Fig. 1.
Presence of SphK2 in mononucleosomes and physical association with histone H3. (A) Nuclei were isolated from MCF-7 cells transfected with vector (white bar) or SphK2 (gray bar) and incubated with [3H]sphingosine (1.5 μM) and ATP (1 mM) for 30 min. Formation of [3H]S1P was determined by differential extraction (22). Abundance of sphingoid bases sphingosine (Sph) and dihydrosphingosine (DHS), and their phosphorylated products, S1P and dihydro-S1P (DHS1P), in nuclei of vector and SphK2-expressing MCF-7 cells (7 × 106) were determined by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS/MS). The data are averages of triplicate determinations and are expressed as picomoles of lipid ± SD. Asterisks indicate statistically significant differences (P < 0.05 by Student’s t test) relative to vector transfectants. (B) Association of SphK2 with chromatin. Equal amounts of cytosol, nucleoplasm, and chromatin fractions from MCF-7 cells were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with SphK2-specific antibody. Antibodies against H3 and tubulin were used as markers for chromatin and cytosol, respectively. Lysate from MCF-7 cells overexpressing SphK2 was included as a positive control (leftmost lane). (C) Association of SphK2 and histone H3. Proteins of nuclear extracts from MCF-7 cells transfected with vector, V5-SphK2, or catalytically inactive V5-SphK2G212E were immunoprecipitated with V5-specific antibody, separated by SDS-PAGE, and probed with antibodies against V5 or H3.