iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells.

To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented "hook effect" of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome.

Salt-inducible kinases (SIKs) regulate TGFß-mediated transcriptional and apoptotic responses.

The signalling pathways initiated by members of the transforming growth factor-ß (TGFß) family of cytokines control many metazoan cellular processes, including proliferation and differentiation, epithelial-mesenchymal transition (EMT) and apoptosis. TGFß signalling is therefore strictly regulated to ensure appropriate context-dependent physiological responses. In an attempt to identify novel regulatory components of the TGFß signalling pathway, we performed a pharmacological screen by using a cell line engineered to report the endogenous transcription of the TGFß-responsive target gene PAI-1. The screen revealed that small molecule inhibitors of salt-inducible kinases (SIKs) attenuate TGFß-mediated transcription of PAI-1 without affecting receptor-mediated SMAD phosphorylation, SMAD complex formation or nuclear translocation. We provide evidence that genetic inactivation of SIK isoforms also attenuates TGFß-dependent transcriptional responses. Pharmacological inhibition of SIKs by using multiple small-molecule inhibitors potentiated apoptotic cell death induced by TGFß stimulation. Our data therefore provide evidence for a novel function of SIKs in modulating TGFß-mediated transcriptional and cellular responses.