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Protein kinases of the Casein Kinase 1 family play a vital role in the regulation of numerous cellular processes. Apart from functions associated with regulation of proliferation, differentiation, or apoptosis, localization of several Casein Kinase 1 isoforms to the centrosome and microtubule asters also implicates regulatory functions in microtubule dynamic processes. Being localized to the spindle apparatus during mitosis Casein Kinase 1 directly modulates microtubule dynamics by phosphorylation of tubulin isoforms. Additionally, site-specific phosphorylation of microtubule-associated proteins can be related to the maintenance of genomic stability but also microtubule stabilization/destabilization, e.g., by hyper-phosphorylation of microtubule-associated protein 1A and RITA1. Consequently, approaches interfering with Casein Kinase 1-mediated microtubule-specific functions might be exploited as therapeutic strategies for the treatment of cancer. Currently pursued strategies include the development of Casein Kinase 1 isoform-specific small molecule inhibitors and therapeutically useful peptides specifically inhibiting kinase-substrate interactions.

Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor – the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.

The Notch signaling pathway is an evolutionary conserved signal transduction cascade present in almost all tissues and is required for embryonic and postnatal development, as well as for stem cell maintenance, but it is also implicated in tumorigenesis including pancreatic cancer and leukemia. The transcription factor RBPJ forms a coactivator complex in the presence of a Notch signal, whereas it represses Notch target genes in the absence of a Notch stimulus. In the pancreas, a specific paralog of RBPJ, called RBPJL, is expressed and found as part of the heterotrimeric PTF1-complex. However, the function of RBPJL in Notch signaling remains elusive. Using molecular modeling, biochemical and functional assays, as well as single-molecule time-lapse imaging, we show that RBPJL and RBPJ, despite limited sequence homology, possess a high degree of structural similarity. RBPJL is specifically expressed in the exocrine pancreas, whereas it is mostly undetectable in pancreatic tumour cell lines. Importantly, RBPJL is not able to interact with Notch−1 to −4 and it does not support Notch-mediated transactivation. However, RBPJL can bind to canonical RBPJ DNA elements and shows migration dynamics comparable to that of RBPJ in the nuclei of living cells. Importantly, RBPJL is able to interact with SHARP/SPEN, the central corepressor of the Notch pathway. In line with this, RBPJL is able to fully reconstitute transcriptional repression at Notch target genes in cells lacking RBPJ. Together, RBPJL can act as an antagonist of RBPJ, which renders cells unresponsive to the activation of Notch.

The Notch pathway is a conserved signaling mechanism that is essential for cell fate decisions during pre and postnatal development. Dysregulated signaling underlies the pathophysiology of numerous human diseases, most notably T-cell acute lymphoblastic leukemia. Receptor-ligand interactions result in changes in gene expression, which are regulated by the transcription factor CSL. CSL forms a complex with the intracellular domain of the Notch receptor and the transcriptional coactivator Mastermind, which is required to activate transcription of all Notch target genes. CSL can also function as repressor by interacting with corepressor proteins, e.g. SHARP in mammals and Hairless in Drosophila melanogaster; however, its role as a transcriptional repressor is not well understood. Here we determine the high-resolution structure of RBPJ, the mouse CSL ortholog, bound to the corepressor SHARP and DNA, which reveals a new mode of corepressor binding to CSL and an interesting example for how ligand binding sites evolve in proteins. Based on the structure, we designed and tested a number of mutants in biophysical, biochemical, and cellular assays to characterize the role of RBPJ as a repressor of Notch target genes. Our cellular studies clearly demonstrate that RBPJ mutants that are deficient for binding SHARP are incapable of repressing transcription from genes responsive to Notch signaling. Altogether, our structure-function studies of the RBPJ-SHARP corepressor complex bound to DNA provide significant insights into the repressor function of RBPJ and identify a new binding pocket on RBPJ that could be targeted for therapeutic benefit. Footnotes * n/a