SummaryThe cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.

We demonstrate a role for protein kinase casein kinase 2 (CK2) in the phosphorylation and regulation of the M3-muscarinic receptor in transfected cells and cerebellar granule neurons. On agonist occupation, specific subsets of receptor phosphoacceptor sites (which include the SASSDEED motif in the third intracellular loop) are phosphorylated by CK2. Receptor phosphorylation mediated by CK2 specifically regulates receptor coupling to the Jun-kinase pathway. Importantly, other phosphorylation-dependent receptor processes are regulated by kinases distinct from CK2. We conclude that G protein–coupled receptors (GPCRs) can be phosphorylated in an agonist-dependent fashion by protein kinases from a diverse range of kinase families, not just the GPCR kinases, and that receptor phosphorylation by a defined kinase determines a specific signalling outcome. Furthermore, we demonstrate that the M3-muscarinic receptor can be differentially phosphorylated in different cell types, indicating that phosphorylation is a flexible regulatory process where the sites that are phosphorylated, and hence the signalling outcome, are dependent on the cell type in which the receptor is expressed.

We investigated the possibility of Ca 2ϩ signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca 2ϩ levels ([Ca 2ϩ ] i ) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca 2ϩ -binding photoprotein apoaequorin. The homeostasis of intracellular Ca 2ϩ in response to increasing external Ca 2ϩ has been studied in this strain. The resting level of free Ca 2ϩ in Anabaena was found to be between 100 and 200 nm. Additions of increasing concentrations of external Ca 2ϩ gave a transient burst of [Ca 2ϩ ] i followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca 2ϩ ] i back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca 2ϩ levels. We also monitored Ca 2ϩ transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was dependent on the way they were induced. The use of inhibitors suggests that heat shock mobilizes cytosolic Ca 2ϩ from both intracellular and extracellular sources, while the Ca 2ϩ source for cold shock signaling is mostly extracellular.