PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor

Abstract

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequencespecific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.The effects of many growth factors and cytokines are mediated by high-affinity binding to receptor tyrosine kinases (RTKs) resulting in autophosphorylation of the cytoplasmic domain (1). Phosphotyrosine residues serve as binding sites for downstream signaling proteins that establish a complex network of interactions within the cell. Modular structures involved in binding of activated RTKs identified so far include Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains, which both bind specifically to phosphorylated tyrosine residues in a sequencespecific fashion (2).Recently, we isolated two Eph-related RTKs, named EphB3 and EphB2 (refs. 3 and 4; B.B., U.H., T.K., and K.S., unpublished data), which both bind specifically to the transmembrane subgroup of Eph-receptor ligands (5, 6). The genetic analysis of EphB2 and EphB3 revealed a physiological requirement of both receptors for pathfinding of specific commissural axons in the central nervous system (7,8). To date, the information about signaling molecules mediating Eph-receptor-specific responses is still limited to a few family members. Interactions have been described for the activated Eph-family members EphA2, EphA4, and EphB1 and the SH2-domain-containing proteins p85 subunit of phosphatidylinositol 3-kinase, the adapter protein SLAP, Grb2, Grb10, and Fyn (8-11). Lately, we found that activated EphB3 interacts with Crk, Fyn, and rasGAP in a SH2-dependent manner (12): Autophosphorylation of Tyr-614 in the juxtamembrane region of the receptor generates a multi-docking-site for these interactions...

Serine protease Omi/HtrA2 targets WARTS kinase to control cell proliferation

“…However, LTS2 could neither interact with the Omi PDZ domain nor be cleaved by STS-activated Omi protease under our experimental conditions (Supplementary Information, Figure S9, S10). Recent analysis of target specificity between the PDZ domain and its binding partner has demonstrated that like the extreme C-terminal three peptides the residues at positions -1, -4 and/or -5 of ligands are important for many PDZ interactions (Songyang et al, 1997;Hock et al, 1998). Given that amino acids at positions -4 and -5 of LTS2 differ from the corresponding locations on WTS (WTS: Arg-AspLeu-Val-Tyr-Val; LTS2: Cys-Gln-Pro-Val-Tyr-Val), the interaction is specific for WTS.…”

The C-terminal PDZ-Ligand of JAGGED1 Is Essential for Cellular Transformation

“…More specifically, we assessed if the PDZ-domain protein AF6 could bind the intracellular domain of JAGGED1, since AF6 was previously reported to interact with six residues (RMEYIV) found at the C terminus of Jagged1 using a directed yeast two-hybrid analysis (13). Furthermore, AF6 is the mammalian homolog of Drosophila canoe which has been genetically linked to the Notch pathway (46).…”

“…3A) (13). To determine if these residues were required for transformation by JAGGED1, we generated a mutant that has a deletion of the six C-terminal residues that comprise the PDZ-ligand (J1 ⌬PL ).…”

“…However, an important question remains to be addressed: Does the signal-transmitting cell itself receive a signal that can be transmitted through the expressed DSL protein? Interestingly, the C terminus of JAGGED1 encodes a putative PDZ (PSD-95/Dlg/Zo-1)-ligand (13). Therefore, a potential role for JAGGED1 in novel PDZ-dependent signaling mechanisms exists.…”

Bidirectional ephrin/Eph signaling in synaptic functions

“…This interaction inhibits the Ras-MAPK pathway and plays important roles in growth cone collapse, cell repulsion and morphogenesis of capillary endothelium. The C-terminal PDZ-binding motif of Eph receptors mediates their interactions with many neuronal PDZ scaffolding proteins, such as glutamate receptor interacting protein (GRIP), protein interacting with C-kinase (PICK1) and AF-6 [4,39,42,81]. Forward signaling through Eph receptors induces changes to the local milieu either through modulating actin dynamics or through the localization of scaffolding molecules in lipid rafts.…”