A modular synthesis of alkynyl-phosphocholine headgroups for labeling sphingomyelin and phosphatidylcholine.

A general route to phospho- and sphingolipids that incorporate an alkyne in the phosphocholine headgroup is described. The strategy preserves the ammonium functionality of the phosphocholine and can be easily modified to introduce desired functional groups at the N-acyl chain. The targets accessible with this strategy provide a bioorthogonal handle for postsynthetic introduction of fluorophores or other labeling agents with aqueous phase chemistry. We report the synthesis of sphingomyelin derivatives that incorporate a fluorophore and an alkyne. The modified sphingolipids retain activity as substrates for sphingomyelinase, making these compounds viable probes of enzymatic activity. Importantly, the strategy allows modification of the lipid across the phosphodiester, making the alkyne a potential probe of sphingomyelinase activity.

Ser-10 phosphorylation of histone H3 and immediate early gene expression in oncogene-transformed mouse fibroblasts.

Stimulation of the Ras-mitogen-activated protein kinase (MAPK) pathway by growth factors, phorbol esters, and oncoproteins results in the phosphorylation of histone H3. Rsk-2 and MSK1 have been reported to be H3 kinases activated by the Ras-MAPK signal transduction pathway. In this study, we used inhibitors of Rsk-2 and MSK1 to decide which of these kinases was responsible for the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of H3 in 10T(1/2) and Ciras-3 (H-ras-transformed 10T(1/2)) mouse fibroblasts. These studies demonstrated that MSK1, but not Rsk-2, was the H3 kinase activated in these cells. Furthermore, assays with Rsk-2 showed that this kinase phosphorylates H2B but not H3 in vitro. H89, a potent MSK1 inhibitor, prevented TPA induction of H3 phosphorylation and diminished the TPA-induced expression of the c-fos and urokinase plasminogen activator genes. We propose that persistent activation of the Ras-MAPK pathway and MSK1 resulting in the elevation of phosphorylated H3 levels may contribute to the aberrant gene expression observed in the oncogene-transformed cells.