Translocation of Sphingosine Kinase 1 to the Plasma Membrane Is Mediated by Calcium- and Integrin-binding Protein 1

Abstract

SK1 (sphingosine kinase 1) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK1 activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK1 from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK1 has remained undefined. Here, we demonstrate a direct role for CIB1 (calcium and integrin-binding protein 1) in this process. We show that CIB1 interacts with SK1 in a Ca 2؉ -dependent manner at the previously identified "calmodulin-binding site" of SK1. We also demonstrate that CIB1 functions as a Ca 2؉ -myristoyl switch, providing a mechanism whereby it translocates SK1 to the plasma membrane. Both small interfering RNA knockdown of CIB1 and the use of a dominant-negative CIB1 we have generated prevent the agonist-dependent translocation of SK1. Furthermore, we demonstrate the requirement of CIB1-mediated translocation of SK1 in controlling cellular sphingosine 1-phosphate generation and associated anti-apoptotic signaling. SK1 (sphingosine kinase 1) catalyzes the formation of sphingosine 1-phosphate (S1P), 2 a bioactive phospholipid that mediates a wide variety of cellular processes. Elevated cellular S1P has been shown to be pro-proliferative and anti-apoptotic (1), and considerable evidence now exists implicating SK1 in cancer. In particular, overexpression of SK1 in NIH3T3 fibroblasts leads to a transformed phenotype and the ability to form tumors in mice, with SK1 activity also involved in oncogenic H-Ras-mediated transformation (2). Furthermore, suppression of cellular SK1 activity by genetic or pharmacologic approaches has been shown to significantly reduce tumor growth in vivo in mice (3-5) and also sensitize tumor cells to other chemotherapeutics (6).We have previously shown that although SK1 has intrinsic catalytic activity (7), its further activation is required for oncogenic signaling (8). This activation, brought about through phosphorylation at Ser-225 by ERK1/2, not only increases the catalytic activity of SK1 but also results in its translocation from the cytoplasm to the plasma membrane (9), which is essential for the oncogenic signaling by this enzyme (8, 10).Although critical in understanding SK1-induced oncogenesis, the mechanisms regulating agonist-induced translocation of SK1 to the plasma membrane are poorly understood. Studies have suggested that SK1 associates with phosphatidylserine in a phosphorylation-dependent manner, providing a possible mechanism for retention of SK1 at the plasma membrane (11). Although this may facilitate retention of SK1 at the plasma membrane, the molecular mechanism mediating the initial rapid agonist-induced translocation of SK1 has not yet been established. Calmodulin (CaM) has been indirectly implicated in this process because W7, a CaM inhibitor, blocked SK1 translocation (12), as did mutation of the CaM-bindin...

An intrinsic lipid-binding interface controls sphingosine kinase 1 function

“…Interestingly, this CIB1 interaction site F197/L198 [24] overlaps with the hydrophobic patch that has been shown to be important for direct membrane binding in a previous study [23] and this work. As suggested by Jarman et al [24], it is possible that CIB1 acts as a "molecular shepherd" to bring SK1 close enough to the membrane but does not influence SK1s intrinsic mechanism for binding to membranes. However, it is also likely that CIB1 can "override" the intrinsic mechanism of SK1 membrane binding and force it to the membrane as a function of the myristoyl-switch.…”

“…Membrane localization of SK1 has also been attributed to interaction with CIB1 [24,25], which acts as a calcium-myristoyl switch. Interestingly, this CIB1 interaction site F197/L198 [24] overlaps with the hydrophobic patch that has been shown to be important for direct membrane binding in a previous study [23] and this work.…”

“…There are currently three different proposed mechanisms to explain SK1 translocation and interaction with membranes. Two of the by guest, on www.jlr.org Downloaded from 4 mechanisms identify certain residues that mediate membrane localization [21,23], while the third explains SK1 localization as being dependent on another protein, calcium/integrin binding protein 1 (CIB1) [24,25]. Interestingly, the protein-protein interaction between SK1 and CIB1 takes place at a hydrophobic site on SK1 which has been identified for membrane interaction.…”

Recruitment of sphingosine kinase to presynaptic terminals by a conserved muscarinic signaling pathway promotes neurotransmitter release

“…To test this, we interfered with the ability of SPHK-1 to translocate to synapses by mutating its calcium/calmodulin (CaM)-binding site. Mutations in the CaM-binding site of mammalian SphK have been reported to decrease translocation of SphK to membranes but to have no effect on its catalytic activity (Sutherland et al 2006;Jarman et al 2010). We found that GFP-tagged SPHK-1 transgenes lacking the functional CaM-binding site [SPHK-1(DCaM) -GFP] were expressed at levels in the cell bodies of neurons similar to wild-type transgenes [cell body fluorescence was 2280 6 243 arbitrary units for SPHK-1(WT)-GFP and 2561 6 222 arbitrary units for SPHK-1(DCaM)-GFP; n = 20].…”

Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells

“…SK (which exists as two isoforms termed SK1 and SK2) is activated by agonists such as antigen, plateletderived growth factor, nerve growth factor, tumor necrosis factor ␣, and epidermal growth factor (EGF), resulting in an increase in intracellular S1P. SK1 activation and/or translocation is regulated by phosphorylation catalyzed by ERK-1/2 and by CIB1 (calcium and integrin-binding protein 1) (14,15). S1P has an important role in breast cancer cells in terms of regulating survival, proliferation, and migration (16 -18).…”