SUMMARYGenetic instability of the mitochondrial genome (mtDNA) plays an important role in human aging and disease. Thus far, it has proven difficult to develop successful treatment strategies for diseases that are caused by mtDNA instability. To address this issue, we developed a model of mtDNA disease in the nematode C. elegans, an animal model that can rapidly be screened for genes and biological pathways that reduce mitochondrial pathology. These worms recapitulate all the major hallmarks of mtDNA disease in humans, including increased mtDNA instability, loss of respiration, reduced neuromuscular function, and a shortened lifespan. We found that these phenotypes could be rescued by intervening in numerous biological pathways, including IGF-1/insulin signaling, mitophagy, and the mitochondrial unfolded protein response, suggesting that it may be possible to ameliorate mtDNA disease through multiple molecular mechanisms.

Discoidin, CUB, and LCCL Domain-containing (DCBLD) 2 is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also upregulated in several cancers and can drive glioblastomas downstream of activated Epidermal Growth Factor Receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-Like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL-SH2 binding. We report Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL-SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promotes DCBLD1 and DCBLD2 binding to the CRKL-SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The non-receptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs. However, we have identified additional FYN- and ABL-dependent phosphorylation sites on DCBLD1 and DCBLD2. This suggests that beyond CRK and CRKL, additional DCBLD interactors may be regulated by FYN and ABL activity. Here, we report a quantitative proteomics approach in which we map the FYN- and ABL-regulated interactomes of DCBLD family members. We found FYN and ABL regulated the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.

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.