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Protein phosphatase PP1 has two active‐site metals (Zn2+/Fe2+) that are essential for catalysis. However, when expressed in bacteria, PP1 has two Mn2+‐ions in its active site, indicating that the incorporation of Zn2+/Fe2+ depends on additional eukaryotic component(s). Here, we used purified, metal‐deficient PP1 to study metal incorporation. Fe2+ was incorporated spontaneously, but Zn2+ was not. Mn2+‐incorporation at physiological pH depended on the co‐expression of PP1 with PPP1R2 (Inhibitor‐2) or PPP1R11 (Inhibitor‐3), or a pre‐incubation of PP1 at pH 4. We also demonstrate that PPP1R2 and PPP1R11 are Zn2+‐binding proteins but are, by themselves, not able to load PP1 with Zn2+. Our data suggest that PPP1R2 and PPP1R11 function as metal chaperones for PP1 but depend on co‐chaperone(s) and/or specific modification(s) for the transfer of associated Zn2+ to PP1. [ABSTRACT FROM AUTHOR]

Protein phosphatase‐1 (PP1) complexed to nuclear inhibitor of PP1 (NIPP1) limits DNA repair through dephosphorylation of NIPP1‐recruited substrates. However, the PP1:NIPP1 holoenzyme is completely inactive under basal conditions, hinting at a DNA damage‐regulated activation mechanism. Here, we report that DNA damage caused the activation of PP1:NIPP1 after a time delay of several hours through phosphorylation of NIPP1 at the C‐terminal tyrosine 335 (Y335) by a Src‐family kinase. PP1:NIPP1 activation partially resulted from the dissociation of the C terminus of NIPP1 from the active site of PP1. In addition, the released Y335‐phosphorylated C terminus interacted with the N terminus of NIPP1 to enhance substrate recruitment by the flanking forkhead‐associated (FHA) domain. Constitutive activation of PP1:NIPP1 by knock‐in of a phospho‐mimicking (Y335E) NIPP1 mutant led to the hypo‐phosphorylation of FHA ligands and an accumulation of DNA double‐strand breaks. Our data indicate that PP1:NIPP1 activation through circularization of NIPP1 is a late response to DNA damage that contributes to the timely recovery from damage repair. [ABSTRACT FROM AUTHOR]

Aims: Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1−/−) deficiency on leukocyte subsets relevant to atherosclerosis. Methods and Results: LDL receptor deficient mice that were transplanted with Sgpl1−/− bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1−/− chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. Conclusions: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution. [ABSTRACT FROM AUTHOR]

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-32P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3- diazole)]-6-aminohexanoyl-sphingenine (NBD-C6-ceramide) as substrate. Its Km value (4 μM) was comparable to that of Nhexanoyl- sphingenine (C6-ceramide). The produced fluorescent NBD-C6-ceramide-1-phosphate was captured by means of solidphase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126; IC50 4 μM) and ceramide analogues (e.g., fenretinide, AMG-9810; IC50 1.1 μM) affect CERK in vitro. [ABSTRACT FROM AUTHOR]

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-(32)P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C(6)-ceramide) as substrate. Its K(m) value (4 μM) was comparable to that of N-hexanoyl-sphingenine (C(6)-ceramide). The produced fluorescent NBD-C(6)-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126; IC(50) 4 μM) and ceramide analogues (e.g., fenretinide, AMG-9810; IC(50) 1.1 μM) affect CERK in vitro.