MALT1 Knockout Cell Lysate

Glycogen synthase kinase 3 beta (GSK3 beta) is a ubiquitously expressed serine/threonine kinase involved in the regulation of various cellular functions, such as energy homoeostasis, cell growth and developmental processes. More recently, GSK3 beta has been identified as a part of a protein complex involved in the regulation of the CARMA1-BCL10-MALT1 complex (CBM complex) formation, which is a key signalling event upon antigen receptor engagement of B and T cells, required for the activation of the NF-kappa B and JNK pathways. However, conflicting reports have been published regarding the role of GSK3 beta for the activation of the NF-kappa B signalling pathways. Therefore, we aimed to determine the impact of GSK3 beta on the NF-kappa B signalling induced upon T cell activation. Blocking GSK3 beta by either pharmacologic inhibitors (SB216763 and SB415286) or by RNAi caused a reduced proteolysis of the MALT1 targets CYLD1, BCL10 and RelB as well as diminished I kappa B alpha degradation, NF-kappa B DNA binding and NF-kappa B activity. This negative effect on NF-kappa B appears to be due to a diminished CBM complex formation caused by a reduced BCL10 phosphorylation. Taken together, we provide here evidence for a novel regulatory mechanism by which GSK3 beta affects NF-kappa B signalling in activated T cells.

Metacaspases, distant relatives of metazoan caspases, have been shown to participate in programmed cell death in plants and in progression of the cell cycle and removal of protein aggregates in unicellular eukaryotes. However, since natural proteolytic substrates have scarcely been identified to date, their roles in these processes remain unclear. Here, we report that the DNA-damage inducible protein 1 (Ddi1) represents a conserved protein substrate for metacaspases belonging to divergent unicellular eukaryotes (trypanosomes and yeasts). We show that although the recognized cleavage sequence is not identical among the different model organisms tested, in all of them the proteolysis consequence is the removal of the ubiquitin-associated domain (UBA) present in the protein. We also demonstrate that Ddi1 cleavage is tightly regulated in vivo as it only takes place in yeast when calcium increases but under specific metabolic conditions. Finally, we show that metacaspase-mediated Ddi1 cleavage reduces the stability of this protein which can certainly impact on the many functions ascribed for it, including shuttle to the proteasome, cell cycle control, late secretory pathway regulation, among others.