Sphingosine kinase (SK) 1 catalyzes the formation of the bioactive lipid sphingosine 1-phosphate, and has been implicated in several biological processes in mammalian cells, including enhanced proliferation, inhibition of apoptosis, and oncogenesis. Human SK (hSK) 1 possesses high instrinsic catalytic activity which can be further increased by a diverse array of cellular agonists. We have shown previously that this activation occurs as a direct consequence of extracellular signal–regulated kinase 1/2–mediated phosphorylation at Ser225, which not only increases catalytic activity, but is also necessary for agonist-induced translocation of hSK1 to the plasma membrane. In this study, we report that the oncogenic effects of overexpressed hSK1 are blocked by mutation of the phosphorylation site despite the phosphorylation-deficient form of the enzyme retaining full instrinsic catalytic activity. This indicates that oncogenic signaling by hSK1 relies on a phosphorylation-dependent function beyond increasing enzyme activity. We demonstrate, through constitutive localization of the phosphorylation-deficient form of hSK1 to the plasma membrane, that hSK1 translocation is the key effect of phosphorylation in oncogenic signaling by this enzyme. Thus, phosphorylation of hSK1 is essential for oncogenic signaling, and is brought about through phosphorylation-induced translocation of hSK1 to the plasma membrane, rather than from enhanced catalytic activity of this enzyme.

Sphingosine kinase 1 (SK1) is an enzyme that catalyzes the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK1 in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK1 by SKi. Moreover, SK1 is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK1 by activating the proteasome. In addition, the proteasomal degradation of SK1a and SK1b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK1b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK1 as an important mechanism controlling cell survival.Sphingosine 1-phosphate (S1P) 5 is a bioactive lipid that has an important role in regulating the growth, survival, and migration of mammalian cells. S1P binds to a family of five GPCR termed S1P n (where n ϭ 1-5) that regulate various effectors, such as MAP kinase (1). S1P is produced by the enzyme sphingosine kinase (SK1 and SK2 isoforms), which catalyzes the phosphorylation of sphingosine to produce S1P (2, 3). There are three N-terminal variants of SK1. SK1a (GenBank TM number: NM_001142601) is a 42.5 kDa protein, while SK1b (GenBank TM number: NM_182965) is a 51 kDa protein identical to SK1a, but with an 86 amino acid N-terminal extension. The third form has a molecular mass of 43.9 kDa and is identical to SK1a except for a 14 amino acid Nterminal extension (GenBank TM number: NM_021972) and migrates with similar mobility as SK1a on SDS-PAGE. The SK1a annotation used here therefore includes SK1a and possibly SK1aϩ14.SK1 has been demonstrated to have an important role in cancer (4). For instance, enforced overexpression of SK1 increases V12-Ras-dependent transformation of cancer cells (5), S1P levels, estrogen-dependent tumorigenesis, and blocks apoptosis of MCF-7 cells induced by anti-cancer drugs (6). SK1/S1P is also required for EGF-induced MCF-7 cell migration, proliferation and survival (7) and breast cancer cell growth (8). High SK1 expression is also correlated with poor prognosis in ER ϩ breast cancer, and SK1 induces a migratory phenotype in response to S1P in MCF-7 cells, via SK1-dependent changes in S1P 3 expression and PAK1/ERK-1/2 regulation (9). There is no evidence that mutations occur in the SK1 gene linked to cancer and therefore, the term non-oncogene addiction has been used to describe its role in cancer progression (10). The S1P signaling pathway has also been implicated in promoting the proliferation of androgen-independent prostate cancer PC-3 cells (11). Moreover, irradiation of a radiation-sensitive cancer cell ...