Biology subjects

McGlade, C. J.

Publications and source records attributed to McGlade, C. J..

3 recordsLinked to original sources

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

Sleuthing biochemical evidence to elucidate unassigned electron density in a CBL/SLAP2 crystal complex

The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL E3 ubiquitin ligase to downregulate antigen, cytokine, and tyrosine kinase receptor signaling. In contrast to phospho-tyrosine dependent binding of CBL substrates through its tyrosine kinase binding domain (TKBD), CBL TKBD associates with the C-terminal tail of SLAP2 in a phospho-independent manner. To understand the distinct nature of this interaction, a purification protocol for SLAP2 in complex with CBL TKBD was established and the complex crystallized. However, determination of the complex crystal structure was hindered by apparent SLAP2 degradation during the crystallization process, such that only CBL TKBD residues could be modeled initially. Close examination of the CBL TKBD structure revealed a unique dimer interface that included two short segments of electron density of unknown origin. To elucidate which residues of SLAP2 to model in this unassigned density, a co-expression system was generated to test SLAP2 deletion mutants and define the minimal SLAP2 binding region. As well, SLAP2 degradation products were analyzed by mass spectrometry. Model building and map generation features of the Phenix software package were employed, leading to successful modeling of the C-terminal tail of SLAP2 in the unassigned electron density segments.

biochemistry↗

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↗