Direct Phosphorylation of SRC Homology 3 Domains by Tyrosine Kinase Receptors Disassembles Ligand-Induced Signaling Networks

Abstract

Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.

Eph receptor signalling: from catalytic to non-catalytic functions

“…5a). The activated Eph kinase domain then phosphorylates downstream substrates, such as adaptor protein Nck1/2 [37]. The two most frequent autophosphorylation sites are located in the JM region, JX1 and JX2, as demonstrated by in vitro kinase assays and in cell proteomic mapping [38].…”

“…The main difficulty in identifying the Eph receptor kinase domain direct downstream substrates is due to the associated SH2 domain-containing SFKs, as both of them are tyrosine kinases with potential overlapping substrates. Nck1/2, an adaptor protein that is involved in cytoskeletal organisation, was recently identified to be a direct substrate of EphA4 in vitro and in cells [37]. The binding between the Nck SH3 domain and its interacting partners was abolished once the conserved tyrosine residue in the Nck SH3 domain is phosphorylated by EphA4.…”

LY75 Ablation Mediates Mesenchymal-Epithelial Transition (MET) in Epithelial Ovarian Cancer (EOC) Cells Associated with DNA Methylation Alterations and Suppression of the Wnt/β-Catenin Pathway

“…Gel bands of interest of the SKOV3-M peptide fraction were digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. Protein digestion and MS analyses were performed at the Proteomics Platform of the CHU de Québec Research Center (Quebec, PQ, Canada), using the Ekspert NanoLC425 HPLC system (Eksigent technologies Dublin, CA, USA) coupled to a 5600+ mass spectrometer (Sciex, Framingham, MA, USA) equipped with a nanoelectrospray ion source, as previously described [94]. MGF peak list files were consecutively created using Protein Pilot version 4.5 software (Sciex, Framingham, MA, USA).…”

Structural insights into the tyrosine phosphorylation–mediated inhibition of SH3 domain–ligand interactions

“…Source data for biochemical experiments that support the findings of this study are available from the corresponding authors upon reasonable request. Addendum-While this manuscript was being prepared for publication, an article by Dionne et al (39) appeared reporting the phosphorylation of TyrC residues in the SH3 domains of NCK by the receptor tyrosine kinase Eph4a. Their findings confirm our conclusions showing that phosphorylation of TyrC inhibits ligand binding and functions as a negative regulatory loop through which receptor tyrosine kinases terminate signaling.…”