Study of serine palmitoyltransferase and de novo synthesis of sphingolipids

Sphingolipids are membrane components in eukaryotes, some prokaryotes and viruses. They play important roles in membrane structure, biological recognition and signal transduction (3-5). Serine palmitoyltransferase (SPT) catalyzes the first unique step of de novo sphingolipid biosynthesis, the condensation of serine and palmitoyl-CoA to form the sphingoid base backbone (6). We have studied the molecular and biological consequences of overexpression of SPT using HEK293 cells stably transfected with plasmids containing the cDNA for SPTLC1 and SPTLC2 (termed "SPT1/2 cells"). The effects of the elevated SPT activity were analyzed by measuring the sphingolipid amounts and types in these cells as well as by following the incorporation of [13C] palmitate into the sphingoid base (and/or fatty acid) backbones, which were analyzed by liquid chromatography, electrospray ionization-tandem mass spectrometry (LC-ESIMS/MS). These studies revealed that most sphingolipid subspecies were elevated in SPT1/2 cells (with disproportionate increases in dihydrosphingolipids and subspecies with stearic acid in the ceramide backbone); however, sphingomyelins were lower. The lower sphingomyelin does not appear to be caused by faster degradation, but possibly by substitution by dihydrosphingomyelins. Despite large increases in ceramide, a growth inhibitory and pro-apoptotic mediator in many cell types, the SPT1/2 cells did not display higher numbers of dead cells but, instead, grew faster than HEK293 cells. Thus, increased de novo sphingolipid biosynthesis via this manipulation has uncovered several new features of this pathway that warrant further investigation.;In the course of these studies, we noted by confocal microscopy that SPT1 is not only found in the endoplasmic reticulum, but also in the nucleus and focal adhesions. This unexpected localization was confirmed in several cell lines, and by finding that SPT1 is co-immunoprecipitated with vinculin using an anti-vinculin antibody. The association of SPT1 with focal adhesions may indicate that it plays a role in cell morphology and migration, and consistent with this hypothesis, focal adhesion staining of SPT1 is seen mainly before cells in culture reach confluence, and reappears when proliferation and migration is reinitiated by a standard scratch-wound healing assay. Furthermore, elimination of SPT1 using SPTLC1 siRNA causes cell rounding that does not appear to be due to interference with de novo sphingolipid biosynthesis. Thus, in addition to its "traditional" role in de novo sphingolipid biosynthesis in the ER, SPT1 is present in other cellular compartments and is required for normal cell morphology and migration.;SPT1 was hypothesized to be present in cells in more than one isoform because GenBank has at least three putative SPT1 transcripts that can be rationalized to be produced by alternative splicing. The predicted amino acid sequence of one isoform (which we term SPTLC1L) is longer than the SPT1 that has heretofore been associated with SPT activity, and this isoform appears to be expressed in Hek cells because a polypeptide of the predicted size was found by antibodies against its unique amino acid sequence. The functions of the alternative isoforms are not yet clear; however, their existence further underscores how much remains to be learned about the biochemistry and regulation this pathway and its components.