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

Anna M Schmoker,Bryan A Ballif,Alicia M Ebert,Hannah E Johnson,Kyle J Kellett,Jaye L Weinert,Ryan M Joy,Marion E Weir

doi:10.1042/bcj20170615

Abstract

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.

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

INTRODUCTION

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)

RESULTS

DISCUSSION

FUNDING INFORMATION