Biology subjects

Simpson, C. D.

Publications and source records attributed to Simpson, C. D..

3 recordsLinked to original sources

bioRxiv2026

Targeting CBL ubiquitin ligase activation to downregulate tyrosine kinase signalling

The CBL E3 ubiquitin ligase is a critical regulator of tyrosine kinase (TK) signalling. CBL activity is regulated by a feedback loop in which an active TK phosphorylates CBL, relieving its autoinhibited conformation, and allowing ubiquitination of substrates, including TKs, leading to their degradation. Binding of the Src Like Adapter Protein 2 (SLAP2) to CBL can also activate autoinhibited CBL and promote substrate ubiquitination. Using an engineered CBL mutant in the SLAP2 binding interface, termed RE CBL, that mimics activation by SLAP2 binding, we characterized the cellular functions of this CBL activation mechanism. Comparison of wildtype and RE CBL interactomes using MiniTurboID showed extensive and overlapping interaction networks, with a discrete subset of proteins, including the known substrate, epidermal growth factor receptor (EGFR), as well as endocytic factors such as EPS15, in higher abundance with RE CBL compared to wildtype. Consistent with these observations, RE CBL interacted more readily with EGFR, enhanced EGFR internalization, and attenuated downstream signalling compared to WT. In Cbl null hematopoietic cells, RE CBL expression reduced sensitivity to cytokines IL-3 and GM-CSF, and decreased activation of the Src-family kinase Lyn. Furthermore, we optimized and conducted a small molecule screen to identify a group of structurally related compounds that, like SLAP2 binding, promoted CBL activation in vitro. Together these findings provide proof of concept for targeting CBL activity to downregulate TK signalling.

biochemistry↗

bioRxiv2021

Isoform-specific functions of Numb in breast cancer progression, metastasis and proteome remodeling.

Deregulated alternative splicing of the endocytic adaptor NUMB resulting in high expression of Exon9in (exon 9-containing) isoforms has been reported in several cancer types. However, the role of Numb isoform expression in tumor progression and the underlying mechanisms remain elusive. Here, we report greater exon 9 inclusion in multiple cancer types including all subtypes of breast cancer, and correlation of higher exon 9 inclusion in patients with worse prognosis. Deletion of Exon9in in breast cancer cells leads to reduced cell growth and a significant decrease of lung metastasis in orthotopic xenograft experiments. Quantitative mass spectrometry revealed downregulation of proteins involved in EMT and ECM organization and remodeling of the endocytic protein network in cells lacking the Exon9in Numb isoforms. Exon 9 deletion also results in reduced surface levels of ITG{beta}5, and downstream signaling to ERK and SRC, consistent with enhance lysosomal targeting mediated by the remaining Exon9sk (exon 9 skipping) Numb isoforms. SIGNIFICANCEExpression of NUMB Exon9in protein isoforms correlate with worse progression free survival, particularly in breast cancer. Our findings also reveal that Exon9in isoforms promote breast cancer progression by relieving Numb mediated down regulation of integrins and implicate Numb alternative splicing as a progression factor in multiple cancer types.

cancer biology↗

bioRxiv2020

Structural basis for phosphorylation independent activation of c-CBLby Src-like adaptor protein 2.

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 -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.

molecular biology↗