Tyrosine-614, the major autophosphorylation site of the receptor tyrosine kinase HEK2, functions as multi-docking site for SH2-domain mediated interactions

Abstract

HEK2 belongs to the family of EPH-related receptor tyrosine kinases (RTK) which are involved in axonal path®nding and the formation of the embryonic body plan. The knowledge about intracellular pathways of signal transduction mediated by EPH-related receptors is still limited. Many of the known key players of cellular signalling contain Src homology 2 (SH2) domains, which recognize phosphotyrosine motifs in RTKs. Thus, we examined the interactions of various SH2-containing molecules like PLC-g1, rasGAP, p85 subunit of PI3-kinase, Src, Fyn, Crk, Nck, Grb2 and Shc with HEK2 using in vitro binding assays, immunoprecipitations and yeast Two-Hybrid assays. We found that rasGAP, Crk and Fyn bind in a SH2-dependent manner to autophosphorylated HEK2. rasGAP, which contains two SH2-and one SH3-domain, was shown to associate with its Nterminal SH2-domain to HEK2. Furthermore, we demonstrated that a single amino acid substitution (Y614F) clearly reduces the phosphotyrosine content of HEK2 and abrogates its ability to bind rasGAP, Crk and Fyn indicating that this residue functions as major phosphorylation and multi-docking site. The conservation of this predicted binding site among various EPH-related RTKs provides evidence that Fyn, Crk and rasGAP are key players in signal transduction of at least a subset of these receptors.

Phosphorylation at Tyr-838 in the kinase domain of EphA8 modulates Fyn binding to the Tyr-615 site by enhancing tyrosine kinase activity

“…Tyr-615 in EphA8 is located in the juxtamembrane domain and all known Eph-related receptors possess the corresponding tyrosine residue in their juxtamembrane domain (Figure 1). Previous studies indicated that phosphorylation at this conserved tyrosine residue mediates high a nity association with the SH2 domains of either the nonreceptor tyrosine kinases or Ras-GAP (Ellis et al, 1996;Holland et al, 1997;Hock et al, 1998;Zisch et al, 1998). Interestingly, EphA8 lacks the tyrosine residue corresponding to Tyr-596 in EphA4, which is also highly conserved and may modulate protein binding to its neighboring tyrosine residue (Figure 1) (Ellis et al, 1996).…”

“…It is not clear how the SH2 domains of nonreceptor tyrosine kinases discriminate between Eph receptors, but it is likely that additional nonconserved amino acids, in addition to the sequence motif identi®ed as the key site of interaction may provide the speci®city observed. It was recently proposed that the sequence divergence at position +5 relative to Tyr-614 in EphB3, the site corresponding to Tyr-615 in EphA8, might be crucial for determining the binding properties of Ras-GAP (Hock et al, 1998). For instance, the presence of an acidic amino acid such as glutamic or aspartic acid at position +5 determined the binding a nity to the amino-terminal SH2 domain of Ras-GAP as demonstrated in the cases of EphB1 and EphB3.…”

“…Previous studies indicated that tyrosine phosphorylated Eph-related receptors directly interact with the SH2 domains of either nonreceptor tyrosine kinases or Ras GAP (Ellis et al, 1996;Holland et al, 1997;Hock et al, 1998;Zisch et al, 1998). To identify the SH2 domains directly interacting with the tyrosine phosphorylated EphA8 receptors, in vitro binding assays were performed using the bacterially expressed GST-SH2 fusion proteins of Fyn, Src, Ras-GAP or the PI-3 kinase p85 subunit.…”

“…Phosphorylation at Tyr-615 in EphA8 mediates association with the Fyn kinase It is likely that phosphorylation at Tyr-615 in EphA8 mediates association with the Fyn kinase, on the basis of the reports that phosphorylation at the corresponding residue in other Eph-related receptors is required for binding to the SH2 domains of nonreceptor tyrosine kinases or Ras-GAP (Ellis et al, 1996;Holland et al, 1997;Hock et al, 1998;Zisch et al, 1998) F165,F838 . The washed beads were assayed by immunoblot using anti-EphA8 antibodies as probe (Figure 5b, top panel).…”

“…During the past few years, several immediate downstream targets of Eph receptors have been identi®ed. Several known signaling proteins containing SH2 domains, such as Fyn, Src, and Ras GTPase-activating protein (Ras GAP) have been shown to interact with the conserved tyrosine phosphorylation site in the juxtamembrane domain of Eph receptors (Ellis et al, 1996;Holland et al, 1997;Hock et al, 1998;Zisch et al, 1998). It has also been reported that direct interaction of EphB1 with the SH2 domain of the adapter protein Nck involves Tyr-594 phosphorylation in EphB1 (Stein et al, 1998b).…”