoma cells

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers, and metastasis is the principal cause of death in ESCC patients. It has been shown that amplification and overexpression of mitotic serine/threonine kinase Aurora-A occur in several types of human tumors, including ESCC. Moreover, increase in expression levels of Aurora-A has been predicted to correlate with the grades of tumor differentiation and invasive capability. However, the mechanisms by which Aurora-A mediates its invasive effects still remain elusive. In this article, we showed that Aurora-A overexpression significantly increased cell migration and invasion as well as secretion and expression of matrix metalloproteinase-2 (MMP-2). Conversely, siRNA-mediated knockdown of Aurora-A expression in human ESCC cells led to inhibition of cell invasiveness as well as secretion and expression of MMP-2. In addition, Aurora-A overexpression increased phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and Akt, and the knockdown of Aurora-A by siRNA decreased the activity of p38 MAPK and Akt. Moreover, the blocking of the activity of above kinases using chemical inhibitors suppressed the ability of Aurora-A to induce MMP-2 secretion and expression as well as cell invasion. These data show that overexpression of Aurora-A contributes to the malignancy development of ESCC by enhancing tumor cell invasion as well as MMP-2 activity and expression, which can occur through signaling pathways involving p38 MAPK and Akt protein kinases. Taken together, these studies provide a molecular basis for promoting the role of Aurora-A in malignancy development of ESCC.     

Antagonistic effect of EGF on FAK phosphorylation/dephosphorylation in a cell

Focal adhesion kinase (FAK) plays a key role in the crosstalk of growth factor- and cell adhesion-mediated signaling pathway. In this study, we found that the quantitative change of phosphorylated FAK was bell-shaped time-dependently by EGF stimulation in immortalized human keratinocyte (HaCaT). EGF enhanced FAK phosphorylation and cell spreading in adhering HaCaT cells with low-phosphorylated FAK. On the other hand, spread HaCaT cells having high-phosphorylated FAK changed to round shapes with FAK dephosphorylation 15 min after EGF stimulation. Pharmacological agents, U0126 and PD98059 (mitogen-activated protein kinases (MAPK) kinases (MEK) inhibitors), and AG1478 (an EGF receptor kinase inhibitor) blocked the cell rounding and FAK dephosphorylation. In addition, the EGFR-MAPK signaling pathway had an influence on cell migration by regulating FAK dephosphorylation of keratinocytes in response of EGF, since the MEK inhibitors and AG1478 suppressed EGF-induced cell migration. However, FAK phosphorylation and HaCaT cell spreading were inhibited only by the antagonist of EGF-EGFR binding but not by the MEK inhibitors and AG1478. Taken together, we suggest that EGF is antagonistically involved in both FAK phosphorylation and dephosphorylation with different mechanisms in a cell.     

Human cytomegalovirus chemokine receptor US28-induced smooth muscle cell migration is mediated by focal adhesion kinase and Src

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK.Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK.Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2.FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1-401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration.     

Aurora-A kinase phosphorylation of Aurora-A kinase interacting protein (AIP) and stabilization of the enzyme-substrate complex

Aurora-A is an oncogenic kinase that plays essential roles in mitosis as well as cell survival. Aurora-A interacting protein (AIP) was identified as a negative regulator of Aurora-A with its ectopic over expression inducing destabilization of Aurora-A protein. Here we present evidence that in human cells, contrary to the earlier report, AIP functions in stabilizing rather than destabilizing Aurora-A. Furthermore, AIP is phosphorylated on Serine 70 by Aurora-A but not Aurora-B and expression of phosphorylation mimic mutant of AIP results in prolonged protein stability compared to unphosphorylatable mutant. We observed that when co-expressed with AIP, protein levels of both Aurora-A and Aurora-B are markedly elevated regardless of their kinase activities and phosphorylation state of AIP. Interaction of Aurora kinases with AIP is necessary for this elevated stability. This phenomenon is commonly detected in several human cancer cell lines used in this study. Depletion of AIP by RNA interference decreased Aurora-A but not Aurora-B in two of the three cell lines analyzed, indicating that under physiological condition, AIP functions in stabilization of Aurora-A but not Aurora-B, though this regulation may be dependent on additional factors as well. Further, AIP siRNA induced cell cycle arrest at G2/M, which is consistent with anticipated loss of function of Aurora-A in these cells. Thus, our study provides the first evidence of a role for AIP in G2/M cell cycle progression by cooperatively regulating protein stabilization of its up-stream regulator, Aurora-A kinase through protein-protein interaction as well as protein phosphorylation.     

Polymeric Immunoglobulin Receptor-mediated Invasion of Streptococcus pneumoniae into Host Cells Requires a Coordinate Signaling of SRC Family of Protein-tyrosine Kinases,ERK, and c-Jun N-terminal Kinase

Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.     

Structure-based design of 2,6,7-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines as Aurora kinases inhibitors

This Letter reports the optimization of a pyrrolopyrimidine series as dual inhibitors of Aurora A/B kinases. This series derived from a pyrazolopyrimidine series previously reported as inhibitors of aurora kinases and CDKs. In an effort to improve the selectivity of this chemotype, we switched to the pyrrolopyrimidine core which allowed functionalization on C-2. In addition, the modeling rationale was based on superimposing the structures of Aurora-A kinase and CDK2 which revealed enough differences leading to a path for selectivity improvement. The synthesis of the new series of pyrrolopyrimidine analogs relied on the development of a different route for the two key intermediates 7 and 19 which led to analogs with both tunable activity against CDK1 and maintained cell potency.     

Dynamic association of a tumor amplified kinase,Aurora-A,with the centrosome and mitotic spindle

Aurora-A kinase, also known as STK15/BTAK kinase, is a member of a serine/threonine kinase superfamily that includes the prototypic yeast Ipl1 and Drosophila aurora kinases as well as other mammalian and non-mammalian aurora kinases involved in the regulation of centrosomes and chromosome segregation. The Aurora-A gene is amplified and overexpressed in a wide variety of human tumors. Aurora-A is centrosome-associated during interphase, and binds the poles and half-spindle during mitosis; its over-expression has been associated with centrosome amplification and multipolar spindles. GFP-Aurora-A was used to mark centrosomes and spindles, and monitor their movements in living cells. Centrosome pairs labeled with GFP-Aurora-A are motile throughout interphase undergoing oscillations and tumbling motions requiring intact microtubules and ATP. Fluorescence recovery after photobleaching (FRAP) was used to examine the relative molecular mobility of GFP-Aurora-A, and GFP-labeled alpha-tubulin, gamma-tubulin, and NuMA. GFP-Aurora-A rapidly exchanges in and out of the centrosome and mitotic spindle (t(1/2) approximately 3 sec); in contrast, both tubulins are relatively immobile indicative of a structural role. GFP-NuMA mobility was intermediate in both interphase nuclei and at the mitotic spindle (t(1/2) approximately 23-30 sec). Deletion mapping identifies a central domain of Aurora-A as essential for its centrosomal localization that is augmented by both the amino and the carboxyl terminal ends of the protein. Interestingly, amino or carboxy terminal deletion mutants that maintained centrosomal targeting exhibited significantly slower molecular exchange. Collectively, these studies contrast the relative cellular dynamics of Aurora-A with other cytoskeletal proteins that share its micro-domains, and identify essential regions required for targeting and dynamics.     

Inhibition of Tumor Vasculogenic Mimicry and Prolongation of Host Survival in Highly Aggressive Gallbladder Cancers by Norcantharidin via Blocking the Ephrin Type a Receptor 2/Focal Adhesion Kinase/Paxillin Signaling Pathway

Vasculogenic mimicry (VM) is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2)/focal adhesion kinase (FAK)/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD) as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D) matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.     

Serum levels of FAK and some of its effectors in adult AML: correlation with prognostic factors and survival

Focal adhesion kinase (FAK), human myofibrillogenesis regulator-1 (MR-1), ephrin receptor type A4 (EphA4), proto-oncogene tyrosine kinase Src (Src), and protein kinase C (PKC) are important markers in proliferation, survival, and migration in some cancers. However, the significance of each is still unclear in different malignancies, including acute myeloid leukemia (AML). Therefore, this study was conducted to investigate their serum levels in Egyptian adult de novo AML patients (n?=?70) against healthy volunteers (n?=?20). We managed to study the correlation between each pair and to investigate their association with diagnosis, prognosis, and survival. Serum levels were analyzed using enzyme-linked immunosorbent assay (ELISA). We found that FAK, MR-1, Src, and PKC serum levels were significantly higher in AML patients compared to control (p?<?0.0001), and this was associated with significantly lower EphA4 level (p?<?0.0001). Interestingly, we also observed a significant negative correlation of FAK (p?=?0.027), MR-1 (p?=?0.003), Src (p?=?0.038), and PKC (p?=?0.03) with patients’ overall survival (OS) while there was a positive significant correlation between EphA4 and OS (p?=?0.007). In conclusion, this study suggests that FAK, MR-1, EphA4, Src, and PKC may be used as early diagnostic and prognostic markers with high sensitivity and specificity in AML patients and thus may be incorporated into the patients’ early diagnostic and prognostic panels.

     

Small molecule inhibitors of the Pyk2 and FAK kinases modulate chemoattractant-induced migration, adhesion and Akt activation in follicular and marginal zone B cells

B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells.     

The EphA4 receptor regulates dendritic spine remodeling by affecting beta1-integrin signaling pathways

Remodeling of dendritic spines is believed to modulate the function of excitatory synapses. We previously reported that the EphA4 receptor tyrosine kinase regulates spine morphology in hippocampal pyramidal neurons, but the signaling pathways involved were not characterized (Murai, K.K., L.N. Nguyen, F. Irie, Y. Yamaguchi, and E.B. Pasquale. 2003. Nat. Neurosci. 6:153-160). In this study, we show that EphA4 activation by ephrin-A3 in hippocampal slices inhibits integrin downstream signaling pathways. EphA4 activation decreases tyrosine phosphorylation of the scaffolding protein Crk-associated substrate (Cas) and the tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) and also reduces the association of Cas with the Src family kinase Fyn and the adaptor Crk. Consistent with this, EphA4 inhibits beta1-integrin activity in neuronal cells. Supporting a functional role for beta1 integrin and Cas inactivation downstream of EphA4, the inhibition of integrin or Cas function induces spine morphological changes similar to those associated with EphA4 activation. Furthermore, preventing beta1-integrin inactivation blocks the effects of EphA4 on spines. Our results support a model in which EphA4 interferes with integrin signaling pathways that stabilize dendritic spines, thus modulating synaptic interactions with the extracellular environment.     

Crystal structure of the EphA4 protein tyrosine kinase domain in the apo- and dasatinib-bound state

The Eph family of receptor tyrosine kinases regulates diverse cellular processes while the over-expression of a member of this family, EphA4, has been reported in a variety of malignant carcinomas. To gain insight into molecular mechanisms and to facilitate structure-based inhibitor design, we solved the crystal structure of the native EphA4 kinase domain in both the apo and dasatinib bound forms. Analysis of the two structures provides insight into structural features of inhibitor binding and revealed a hydrophobic back-pocket in the ATP- binding site of EphA4 which was previously unidentified. The structures suggest a route towards development of novel and specific inhibitors.