Research Article
SLAP2 Adaptor Binding Disrupts c-CBL Autoinhibition to Activate Ubiquitin Ligase Function

https://doi.org/10.1016/j.jmb.2021.166880Get rights and content
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Highlights

  • Src-like adaptors (SLAP/SLAP2) bind the E3 ligase CBL and regulate tyrosine kinase signaling.

  • Crystal structure reveals SLAP2 binds in a region important for CBL autoinhibited conformation.

  • Binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL.

  • The SLAP2-CBL protein-protein interaction provides an alternative CBL activation mechanism.

Abstract

CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

Keywords

Src-like adaptor protein
CBL
protein-protein interaction
ubiquitin ligase
X-ray crystal structure
activation mechanism

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