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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]

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]