In this study, we have reported findings that assessed roles of different tyrosine residues of EphA3 in the regulation of kinase activity, cell migration, neurite outgrowth, and growth cone collapse. In particular, these studies identified two distinct tyrosine residues that co-operate to regulate these functions.
Our analyses also show that phosphorylation of the juxtamembrane tyrosine 596 is necessary for EphA3 activation. Replacing Y596 with phenylalanine inactivates EphA3. Both autophosphorylation assay and tyrosine kinase activity assay using the exogenous substrate enolase yielded results that are consistent with this conclusion ( & ). In further support of this conclusion, the tyrosine kinase activity is fully restored by a glutamic acid replacement in the tyrosine 596 mutant Y596E (), which mimics the negative charge of phosphorylated tyrosine at this position (28
). The Y596E mutant displayed a relatively weaker tyrosine autophosphorylation of EphA3 receptor than the wild type, suggesting that Y596 is one of the major phosphorylation sites (). However, a study by Lawrenson and colleagues (27
) reported no loss of tyrosine kinase activity when tyrosine 596 is mutated to phenylalanine in human EphA3 as measured by autophosphorylation. The cause of this discrepancy is not known at present, which could be due to sequence variations between these two molecules. A critical role of the juxtamembrane domain tyrosine residues in regulating the kinase activity is supported by studies of other Eph receptors (28
). Mutagenesis of the corresponding tyrosine at position 605 of EphB2 (corresponding to Y596 in EphA3) resulted in a reduction in both the kinase activity and autophosphorylation levels (28
), consistent with our observations reported here. Interestingly, mutation of a second juxtamembrane region tyrosine of EphB2 (residue 611, corresponding to Y602 in EphA3) also reduces the kinase activity. Mutation of both residues led to complete inactivation of EphB2 kinase activity (28
). In contrast to EphB2, mutation of the Y602 in EphA3 (corresponding to tyrosine 611 in EphB2) does not affect tyrosine kinase activity (). A more recent study provided new evidence to support our conclusion that Y596 is important for kinase activity and Y602 act as a docking site (40
). In this study, Davis et al reported that Y602 and its neighboring residues are solvent exposed instead of packed inside as in the case of EphB2, suggesting that Y602 functions differently (40
). Thus, although some variations exist among different Eph receptors, phosphorylation of the juxtamembrane tyrosine residues is necessary for the activation of the Eph receptors.
The current study identified two cytoplasmic tyrosine residues of EphA3 that together regulate cell migration, neurite outgrowth, and growth cone collapse, Y602 and Y779. Mutation of either of these two residues alone leads to a partial reduction in EphA3 biological activity (, , and ). Mutation of both residues leads to a complete loss of biological activity, although the tyrosine kinase activity of the receptor remains intact (, , ). Phosphorylated tyrosine residues have been known to serve as docking sites for SH2-domain-harboring proteins to allow transduction of signals downstream (27
). Since the kinase activity in these mutants was not altered (), deficiency in EphA3 activity is most likely due to a loss of phosphotyrosine docking sites to link with downstream signaling pathways.
A previous study by Lawrenson et al. also identified Y602 and Y779 as required in a cell rounding assay (27
). In the absence of either Y602 or Y779, EphA3 receptor completely loses its ability to induce cell rounding. Our present study confirms the importance of these two tyrosine residues. In addition, the current study extended the previous analysis in several important aspects. First, the assays used in this study better correlate with cellular behavior in vivo. The wound healing and transwell assays directly measure cell migration, an ubiquitous process during embryogenesis and cancer formation. The neurite outgrowth and growth cone collapse assays mimic behavior of growing neurites when they encounter negative guidance cues in the developing nervous system or during axon regeneration (41
). Although very useful, the cell-rounding assay used previously did not easily confirm with behavior in vivo. Second, we showed here that tyrosine residues other than 596, 602, and 779 are not required for the inhibition of cell migration, process retraction, and growth cone collapse, although it is possible that they may play roles in other biological processes. Third, only a partial loss of EphA3 function was observed when a single mutation was introduced. This difference may be due to a higher sensitivity of the assays used in this study which allows detection of more subtle differences. These observations suggest that different downstream pathways coupled to different phosphotyrosine residues collaborate together to mediate EphA3 functions.
Several different SH2-domain containing proteins have been shown to bind to the corresponding juxtamembrane domain tyrosine residues on Eph receptors including RasGAP, Src, Nck and Vav (42
). Among these proteins, RasGAP has been shown to inhibit R-ras activity and regulate cell migration and growth cone collapse (38
). Src family kinases are also required for growth cone collapse (31
). In addition, our previously published study showed that Nck1 binds to phosphorylated Y602 directly and is involved in the regulation of cell migration (44
). Nck has also been shown to interact strongly with R-Ras proline-rich region (48
) even though the role of Nck in R-Ras mediated integrin activation is still not clear (49
). Thus, the inhibitory effects mediated by Y602 may be due to the interaction with either RasGAP, Src, Nck1, or Vav proteins.
The phosphorylated Y779 has been shown to bind to SH2-domain-containing Crk adaptor protein (27
). Interaction of Crk with Y779 is required for cell rounding effects induced by EphA3 activation (27
), and RhoA activation depends on Crk activity (50
). Activation of the small GTPase RhoA is a downstream event required by growth cone collapse induced by ephrin-A5 through the activation of EphA receptors (51
). Therefore, the partial loss of activity of EphA3 mutant Y779F may be due to the inability to activate RhoA. Taken together, our studies show that multiple pathways coupled to Y602 and Y779 contribute to EphA3-mediated regulation of cell migration and axon guidance. Since EphA3 mutations are found in several different types of human cancer (52
), information from the present study may also contribute to the understanding of mechanism of tumorigenesis.