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.

FIGURE 1.

Characterization of the CIB1-SK1 interaction. A, SK1 association with CIB1 was examined by coimmunoprecipitation using lysates from HEK293T cells expressing HA-tagged CIB1 and FLAG-tagged SK1 either individually or together. Expression of these constructs was confirmed via Western blot (lysates). CIB1 was immunoprecipitated (IP) via its HA tag and associated SK1 detected by Western blot. IgG is the light chain of the anti-HA antibody used in the immunoprecipitation. B, recombinant GST or GST-CIB1 was incubated with cell lysates from untransfected DU145 cells (lysate). Endogenous SK1 pulled down by GST-CIB1 but not GST alone was detected using anti-SK1 antibodies via Western blot. C, to demonstrate an interaction between endogenous CIB1 and SK1, endogenous SK1 was immunoprecipitated from cell lysates of MCF7 cells (lysate) using anti-SK1 antibodies and protein A MicroBeads. Lysate containing protein A MicroBeads but no anti-SK1 antibodies was used as a negative control (IP control). CIB1 associated with the anti-SK1 immunocomplexes was detected via Western blot. Dividing lines indicate where lanes from the same Western blot have been spliced to simplify viewing. D, binding of SK1 to CIB1 was further examined using recombinant GST-CIB1 to bind recombinant His₆-tagged SK1 (load) in the presence of 2 mm CaCl₂, MgCl₂, or EGTA. E, Ca²⁺ concentration dependence of this interaction was determined by performing similar pulldowns with GST-CIB1 and recombinant His₆-tagged SK1 under the range of indicated CaCl₂ concentrations. F, ability of CIB1 to bind nonphosphorylated SK1 was tested using recombinant GST-CIB1 and lysates from HEK293T cells expressing either wild type (WT) SK1 or SK1^S225A (lysates). Total SK1 was detected using anti-FLAG antibodies, whereas phospho-SK1 was detected using anti-phospho-SK1 antibodies. All data are representative of at least three independent experiments.

FIGURE 2.

CIB1 interacts with the CaM-binding site of SK1. A, ability of CIB1 to bind SK1 mutated in the CaM-binding site was examined using recombinant GST-CIB1 and lysates from HEK293T cells expressing FLAG-tagged wild type (WT) SK1 or SK1^F197A/L198Q. B, to determine the effect of W7 on phorbol ester-induced SK1 plasma membrane translocation, HeLa cells expressing GFP-SK1 were stimulated with PMA either with or without pretreatment with W7. GFP-SK1 was visualized through fluorescence microscopy. Membrane fluorescence quantitation data represents mean ± S.E. Statistical significance was calculated by an unpaired t test. *, p < 0.0001. C, GST-CIB1 was used to pull down recombinant His₆-tagged SK1 in the presence or absence of 100 μm W7. All data are representative of at least three independent experiments.

FIGURE 3.

CIB1 acts like a Ca²⁺-myristoyl switch protein. A, myristoylation status of CIB1 was determined by metabolically labeling HEK293T cells expressing CIB1 either HA-tagged at the C terminus (CIB1) or N terminus (HA-CIB1) or CIB1^G2A (also HA-tagged at the C terminus) with [³H]myristic acid. CIB1 protein was immunoprecipitated (IP) from these cell extracts using anti-HA antibodies, proteins separated by SDS-PAGE, and the ³H signal was detected by fluorography. IgG is the light chain of the anti-HA antibody used in the immunoprecipitation. IB, immunoblot. B and C, fluorescence microscopy of HeLa cells expressing either CIB1 or nonmyristoylated CIB1 (HA-CIB1) detected by anti-HA antibodies following stimulation with ionomycin (iono) (B), thapsigargin (thap) (C), PMA, or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/AM (BAPTA) and PMA (D). Membrane fluorescence quantitation data represent mean ± S.E. Statistical significance was calculated by an unpaired t test. *, p < 0.02; **, p < 0.0001.

FIGURE 4.

CIB1 and SK1 colocalize at the plasma membrane following agonist stimulation. Fluorescence microscopy of HeLa cells coexpressing CIB1 and SK1 following PMA stimulation shows colocalization of CIB1 (red) and SK1 (green) at the plasma membrane after agonist stimulation. CIB1 was detected using anti-HA antibodies, and SK1 was fused to GFP.

FIGURE 5.

CIB1 mediates agonist-dependent translocation of SK1 to the plasma membrane. A, Western blot of HeLa cell extracts expressing GFP-SK1 and either a control siRNA (Validated Stealth^TM negative control) or CIB1 siRNA demonstrating a knockdown of endogenous CIB1 protein. Tubulin levels show protein loading. B, fluorescence microscopy of HeLa cells coexpressing GFP-SK1 (green) and control siRNA or CIB1 siRNA with and without PMA stimulation. Experiments were performed with two independent CIB1 siRNAs, each generating comparable results. Membrane fluorescence quantitation data represent means ± S.E. Statistical significance was calculated by an unpaired t test. C, ERK1/2 phosphorylation was monitored in either control siRNA or CIB1 siRNA-transfected cells with or without PMA stimulation by Western blot. Dashed lines indicate where lanes from the same Western blot have been spliced to simplify viewing. D, S1P levels in either control or CIB1 siRNA transfected cells with or without PMA stimulation. Data represent the mean ± S.D. of three independent experiments, with p values calculated by an unpaired t test. ***, p < 0.0001; **, p < 0.02; and *, p < 0.05.

FIGURE 6.

Expression of nonmyristoylated CIB1 blocks the translocation of endogenous SK1 to the plasma membrane. Fluorescence microscopy of either untransfected HeLa cells or cells expressing HA-CIB1 (red) with or without PMA stimulation. Endogenous SK1 (green) was detected using anti-SK1 antibodies. Membrane fluorescence quantitation data represent mean ± S.E. Statistical significance was calculated by an unpaired t test. *, p < 0.002.

FIGURE 7.

CIB1 knockdown or use of a dominant-negative CIB1 enhances cell susceptibility to TNFα-induced apoptosis through the NF-κB pathway. A, to examine the role of CIB1 in TNFα-mediated cell survival, apoptosis was measured in HeLa cells transfected with either control or CIB1 siRNA and treated with TNFα and CHX for 18 h. Apoptosis was measured through the percentage of floating cells with ∼99% of these floating cells showing positive cell surface staining for the apoptosis marker annexin V. Data are the mean percentage apoptosis ± S.D. of three independent experiments. Statistical significance was calculated by an unpaired t test. *, p < 0.01. B, to examine whether the myristoylation of CIB1 was required for this anti-apoptotic signaling, either wild type CIB1 or nonmyristoylated CIB1^G2A were expressed in HeLa cells, and apoptosis was measured after TNFα and CHX for 18 h. Results show the percentage of annexin V-positive floating cells. Caspase 3/7 assays showed similar results (data not shown). Data are the mean percentage increase ± S.D. of three independent experiments calculated relative to nil-treated vector cells. Statistical significance was calculated by an unpaired t test. *, p < 0.02. C, HeLa cells transfected with either control or CIB1 siRNA were stimulated with TNFα for 30 min and IκB-α levels in cell lysates detected by Western blotting. D, HeLa cells were transfected with either control siRNA or CIB1 siRNA in combination with either pIgKluc, for NF-κB-dependent expression of firefly luciferase, or control pTK81 vector lacking the NF-κB-binding sites. pRL-TK (encoding Renilla luciferase) was included in each transfection to standardize transfection efficiency. Two days following transfection, cells were stimulated with 0.5 ng/ml TNFα for 4 h, and a Dual-Luciferase reporter assay was carried out. Firefly and Renilla luciferase activity was standardized and calculated relative to cells expressing pTK81. Data are mean ± S.D. of six independent experiments, with statistical significance calculated by an unpaired t test. *, p < 0.0001. **, p < 0.002.