Tyrosine phosphorylation of the orphan receptor ESDN/DCBLD2 serves as a scaffold for the signaling adaptor CrkL

Abstract

A quantitative proteomics screen to identify substrates of the Src family of tyrosine kinases (SFKs) whose phosphorylation promotes CrkL-SH2 binding identified the known Crk-associated substrate (Cas) of Src as well as the orphan receptor ESDN. Mutagenesis analysis of ESDN’s seven intracellular tyrosines in YxxP motifs found several contribute to the binding of ESDN to the SH2 domains of both CrkL and a representative SFK Fyn. Quantitative mass spectrometry showed that at least three of these (Y565, Y621 and Y750), as well as non-YxxP Y715, are reversibly phosphorylated. SFK activity was shown to be sufficient, but not required for the interaction between ESDN and the CrkL-SH2 domain. Finally, antibody-mediated ESDN clustering induces ESDN tyrosine phosphorylation and CrkL-SH2 binding.

Dynamic multi-site phosphorylation by Fyn and Abl drives the interaction between CRKL and the novel scaffolding receptors DCBLD1 and DCBLD2

“…The human c-Abl construct, with a C-terminal Flag tag was kindly gifted by A. Howe (U. of Vermont), originally constructed in the Kufe lab (Harvard Medical School) [ 23 ]. DCBLD2 mutant constructs: DCBLD2-Y1F (Tyr750Phe), DCBLD2-Y3F (Tyr750Phe, Tyr732Phe, Tyr565Phe), and DCBLD2-Y7F (Tyr750Phe, Tyr732Phe, Tyr677Phe, Tyr666Phe, Tyr655Phe, Tyr621Phe, Tyr565Phe) were described previously [ 6 ]. The C-terminal Myc- and Flag-tagged DCBLD1 WT and mutant DCBLD1-Y8F (Tyr540Phe, Tyr578Phe, Tyr589Phe, Tyr600Phe, Tyr621Phe, Tyr652Phe, Tyr665Phe, and Tyr696Phe) constructs in pCMV6 vectors were synthesized by Bio Basic Inc. (Markham, ON).…”

“…Previously, we reported a proteomics screen for novel Src Family Kinase (SFK) substrates that, when phosphorylated, would bind to the CRKL SH2 domain [ 6 ]. This screen identified the transmembrane protein D iscoidin, C U B , and L CCL d omain-containing 2 (DCBLD2; also e ndothelial and s mooth muscle cell- d erived n europilin-like, ESDN) as a novel phosphotyrosine-dependent CRKL-SH2 binding partner.…”

“…Furthermore, it was determined that SFKs were sufficient for, but not the only tyrosine kinases capable of, inducing the interaction of DCBLD2 with CRKL. Our initial study also identified four specific sites on DCBLD2 of regulated tyrosine phosphorylation, three of which were in YxxP motifs [ 6 ].…”

“…These YxxP motifs are not only highly conserved among DCBLD2 orthologs, but they are also highly conserved in their related paralog, DCBLD1, which harbors eight YxxP motifs ( Figure 1 ). While to date almost all studies involving DCBLD2 have importantly focused on the interaction of DCBLD2 with RTKs, we have proposed that the clustering of DCBLD proteins by unknown ligands could be an important RTK-independent mechanism of DCBLD signaling [ 6 ]. In order to understand how these conserved motifs might participate in non-RTK or RTK signaling pathways it is important to establish the contribution of specific kinases to the phosphorylation of specific YxxP motifs on DCBLD proteins.…”

The neuropilin-like protein ESDN regulates insulin signaling and sensitivity

“…The extracellular domain of ESDN consists of complement C1r/C1s, Uegf, Bmp1 (CUB); LCCL; and discoidin (FV/VIII) domains. Unlike neuropilins, which are composed of two CUB, two discoidin, a MAM, and a short intracellular domain, ESDN has a relatively long intracellular domain that can function as a scaffold for CT10 regulator of kinase like (4). It is conceivable that ESDN intracellular domain can similarly be involved in its interactions with IR and adaptor proteins, APS and Grb10.…”