Biology subjects

Vit, G.

Publications and source records attributed to Vit, G..

3 recordsLinked to original sources

bioRxiv2026

A chemogenetic CRISPR knockout screen identifies a ubiquitin-mediated calcineurin-BMP pathway relay in T cell activation

T cell activation is dependent on calcineurin signalling, yet how this pathway integrates with other regulatory systems remains incompletely understood. Here, we exploit the dual pharmacology of FK506, which inhibits calcineurin while also releasing FKBP12-mediated repression of BMP receptors, to dissect signalling crosstalk during T cell responses. A comparative genome-wide CRISPR knockout screen using FK506 and Cyclosporin A revealed that FK506 uniquely engages BMP pathway components and ubiquitin regulatory networks. Functional analyses in Jurkat and primary human T cells showed that FK506 induces BMP receptor-dependent activation of SMAD1/5/8 and triggers a rapid remodelling of K48- and K29-linked ubiquitin chains. Proteomics further demonstrated selective ubiquitination of immune signalling proteins and ubiquitin regulators, linking BMP pathway activation to proteasome-dependent turnover and non-proteolytic ubiquitin signalling. In primary T cells, BMP receptor signalling enhance calcineurin-driven activation, while changes in ubiquitin conjugation modulate this response, thereby establishing an integrated calcineurin-BMP-ubiquitin relay. Together, our results uncover a context-dependent signalling axis in which BMP activation and ubiquitin dynamics fine-tune calcineurin-mediated T cell signalling.

cell biology↗

bioRxiv2025

A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

Serine/Threonine phosphoprotein phosphatases (PPPs, PP1-PP7) are conserved metalloenzymes and central to intracellular signaling in eukaryotes, but the details of their regulation is poorly understood. To address this, we performed genome-wide CRISPR knockout and focused base editor screens in PPP perturbed conditions to establish a high-resolution functional map of PPP regulation that pinpoints novel regulatory mechanisms. Through this, we identify the orphan reductase CYB5R4 as an evolutionarily conserved activator of PP4 and PP6, but not the closely related PP2A. Heme binding is essential for CYB5R4 function and mechanistically involves the reduction of the metal ions in the active site. Importantly, CYB5R4-mediated activation of PP4 is critical for cell viability when cells are treated with DNA damage-inducing agents known to cause oxidative stress. The discovery of a dedicated PPP reductase points to shared regulatory principles with protein tyrosine phosphatases, where specific enzymes dictate activity by regulating the active site redox state. In sum, our work provides a resource for understanding PPP function and the regulation of intracellular signaling.

cell biology↗

bioRxiv2021

Cellular toxicity of iHAP1 and DT-061 does not occur through PP2A-B56 targeting

PP2A is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The small molecule compounds iHAP1 and DT-061 have recently been reported by Leonard et al. (2020) and Morita et al. (2020) in Cell to selectively stabilize specific PP2A-B56 complexes which mediate cell killing. Here, we show that this is not the case and question key findings in these papers. Through genome wide CRISPR-Cas9 screens, we uncover the biological pathways targeted by these compounds. We find that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the endoplasmic reticulum and we directly visualize DT-061 in cytoplasmic granules that co-localize with Golgi markers. Our work demonstrates that iHAP1 and DT-061 cannot be used for dissecting PP2A-B56 biology.

cell biology↗