Biology subjects

Chartier, C. A.

Publications and source records attributed to Chartier, C. A..

2 recordsLinked to original sources

bioRxiv2024

Allosteric regulation of the tyrosine phosphatase PTP1B by a protein-protein interaction

The rapid identification of protein-protein interactions has been significantly enabled by mass spectrometry (MS) proteomics-based methods, including affinity purification-MS, crosslinking-MS, and proximity-labeling proteomics. While these methods can reveal networks of interacting proteins, they cannot reveal how specific protein-protein interactions alter protein function or cell signaling. For instance, when two proteins interact, there can be emergent signaling processes driven purely by the individual activities of those proteins being co-localized. Alternatively, protein-protein interactions can allosterically regulate function, enhancing or suppressing activity in response to binding. In this work, we investigate the interaction between the tyrosine phosphatase PTP1B and the adaptor protein Grb2, which have been annotated as binding partners in a number of proteomics studies. This interaction has been postulated to co-localize PTP1B with its substrate IRS-1 by forming a ternary complex, thereby enhancing the dephosphorylation of IRS-1 to suppress insulin signaling. Here, we report that Grb2 binding to PTP1B also allosterically enhances PTP1B catalytic activity. We show that this interaction is dependent on the proline-rich region of PTP1B, which interacts with the C-terminal SH3 domain of Grb2. Using NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) we show that Grb2 binding alters PTP1B structure and/or dynamics. Finally, we use MS proteomics to identify other interactors of the PTP1B proline-rich region that may also regulate PTP1B function similarly to Grb2. This work presents one of the first examples of a protein allosterically regulating the enzymatic activity of PTP1B and lays the foundation for discovering new mechanisms of PTP1B regulation in cell signaling. Significance StatementProtein-protein interactions are critical for cell signaling. The interaction between the phosphatase PTP1B and adaptor protein Grb2 co-localizes PTP1B with its substrates, thereby enhancing their dephosphorylation. We show that Grb2 binding also directly modulates PTP1B activity through an allosteric mechanism involving the proline-rich region of PTP1B. Our study reveals a novel mode of PTP1B regulation through a protein-protein interaction that is likely to be exploited by other cellular interactors of this important signaling enzyme.

biochemistry↗

bioRxiv2023

The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

Mutations in the tyrosine phosphatase SHP2 are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting auto-inhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C- terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that, while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8-10 [A] from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation. Significance StatementThe protein tyrosine phosphatase SHP2 is mutated in a variety of human diseases, including several cancers and developmental disorders. Most mutations in SHP2 hyperactivate the enzyme by destabilizing its auto-inhibited state, but several disease-associated mutations do not conform to this mechanism. We show that one such mutation, T42A, alters the ligand binding specificity of the N-terminal regulatory domain of SHP2, causing the mutant phosphatase to be more readily activated by certain upstream signals than the wild-type phosphatase. Our findings reveal a novel mode of SHP2 dysregulation that will improve our understanding of pathogenic signaling. Our study also illustrates how mutations distal to the specificity-determining region of a protein can alter ligand binding specificity.

biochemistry↗