title: Effect of a membrane-targeted sphingosine kinase 1 on cell proliferation and survival.
abstract: Numerous extracellular stimuli activate SK1 (sphingosine kinase type 1) to catalyse the production of sphingosine 1-phosphate, a bioactive lipid that functions as both an extracellular ligand for a family of G-protein-linked receptors and as a putative intracellular messenger. Phorbol esters, calcium or immunoglobulin receptors stimulate SK1 by promoting its translocation to the plasma membrane, which brings it into proximity both to its substrate (i.e. sphingosine) and to activating acidic phospholipids (e.g. phosphatidylserine). To evaluate the consequence of SK translocation, we generated an SK1-derivative tagged with a myristoylation sequence (Myr-SK1) on its N-terminus and overexpressed the construct in 3T3-L1 fibroblasts using recombinant retrovirus. Myr-SK1 overexpression increased SK activity by more than 50-fold in crude membranes, while only stimulating cytoplasmic SK activity by 4-fold. In contrast, the overexpression of WT-SK1 (wild-type SK1), as well as that of a construct containing a false myristoylation sequence (A2-Myr-SK1), markedly increased SK activity in both membrane and cytoplasmic compartments. Immunofluorescence confirmed that Myr-SK1 preferentially localized at the plasma membrane, whereas WT-SK1 and A2-Myr-SK1 partitioned in cytoplasmic/perinuclear cellular regions. Surprisingly, Myr-SK1 overexpression significantly decreased the rates of cell proliferation by delaying exit from G0/G1 phase. Moreover, expression of Myr-SK1 but not WT-SK1 or A2-Myr-SK1 protected cells from apoptosis induced by serum withdrawal. Collectively, these findings reveal that altering the subcellular location of SK1 has marked effects on cell function, with plasma membrane-associated SK having a potent inhibitory effect on the G1-S phase transition.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.
authors.forename: Farida
authors.initials: F
authors.lastname: Safadi-Chamberlain
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Wang
authors.forename: Shawn G
authors.initials: SG
authors.lastname: Payne
authors.forename: Chang-Uk
authors.initials: CU
authors.lastname: Lim
authors.forename: Suzanne
authors.initials: S
authors.lastname: Stratford
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Chavez
authors.forename: Michael H
authors.initials: MH
authors.lastname: Fox
authors.forename: Sarah
authors.initials: S
authors.lastname: Spiegel
authors.forename: Scott A
authors.initials: SA
authors.lastname: Summers
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D051379:Mice; D019814:Myristic Acid / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099157:Sphingosine Kinase
chemical_list: D016895:Culture Media, Serum-Free; D019814:Myristic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); D000099157:Sphingosine Kinase