Ligand-independent stimulation and ligand-dependent inhibition of cell migration by EphA2 kinase
We investigated how EphA2 overexpression may regulate chemotactic cell migration and invasion in the absence and presence of its ligand ephrin-A1 using glioma cells as a model system. Examination of public microarray database also revealed overexpression of EphA2 at mRNA levels in GBM (www.oncomine.org
). To mimic the in vivo situation, we overexpressed EphA2 in U373 glioma cells. shows that U373 cells express moderate levels of endogenous EphA2 (), and infection with EphA2-expressing retrovirus increased the level by about one fold. In keeping with the lack of ligand expression, both endogenous and ectopic EphA2 in U373 cells showed low basal activation. Stimulation with exogenous ephrin-A1 caused rapid EphA2 activation, which was followed by degradation of the receptor itself, characteristic of most RTKs including Eph kinases.
EphA2 possesses both ligand-independent and ligand-dependent functions in regulating growth factor-induced chemotaxis
Overexpression of EphA2 alone, in the absence of ligand stimulation, significantly enhanced serum-induced migration of U373 cells in a Boyden chamber cell migration assay (). In contrast, activation of EphA2 with its ligand ephrin-A1 significantly inhibited the chemotaxis of both vector control and EphA2-overexpressing cells. These data suggest that EphA2 has both ligand-independent stimulatory effects and ligand-dependent inhibitory effects on chemotactic cell migration. The diametrically opposite properties of EphA2 in regulating cell migration were also observed in other cell types, including HEK 293, U87, A172 and PC-3M cells (see below).
In a reverse experiment, shRNA knocking down of EphA2 expression in U373 cells () led to a significant reduction in ligand-independent chemotaxis toward serum (). The residual EphA2 on U373 cells was still able to mediate inhibition of cell migration upon ligand stimulation (). Similarly, shRNA knockdown of EphA2 in PC-3M prostate cancer cells significantly reduced ligand-independent chemotaxis (), whereas overexpression of EphA2 promoted it (see below).
Extensive previous investigations have established that a major function of Eph kinases is the ligand-dependent repulsion of migrating cells and axons both in vitro and in vivo (Pasquale, 2005
). Our data demonstrate that EphA2 receptor can also promote cell migration in cooperation with growth factors in a ligand-independent manner.
EphA2 is both a substrate and a negative regulator of Akt
In exploring the molecular mechanisms that mediate ligand-independent promotion of chemotaxis by EphA2, we screened several signaling pathways. Among them, PI3K/Akt pathways stood out by robustly responding to serum in migrating U373 cells. As shown in , Akt became highly phosphorylated at both T308 and S473 sites upon serum stimulation. Cotreatment with ephrin-A1 completely blocked Akt activation. To test possible direct crosstalk between Akt and EphA2, we precipitated EphA2 and probed it with an antibody recognizing the consensus Akt substrate sites (Akt-pSub). The antibody detected a strong band at 125 kDa on EphA2 precipitates prepared from serum-stimulated cells (), indicating that EphA2 could be a substrate for Akt. To confirm this unexpected finding, immunoprecipitation was performed with Akt-pSub antibody, and the precipitates were immunoblotted for EphA2. EphA2 could be readily detected in the Akt-pSub immunoprecipitates (Fig. S1A
). Ephrin-A1 cotreatment led to complete inhibition of EphA2 phosphorylation at Akt substrate sites (), which correlated with the abolishment of Akt activation ().
To verify the specificity of serum-induced S/T phosphorylation of EphA2 to Akt activity, we performed the same experiments on cells pretreated with PI3K inhibitor, LY294002. Inhibition of PI3K/Akt pathway by LY294002 significantly reduced the phosphorylation of EphA2 at Akt substrate sites (). To further demonstrate the Akt-EphA2 crosstalk, dominant negative (DN) Akt was expressed in U373 cells. As shown in , serum-induced S/T phosphorylation of EphA2 was attenuated by the expression of DN Akt. Taken together, these results identify a reciprocal regulatory loop between EphA2 and Akt, with unligated EphA2 functioning as a downstream substrate and effector of Akt kinase, but the ligand-activated EphA2 being an upstream negative regulator to turn off Akt and cause dephosphorylation of EphA2 at the Akt substrate site.
To examine where Akt-EphA2 crosstalk may take place in migrating cells, we investigated the subcellular localization of EphA2 relative to p-Akt. shows that under serum-starved condition, p-Akt was low and uniformly distributed in cells with no obvious colocalization with EphA2 (, top panels). Within 10 min after serum treatment, p-Akt was upregulated and became colocalized with EphA2 in lamellipodia at the leading edge. Cotreatment with ephrin-A1 caused retraction of lamellipodia and disappearance of p-Akt from the leading edge. Akt-pSub was also localized at the migrating front together with EphA2, but not at cell-cell junction sites where EphA2 was also present at high levels (Fig. S1B
), suggesting that Akt-phosphorylated EphA2 is preferentially targeted to migrating front. Thus, in response to growth factor stimulation, the unligated EphA2 is phosphorylated at the leading edge by the active Akt; upon addition of exogenous ligand, EphA2 is activated, leading to “repulsion” of the migrating cells.
Serine 897 of EphA2 is the major substrate site for Akt
Sequence analysis of EphA2 revealed several potential S/T sites that resemble the consensus motif of known Akt substrates (Alessi et al., 1996
; Manning and Cantley, 2007
) (). To map the Akt phosphorylation site(s) in EphA2, site-directed mutagenesis was carried out to replace all five potential S/T residues with alanine in the cytoplasmic tail. A site in the ectodomain (S479) was also included as a control. The resultant mutants were initially tested in HEK 293 cells. As we have reported previously, HEK 293 cells express very low levels of endogenous EphA2 (Miao et al., 2001
), providing a low background to evaluate the mutants. In response to serum stimulation, cells expressing wild type (WT)-EphA2 or EphA2 with single mutation at S479, S636, T795, T838, and S910 were equally phosphorylated at Akt substrate sites. In contrast, S897A mutation rendered EphA2 completely resistant to serum-induced phosphorylation at Akt substrate sites (). Identical results were obtained from three independent transfections. Therefore, our studies mapped serine 897 as the major site for Akt kinase phosphorylation in EphA2. Structurally, serine 897 is located in the linker region between EphA2 kinase domain and sterile α motif (SAM), which is likely to be exposed and accessible to Akt phosphorylation.
Serine 897 of EphA2 is the major substrate site for Akt activity, which is essential for ligand-independent promotion of cell migration
Akt-mediated serine 897 phosphorylation is the major mechanism responsible for ligand-independent stimulation of cell migration by EphA2
To determine the functional significance of phosphorylation at S897, HEK 293 cells expressing WT-, S897A-EphA2 or control vector were characterized. A phospho-mimetic mutation was also created by replacing S897 with aspartic acid (S897D) and tested. Immunofluorescence and biochemical analysis showed that the exogenous WT-, S897A- and S897D-EphA2 were homogenously overexpressed in HEK 293 cells (). Similar to what we observed in U373 glioma cells (), overexpression of WT-EphA2 increased serum-induced chemotaxis of HEK 293 cells (). Interestingly, both S897A and S897D mutations completely abolished the cell migration-promoting effect by EphA2 (). These results suggest that Akt phosphorylation of S897-EphA2 is largely responsible for the ligand-independent stimulation of cell motility by EphA2 in HEK 293 cells. The fact that phospho-mimetic mutation (S897D) had the same effects as S897A indicates that phosphorylation, but not the negative charge-induced conformational change is needed to promote cell migration.
Catalytic activity of EphA2 is required for ligand-dependent inhibition, but not for ligand-independent promotion of cell migration
To investigate the role of the intrinsic tyrosine kinase activity of EphA2, a kinase deficient EphA2 with D739N mutation was tested in HEK 293 cells. The mutant was still fully capable of simulating chemotaxis toward serum when overexpressed (Fig. S2
). However, it lost the ligand-dependent inhibition of cell migration. Thus the intrinsic catalytic activity of EphA2 is required for ligand-dependent inhibition but not ligand-independent promotion of cell migration. These and additional data described below show that the two processes are mutually exclusive and involve distinct signaling mechanisms. In the context of ligand-independent promotion of cell migration, EphA2 is serving as a substrate of Akt kinase when and if its own catalytic function is kept inactive. To keep the dual functions distinct, the term “kinase” will be avoided when ligand-independent functions of EphA2 are under discussion.
Ligand-independent promotion of cell migration by EphA2 in glioma and prostate cancer cells requires S897 phosphorylation and is sensitized by PTEN deletion
Next we investigated the effects of S897A on ligand-independent chemotaxis in a panel of human tumor cell lines, including U373, U87, A172, LN229 and T98G glioma cells, as well as PC-3M prostate cancer cells. As expected, the PTEN-null U373, U87, and A172 cells generally had more active Akt than PTEN-wild type (WT) T98G and LN229 cells (Fig. S3
). Hyperactive Akt was also observed in the PTEN-null PC-3M cells (Fig. S4
) as well as in parental PC-3 cells (Shukla et al., 2007
). WT- and S897A-EphA2 were introduced into these cells via retroviral infection (). Overexpression of WT-EphA2 enhanced serum-stimulated chemotaxis compared to vector control in PTEN-null U373, U87, A172 and PC-3M cells (). S897A mutation blocked most of the stimulatory effects in these cells. Interestingly, U373 cells expressing S897A-EphA2 displayed lower basal levels of serum-induced chemotaxis than vector control cells. The seemingly dominant negative effects were not observed in U87, A172 and PC-3M cells. The differences may reflect the cell type-specific signaling networks regulating chemotaxis.
Serum-induced chemotaxis in glioma and prostate cancer cells is dependent on Akt-mediated phosphorylation of EphA2 at S897
Together these data indicate that PTEN-null cells with hyperactive Akt may have become more dependent on Akt-EphA2 crosstalk to promote cell motility. To test this possibility, we restored PTEN expression in U373 cells, which caused a significant reduction in serum-induced Akt activities (). In both chemotactic cell migration and scratch wound assays, restoration of PTEN expression dramatically inhibited cell migration (), supporting important roles of PTEN loss in promoting malignant behaviors of glioma cells. However, other mechanisms in addition to loss of PTEN may also permit EphA2 to promote chemotactic cell migration, since in PTEN-WT HEK 293 cells EphA2 overexpression was still capable of promoting migration (). Notably hyperactivation of Akt can be achieved by a variety of mechanisms including loss of PTEN, RTK amplification/mutation, Akt amplification, and Ras activation. Further studies are needed to identify the alternative mechanisms.
Serum-induced glioma cell invasion through MatriGel also requires Akt phosphorylation of EphA2 on S897
Invasion through the surrounding normal tissues is a hallmark of malignant progression, which is particularly deadly for glioma patients. We used U87, U373 and A172 cell lines to test how EphA2 might regulate serum-induced glioma cell invasion. The multiplicity of the cell lines with a shared genetic alteration in PTEN status makes them good models for assessing general applicability of our findings. We found that, in the absence of ligand stimulation, overexpression of WT-EphA2 strongly promoted MatriGel invasion by all three cell lines (, open bars). The S897A mutation largely abolished the stimulatory effects. Together with the data from the last section, we conclude that EphA2 functions as a ligand-independent positive regulator of chemotactic migration as well as invasion, and Akt-mediated phosphorylation of EphA2 on S897 is required for both effects.
EphA2 promotes MatriGel invasion, and S897A mutation does not affect ligand-dependent inhibition of cell migration and invasion
Ligand-dependent inhibition of cell migration and invasion by EphA2 is not affected by S897A mutation
Next we investigated if S897 of EphA2 is involved in ligand-dependent inhibition of cell migration and invasion. Similar to what was found in U373 cells (), chemotactic migration of A172 and U87 cells expressing WT-EphA2 or vector control was significantly inhibited by ephrin-A1 (). Interestingly, S897A EphA2 was still capable of mediating ephrin-A1-induced inhibition of migration in all three cell lines, despite its ability to abolish most ligand-independent promotion of cell migration. Indeed, the degrees of ligand-dependent inhibition were indistinguishable between WT- and S897A-EphA2 cells. Similarly, ephrin-A1 treatment also significantly blocked MatriGel invasion of cells expressing either WT- or S897A-EphA2 (). One consistent difference across all three cell lines was that both ligand-independent promotion and ligand-dependent inhibition were more pronounced in the invasion assay than migration assay ().
Time-lapse imaging was employed to monitor cell morphology and chemokinetic movement of cells on two-dimensional culture plate. In keeping with the increased cell motility, U373 cells expressing WT-EphA2 displayed elongated and polarized morphology with a large protruding lamellipodium (), and were more motile compared with vector control cells (Supplementary Movies
). In contrast, cells expressing S897A-EphA2 showed a “pancake-like” morphology in culture dishes and were largely stationary during the course of the observation. Ephrin-A1 treatment minimized chemokinetic movement in all three cell types.
Multiple growth factors stimulate S897 phosphorylation of EphA2
To better understand the function of S897 phosphorylation of EphA2, we generated and characterized a rabbit polyclonal antibody against a synthetic phospho-peptide surrounding S897. We validated the specificity of the antibody in HEK 293 cells expressing WT- or S897A-EphA2. Immunoblot of total cell lysates with the antibody detected a strong band at the same molecular weight as EphA2 in the serum-stimulated WT-EphA2-expressing cells, but not in cells expressing S897A-EphA2 (). Costimulation with serum and ephrin-A1 abolished the phospho-S897 (pS897)-EphA2 signal. A robust pS897 band co-migrating with EphA2 was also detected in EphA2 immunoprecipitates (). Conversely, EphA2 was detected in the materials precipitated with the anti-pS897-EphA2 antibody (Fig. S5
). Treatment of cell lysates with recombinant S/T protein phosphatase 2A (PP2A) caused dephosphorylation of pS897-EphA2 (). Finally treatment of cells with SH-5, an Akt inhibitor, suppressed S897 phosphorylation (Fig. S6A
); as did LY294002, an inhibitor of PI3K (Fig. S6B
). Taken together, this series of experiments confirmed specificity of the antibody. Moreover, the results further established EphA2 as an effector molecule for PI3K/Akt signaling cascade.
Multiple growth factors can induce S897 phosphorylation of EphA2, which is required for EphA2 localization to the leading edge and for cell polarization
To determine which growth factors induce phosphorylation of EphA2 on S897, we stimulated U373 glioma cells with a panel of growth factors including EGF, bFGF, PDGF, and HGF, which have all been implicated in human GBM. Serum was used as a positive control. We found that all growth factors were capable of inducing pS897-EphA2, which was correlated with Akt activation (). In cell migration assays, all four growth factors were potent chemoattractants for U373 cells (). The number of migrating cells correlated with the degree of S897 phosphorylation and Akt activation. Costimulation of cells with ephrin-A1-Fc significantly inhibited all growth factor-induced pS897-EphA2 and chemotaxis, concomitant with Akt inactivation (). The degree of ephrin-A1-dependent inhibition of Akt varied among different growth factors, with strongest inhibition for bFGF; significant but less inhibition was seen with EGF and HGF (). These results suggest that phosphorylation of EphA2 at S897 is a common mediator of growth factor-induced cell migration, which could have important implications in understanding the molecular basis of malignant progression for GBM and other tumor types.
LPA in serum is known to activate Akt and induce cell migration. However, in U373 cells, LPA is a weaker inducer of Akt activation and chemotactic cell migration, which was not affected by ephrin-A1 (). Thus LPA in serum did not contribute significantly to either Akt activation or S897 phosphorylation in this system.
Next we examined whether kinase activities of the growth factor receptors are required for activation of Akt and phosphorylation of EphA2 on S897. Two inhibitors of FGFR abolished the stimulatory effects of bFGF (). Although a small decrease was observed, serum-induced activation of Akt and pS897-EphA2 was not significantly reduced by FGFR inhibitors (), consistent with the presence of multiple growth factors in serum.
S897 phosphorylation of EphA2 is required for dendritic actin cytoskeleton assembly and lamellipodia formation at the leading edge of migrating cells
Immunofluorescence staining of U373 expressing WT-EphA2 revealed that pS897-EphA2 was localized to the migrating front with dendritic actin in lamellipodia or tips of F-actin fibers that ran perpendicular to the wound (). As expected, cells expressing mutant S897A-EphA2 showed little staining for phospho-S897 (, lower panel). Staining for total EphA2 showed that the mutant S897A-EphA2 was primarily localized to the cell-cell junctions instead (). Interestingly, prominent actin stress fibers were detected in cells expressing mutant S897A-EphA2 that ran parallel to the wound (). This is analogous to rearrangement of F-actin following stimulation with ephrin-A1, that caused F-actin to switch from a predominantly dendritic pattern in lamellipodia to stress fibers that aligned with the direction of wounding (Fig. S7
). These results suggest that phosphorylation of EphA2 at S897 by Akt is critical for EphA2 localization at cell leading edge, which is required to promote assembly of actin cytoskeleton and extension of lamellipodia.
In human astrocytoma, S897 phosphorylation of EphA2 is correlated with malignant progression and overlaps with active Akt
PI3K/Akt is activated in the vast majority of human GBM (TCGA Research Network, 2008
; Parsons et al., 2008
), and glioma cells induced to migrate are known to have further elevated Akt activities (Joy et al., 2003
). We hypothesize that if Akt phosphorylation of S897-EphA2 facilitates glioma cell migration in vitro, S897 phosphorylation of EphA2 could be elevated during malignant progression of human brain tumors. To test this, we performed immunohistochemical (IHC) staining on paraffin-embedded sections from normal human brains and human gliomas of different grades using the phospho-specific antibody characterized above. We first tested the specificity of the antibody in IHC staining. The antibody detected pS897 signals in a grade IV GBM. On adjacent sections, the signal was blocked by the immunizing phospho-peptide but not the unphosphorylated peptide (), suggesting the antibody specifically recognized p897-EphA2 in tumor sections.
Levels of pS897-EphA2 in human glioma specimens are correlated with tumor grades and overlap with active Akt
As shown in , normal brains from five individuals were largely negative for pS897-EphA2, whereas one case of grade I () and two cases of grade III astrocytomas () showed low to moderate pS897-EphA2 levels. In contrast, 19 of 21 cases of grade IV astrocytomas (GBM) samples contained regions displaying high levels of pS897-EphA2 (). The areas affected varied among different tumors, comprising 5% to over 90% of the non-necrotic regions. The 90% positive rate is consistent with a recent large scale study where Akt is activated in 88% of human GBM cases (TCGA Research Network, 2008
Interestingly the strongest staining for pS897-EphA2 was frequently localized in regions known to be enriched for growth factors and invasive cells in grade IV astrocytomas or GBM. Clusters of strongly positive tumors cells were often observed next to necrotic areas, one of the WHO-designated hallmarks of grade IV astrocytomas () (Miller and Perry, 2007
; Furnari et al., 2007
). The distinctive membrane staining pattern of pS897-EphA2 could be visualized in the high magnification images (). Phospho-S897-EphA2 was also abundantly detected in the perivascular regions (), or at the interface between vasculature and necrosis (). Invasive glioma cells often migrate along blood vessels, which is referred to as perivascular satellitosis, one of morphological signatures of invasive GBM collectively known as Scherer’s secondary structures (Scherer, 1938
; Furnari et al., 2007
). Phospho-S897-EphA2 was also detected in a tumor that has infiltrated meninges adjacent to normal brain (), further supporting its role in promoting tumor cell migration. On multiple sections pS897-EphA2 was found at high levels in cells comprising palisading necrosis (). In GBMs, necrotic foci are often surrounded by pseudopalisading cells, which are thought to be actively migrating and are critical for tumor progression (Brat et al., 2004
; Wippold et al., 2006
). Finally, pS897-EphA2 was found in microvascular proliferation resulting from active angiogenesis in GBM (), suggesting that pS897-EphA2 could also play a role in promoting endothelial cell migration and invasion during neo-angiogenesis.
One prediction based on our data is that pS897-EphA2 and pS473-Akt are likely to reside in the same cells in vivo. To directly examine this possibility, we stained the same set of GBM specimens for pS473-Akt. Interestingly, in most regions where there was strong pS473-Akt, there was also robust staining for pS897-EphA2 on adjacent sections (). The spatial colocalization was most pronounced in tumor cells surrounding blood vessels ( on adjacent slides with ) or necrotic regions (). In about a third of the specimens, pS473-Akt signal was detectable but much weaker than pS897-EphA2, possibly due to the more labile nature of the pS473-Akt epitope, suggesting that p897-EphA2 could serve as surrogate marker for Akt activation. Taken together, the results demonstrate that phosphorylation of EphA2 at S897 by Akt is correlated with malignant progression of human astrocytoma. We propose that the crosstalk between RTK/PI3K/Akt and EphA2 may contribute to malignant progression of GBM and potentially other types of human cancers.