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Oligopeptide Competition Assay for Phosphorylation Site Determination
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Dynamic multi-site phosphorylation by Fyn and Abl drives the interaction between CRKL and the novel scaffolding

Anna M Schmoker1, Jaye L Weinert1, Kyle J Kellett1

  • 1Department of Biology, University of Vermont, 109 Carrigan Drive, 120A Marsh Life Sciences, Burlington, VT 05405, U.S.A.

The Biochemical Journal
|October 14, 2017
PubMed

Insights

Discoidin domain-containing proteins DCBLD1 and DCBLD2 utilize conserved YxxP motifs for binding signaling adaptors CRK and CRKL. Kinases Src family kinases (SFKs) and Abl differentially regulate this interaction, providing insights into receptor signaling.

Area of Science:

  • Molecular and Cellular Biology
  • Cancer Biology
  • Signal Transduction

Background:

  • Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like receptor implicated in vascular remodeling, neuronal positioning, and cancer, particularly glioblastoma.
  • While DCBLD2's role in growth factor signaling is partially understood, the conserved signaling mechanisms of the DCBLD family remain largely unexplored.
  • Previous work identified tyrosine phosphorylation sites in DCBLD2's YxxP motifs, crucial for binding signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like).

Purpose of the Study:

  • To investigate the conserved signaling features of DCBLD family members, focusing on the role of YxxP motifs in DCBLD1 and DCBLD2.
  • To determine how Src family kinases (SFKs) and Abl influence the interaction between DCBLD1/DCBLD2 and the CRKL-SH2 domain.
  • To map site-specific phosphorylation events on DCBLD1 and DCBLD2 by SFKs and Abl.

Main Methods:

  • Investigated the requirement of YxxP motifs in DCBLD1 for CRKL-SH2 (Src homology 2) domain binding.
  • Assessed the differential effects of SFKs and Abl on the interaction between DCBLD1/DCBLD2 and the CRKL-SH2 domain.
  • Utilized high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) to quantify phosphorylation sites and map kinase specificities.

Main Results:

  • Confirmed that DCBLD1 YxxP motifs are essential for CRKL-SH2 binding, similar to DCBLD2.
  • Demonstrated that both SFKs and Abl promote CRKL-SH2 binding to DCBLD1 and DCBLD2, with Abl showing a more pronounced effect on DCBLD1.
  • Successfully mapped site-specific phosphorylation preferences of SFKs and Abl on DCBLD1 and DCBLD2 YxxP sites.

Conclusions:

  • The conserved YxxP motifs in DCBLD1 and DCBLD2 are critical for their interaction with CRKL.
  • SFKs and Abl play distinct roles in modulating DCBLD1 and DCBLD2 signaling through CRKL.
  • These findings establish a foundation for understanding the signaling pathways regulated by DCBLD family members.
Keywords:
AblCrkLDcbldFynphosphorylationquantitative mass spectrometry

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