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

Abstract

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated 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 (Src homology 2) binding. We report that 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 promote 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.

• Authors: Anna M Schmoker, Jaye L Weinert, Kyle J Kellett, Hannah E Johnson, Ryan M Joy, Marion E Weir, Alicia M Ebert, Bryan A Ballif
• Journal: The Biochemical journal (Biochem J) Vol. 474 Issue 23 Pg. 3963-3984 (11 21 2017) ISSN: 1470-8728 [Electronic] England
• PMID: 29025973 (Publication Type: Journal Article)
• Copyright: © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Chemical References

• Adaptor Proteins, Signal Transducing
• CRKL protein
• DCBLD2 protein, human
• Membrane Proteins
• Nuclear Proteins
• Oncogene Proteins v-abl
• Protein Isoforms
• Recombinant Proteins
• FYN protein, human
• Proto-Oncogene Proteins c-fyn

Topics

• Adaptor Proteins, Signal Transducing (chemistry, genetics, metabolism)
• Amino Acid Sequence
• Animals
• Binding Sites
• Cloning, Molecular
• Conserved Sequence
• Escherichia coli (genetics, metabolism)
• Gene Expression
• HEK293 Cells
• Humans
• Membrane Proteins (chemistry, genetics, metabolism)
• Mice
• Nuclear Proteins (chemistry, genetics, metabolism)
• Oncogene Proteins v-abl (chemistry, metabolism)
• Phosphorylation
• Plasmids (chemistry, metabolism)
• Protein Binding
• Protein Interaction Domains and Motifs
• Protein Isoforms (chemistry, genetics, metabolism)
• Proto-Oncogene Proteins c-fyn (chemistry, metabolism)
• Rats
• Recombinant Proteins (chemistry, genetics, metabolism)
• Sequence Alignment
• Sequence Homology, Amino Acid
• Zebrafish