Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates

Expert consensus document:Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the "European Network for the Study of Cholangiocarcinoma" (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted

Insights into gli factors ubiquitylation methods

The Hedgehog (Hh) signaling pathway governs cell growth and tissue development. Malfunctioning of several Hh pathway components, including the key transcriptional effector Gli proteins, is responsible for the onset of several tumors. Gli proteins activity is finely controlled by multilayered regulatory mechanisms, the most prominent of which is their proteasome-dependent proteolytic cleavage or massive ubiquitin-mediated proteolysis. Here, we described multiple procedures to determine whether a Gli protein is ubiquitylated both in a cellular context and in vitro, in basal conditions or by different E3 ubiquitin ligases and whether these processes are associated to Gli proteasome degradation

Numb is a suppressor of Hedgehog signalling and targets Gli1 for Itch- dependent ubiquitination

The developmental protein Numb is a major determinant of binary cell fates. It is also required for the differentiation of cerebellar granule cell progenitors (GCPs) at a stage of development responsive to the morphogenic glycoprotein Hedehog. Hedgehog signalling is crucial for the physiological maintenance and self-renewal of neural stem cells and its deregulation is responsible for their progression towards tumorigenesis. The mechanisms that inhibit this pathway during the differentiation stage are poorly understood. Here, we identify Numb as a Hedgehog-pathway inhibitor that is downregulated in early GCPs and GCP-derived cancer cells. We demonstrate that the Hedgehog transcription factor Gli1 is targeted by Numb for Itch-dependent ubiquitination, which suppresses Hedgehog signals, thus arresting growth and promoting cell differentiation. This novel Numb-dependent regulatory loop may limit the extent and duration of Hedgehog signalling during neural-progenitor differentiation, and its subversion may be a relevant event in brain tumorigenesis

Jagged 1 is a major Notch ligand along cholangiocarcinoma development in mice and humans

Intrahepatic cholangiocarcinoma (ICC) is a rare yet deadly malignancy with limited treatment options. Activation of the Notch signalling cascade has been implicated in cholangiocarcinogenesis. However, while several studies focused on the Notch receptors required for ICC development, little is known about the upstream inducers responsible for their activation. Here, we show that the Jagged 1 (Jag1) ligand is almost ubiquitously upregulated in human ICC samples when compared with corresponding non-tumorous counterparts. Furthermore, we found that while overexpression of Jag1 alone does not lead to liver tumour development, overexpression of Jag1 synergizes with activated AKT signalling to promote liver carcinogenesis in AKT/Jag1 mice. Histologically, tumours consisted exclusively of ICC, with hepatocellular tumours not occurring in AKT/Jag1 mice. Furthermore, tumours from AKT/Jag1 mice exhibited extensive desmoplastic reaction, an important feature of human ICC. At the molecular level, we found that both AKT/mTOR and Notch cascades are activated in AKT/Jag1 ICC tissues, and that the Notch signalling is necessary for ICC development in AKT/Jag1 mice. In human ICC cell lines, silencing of Jag1 via specific small interfering RNA reduces proliferation and increases apoptosis. Finally, combined inhibition of AKT and Notch pathways is highly detrimental for the in vitro growth of ICC cell lines. In summary, our study demonstrates that Jag1 is an important upstream inducer of the Notch signalling in human and mouse ICC. Targeting Jag1 might represent a novel therapeutic strategy for the treatment of this deadly disease.NIH [R01CA136606, R01CA190606]; Deutsche Forschungsgemeinschaft DFG [RI2695/1-1]; China Scholarship Council [201408525080]; [P30DK026743]SCI(E)[email protected]; [email protected]