The identification of myriocin-binding proteins.
BACKGROUND
Myriocin is a natural product that potently induces apoptosis of a murine cytotoxic T lymphocyte cell line (CTLL-2) and inhibits a serine palmitoyltransferase (SPT) activity that has been detected in cell extracts and is thought to initiate sphingolipid biosynthesis. Because SPT has never been biochemically purified and a comprehensive appraisal of myriocin-binding proteins has not been conducted, we isolated specific targets using myriocin-based affinity chromatography.


RESULTS
Myriocin derivatives were synthesized and evaluated using CTLL-2 proliferation and SPT activity assays. Guided by these results, affinity chromatography matrices were prepared and two specific myriocin-binding proteins were isolated from CTLL-2 lysates. Analyses of these polypeptides establish conclusively that they are murine LCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis.


CONCLUSIONS
The ability of myriocin-containing matrices to bind factors that have SPT activity and the exclusive isolation of LCB1 and LCB2 as myriocin-binding proteins demonstrates that the two proteins are directly responsible for SPT activity and that myriocin acts directly upon these polypeptides.
Chen
J K
JK
Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Lane
W S
WS
Schreiber
S L
SL
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D005229:Fatty Acids, Monounsaturated / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D051102:Serine C-Palmitoyltransferase; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D005229:Fatty Acids, Monounsaturated; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase; C495088:Sptlc1 protein, mouse; C001996:thermozymocidin