Abstract
Discoidin, CUB, and LCCL domain containing 1 (DCBLD1) and DCBLD2 compose a family of orphan transmembrane scaffolding receptors that possess similar domain structure to that of neuropilins, critical co‐receptors for neuronal guidance cues. While DCBLD1 remains largely uncharacterized in the literature, DCBLD2 has both known and anticipated roles in vascular remodeling and neuronal positioning. Further, DCBLD2 is up‐regulated in several cancers and is required for hyperactive epidermal growth factor receptor (EGFR)‐driven tumorigenesis. While a few studies have demonstrated both positive and negative regulatory roles of DCBLD2 in growth factor receptor signaling, the conserved features of DCBLD family members that drive their molecular activities remain undefined. Previously, we identified DCBLD2 as a Src family kinase substrate that, when phosphorylated on tyrosines in intracellular YXXP motifs, would bind the Src homology 2 (SH2) domain of the signaling adaptor CRKL (CT10 regulator of kinase‐like). These intracellular YXXP motifs are highly conserved, both across vertebrates and between DCBLD family members. Similarly, we demonstrate here that DCBLD1 YXXP motifs are also required to interact with the CRKL–SH2 domain, distinguishing this motif as a conserved signaling feature of DCBLD family proteins. We show that Src family kinases (SFKs) and Abl differentially mediate the CRKL/DCBLD interaction. Although SFKs and Abl each are able to induce both DCBLD family members to bind the CRKL–SH2 domain, the effect of Abl is stronger for DCBLD1. Using LC‐MS/MS, we quantified relative changes in phosphorylation at several YXXP sites in DCBLD1 and DCBLD2 using both stable isotope labeled standards and a label free method, allowing us to map site‐specific preferences for SFKs and Abl. In addition to these published findings, we have characterized phosphorylation of additional tyrosine residues and have identified proteins differentially induced to bind DCBLD family members from a variety of cell types. We propose two mechanisms for DCBLD signaling, namely, a co‐receptor mechanism in tandem with growth factor receptors and a ligand‐induced clustering mechanism. Together, these findings provide a platform to elucidate the mechanisms the drive the biological activities of these unexplored receptors.Support or Funding InformationU.S. National Science Foundation IOS grants [1021795 and 1656510]; Vermont Genetics Network through U.S. National Institutes of Health Grant [8P20GM103449] from the INBRE program of the NIGMS; U.S. National Institutes of Health Grant [5P20RR016435] from the COBRE program of the NIGMS; University of Vermont College of Arts and Sciences Small Grant Research Award; University of Vermont College of Arts and Sciences Undergraduate APLE research AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Highlights
Proper neurodevelopment requires precise temporal and spatial regulation of a complex array of signaling molecules, the regulation of which remains largely uncharacterized
We identified tyrosine phosphorylation sites in YxxP motifs within the DCBLD2 intracellular domain and found that these motifs were essential for the phosphorylationdependent binding of the signaling adaptor CRKL [6]
We previously-reported the importance of DCBLD2 YxxP motifs in the interaction with the CRKL-Src homology 2 (SH2) domain [6]
Summary
Proper neurodevelopment requires precise temporal and spatial regulation of a complex array of signaling molecules, the regulation of which remains largely uncharacterized. In this study we used biochemical methods and several quantitative mass spectrometry approaches to characterize the phosphorylation of DCBLD proteins by the non-receptor tyrosine kinases of the Src and Abl families, both known to target YxxP motifs [16], and both known to be activated by both RTK-dependent and RTKindependent signaling mechanisms [17,18,19,20,21]. Beads were washed three times with BCLB and proteins were eluted and denatured in 25 μL of sample buffer at 95 °C for 5 min prior to analysis via SDS-PAGE and Western blotting. It was predicted that the maximum number of unphosphorylated peptides would be found in the unstimulated condition, and that of phosphorylated peptides would be observed in the co-expression of Fyn and c-Abl with DCBLD(X) in kinase expression experiments, or in the H2O2-stimulated condition in the case of inhibitor treatments. Statistical analyses were performed in JMP Pro 12 Statistical Software (SAS Institute, Cary, NY, USA)
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