Mechanisms that alter protein phosphatase 2A (PP2A)-dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site-directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo. Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT-I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1−/− MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.

Background: Pro-TNFα is transformed into the active/soluble form through proteolysis by TNFα-converting enzyme (TACE).Results: Genetic ablation of ceramide kinase induces an increase in TACE activity and secreted TNFα.Conclusion: Ceramide 1-phosphate (C1P) negatively regulates the activity of TACE.Significance: The TACE/C1P interaction is a viable drug target for the treatment of heart disease and sepsis.

The sphingolipid ceramide‐1‐phosphate (C1P) plays a critical role in the cellular signaling that mediates inflammation, cell proliferation and phagocytosis. C1P has been shown to increase the activity of cytosolic phospholipase A2 α (cPLA2α) as well regulate its translocation to cellular membranes, a process that promotes inflammation through the production of arachidonic acid. In this study we have resolved the first C1P binding site of a peripheral protein. Using NMR we demonstrate the cPLA2α C2 domain interaction site for C1P by mapping chemical shifts. Subsequently, this novel‐binding site is confirmed with in vitro biophysical and biochemical analysis as well as molecular dynamics simulations. To search the human genome for other C1P binding proteins we have performed proteomic studies to began high throughput characterization of the conserved nature of C1P binding. Funding source: American Heart Association SDG0735350N (R.V.S)

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Background: Extracellular ceramide 1-phosphate is presumed to interact with extracellular proteins to mediate cellular invasion. These proteins are unidentified. Results: C-1-P interacts with both annexin a2 and p11 proteins. C-1-P-mediated vascular endothelial cell invasion requires expression of these proteins. Conclusion: Extracellular C-1-P mediates invasion via an interaction with the annexin a2-p11 heterotetramer. Significance: Gradients of C-1-P may guide vascular endothelial cell invasion during wound healing.

Background: PKC plays a key role in T lymphocyte activation, but its regulatory mechanism is not understood. Results: Phosphotyrosine binds the PKC C2 domain and activates PKC. Conclusion:The PKC C2 domain-phosphotyrosine binding is important for PKC activation in T cells. Significance: This study provides new mechanistic insight into the regulation of PKC in T cells.

The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.

Group IV phospholipase A2α (cPLA2α) regulates the production of prostaglandins and leukotrienes via the formation of arachidonic acid from membrane phospholipids. The targeting and membrane binding of cPLA2α to the Golgi involves the N-terminal C2 domain, whereas the catalytic domain produces arachidonic acid. Although most studies of cPLA2α concern its catalytic activity, it is also linked to homeostatic processes involving the generation of vesicles that traffic material from the Golgi to the plasma membrane. Here we investigated how membrane curvature influences the homeostatic role of cPLA2α in vesicular trafficking. The cPLA2α C2 domain is known to induce changes in positive membrane curvature, a process which is dependent on cPLA2α membrane penetration. We showed that cPLA2α undergoes C2 domain-dependent oligomerization on membranes in vitro and in cells. We found that the association of the cPLA2α C2 domain with membranes is limited to membranes with positive curvature, and enhanced C2 domain oligomerization was observed on vesicles ~50 nm in diameter. We demonstrated that the cPLA2α C2 domain localizes to cholesterol enriched Golgi-derived vesicles independently of cPLA2α catalytic activity. Moreover, we demonstrate the C2 domain selectively localizes to lipid droplets whereas the full-length enzyme to a much lesser extent. Our results therefore provide novel insight into the molecular forces that mediate C2 domain-dependent membrane localization in vitro and in cells.