Abstract
Accumulation of phosphatidylserine in the inner leaflet of the plasma membrane is a hallmark of eukaryotes. Sublethal levels of staurosporine and related compounds deplete phosphatidylserine from the plasma membrane and abrogate K-Ras signaling. Here, we report that low-dose staurosporine and related compounds increase sphingomyelin mass. Mass-spectrometry and metabolic tracer analysis revealed an increase in both the levels and rate of synthesis of sphingomyelin in response to sublethal staurosporine. Mechanistically, it was determined that the abundance of the ORMDL proteins, which negatively regulate serine-palmitoyltransferase, are decreased by low-dose staurosporine. Finally, inhibition of ceramide synthesis, and thus sphingomyelin, prevented the displacement of phosphatidylserine and cholesterol from the inner leaflet of the plasma membrane. The results establish that an optimal level of sphingomyelin is required to maintain the distribution of phosphatidylserine and cholesterol in the plasma membrane and further demonstrate a complex relationship between the trafficking of phosphatidylserine and sphingomyelin.
Highlights
The plasma membrane provides many essential functions for cell viability but acts primarily as a protective barrier and a conduit for entry
Cholesterol accessibility in the exofacial leaflet of the plasma membrane was increased upon treatment with STS, and supplementation with PtdSer restored this phenotype in STS-Chinese hamster ovary (CHO) cells[11] (Fig. 1c,d)
These data showed that, in STS-treated CHO (STS-CHO) cells, plasmalemmal cholesterol is preferentially accumulated in the exofacial leaflet of the plasma membrane with more cholesterol detectable in the cytosolic leaflet of organelles
Summary
The plasma membrane provides many essential functions for cell viability but acts primarily as a protective barrier and a conduit for entry. Treatment of cells with sublethal concentrations of STS caused a PtdSer biosensor[9] to relocalize from the plasma membrane to intracellular compartments including recycling endosomes in MDCK cells[20]. This result suggests that STS impairs the endosomal sorting and recycling of PtdSer[20], the molecular mechanism of this relocalization and the impaired retrieval of PtdSer to the PM remain unclear. We found that Chinese hamster ovary (CHO) cells had increased levels of SM after treatment with low dose STS. This elevation in SM mass accompanies the displacement of PtdSer from the plasma membrane. Perturbation of PtdSer distribution was prevented by inhibition of SM synthesis, indicating a highly coordinated mechanism to maintain the PM composition that is dependent on SM synthesis and transport
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