We have studied the molecular and biological consequences of overexpression of serine palmiotyltransferase (SPT) using HEK293 cells stably transfected with SPTLC1 and SPTLC2 (termed "SPT1/2 cells"). The effects of the elevated SPT activity were analyzed by liquid chromatography, electrospray ionization tandem mass spectrometry. Most sphingolipid subspecies were elevated in SPT1/2 cells, with disproportionately higher dihydrosphingolipids and ceramides with stearic acid. Sphingomyelins were lower, however, which does not appear to be due to faster degradation, but possibly by substitution by dihydrosphingomyelins. Despite large increases in potentially growth inhibitory and lethal ceramides, SPT1/2 cells grow faster than HEK293 cells. We also noted by confocal microscopy that endogenous SPT1 is not only in the endoplasmic reticulum, but also in the nucleus and focal adhesions, which was confirmed by elimination of SPT1 using SPTLC1 siRNA and co-immunoprecipitation of SPT1 with vinculin. The appearance of SPT1 in focal adhesions is lost when cells reach confluence and reappears after a scratch assay to reinitiate migration; furthermore, SPTLC1 siRNA causes cell rounding. 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. It is possible that some of the previously unnoticed properties of SPT1 are due to alternative isoforms because we have found at least one splice variant that is expressed in HEK293 cells.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/28095 |
| Date | 06 April 2009 |
| Creators | Wei, Jia |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
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