共查询到20条相似文献,搜索用时 15 毫秒
1.
Pitson SM Moretti PA Zebol JR Xia P Gamble JR Vadas MA D'Andrea RJ Wattenberg BW 《The Journal of biological chemistry》2000,275(43):33945-33950
Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNFalpha), interleukin-1beta, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK(G82D)). Basal SK activity was unaffected by expression of hSK(G82D). Expression of hSK(G82D) had no effect on TNFalpha-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFalpha activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation. 相似文献
2.
《Critical reviews in biochemistry and molecular biology》2013,48(5):365-375
Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well. 相似文献
3.
Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well. 相似文献
4.
Johnson KR Becker KP Facchinetti MM Hannun YA Obeid LM 《The Journal of biological chemistry》2002,277(38):35257-35262
Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C(6)-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling. 相似文献
5.
Siow DL Anderson CD Berdyshev EV Skobeleva A Natarajan V Pitson SM Wattenberg BW 《Advances in enzyme regulation》2011,51(1):229-244
The sphingosine kinases (sphingosine kinase-1 and -2) have been implicated in a variety of physiological functions. Discerning their mechanism of action is complicated because in addition to producing the potent lipid second messenger sphingosine-1-phosphate, sphingosine kinases, both by producing sphingosine-1-phosphate and consuming sphingosine, have profound effects on sphingolipid metabolism. Sphingosine kinase-1 translocates to the plasma membrane upon agonist stimulation and this translocation is essential for the pro-oncogenic properties of this enzyme. Many of the enzymes of sphingolipid metabolism, including the enzymes that degrade sphingosine-1-phosphate, are membrane bound with restricted subcellular distributions. In the work described here we explore how subcellular localization of sphingosine kinase-1 affects the downstream metabolism of sphingosine-1-phosphate and the access of sphingosine kinase to its substrates. We find, surprisingly, that restricting sphingosine kinase to either the plasma membrane or the endoplasmic reticulum has a negligible effect on the rate of degradation of the sphingosine-1-phosphate that is produced. This suggests that sphingosine-1-phosphate is rapidly transported between membranes. However we also find that cytosolic or endoplasmic-reticulum targeted sphingosine kinase expressed at elevated levels produces extremely high levels of dihydrosphingosine-1-phosphate. Dihydrosphingosine is a proximal precursor in ceramide biosynthesis. Our data indicate that sphingosine kinase can divert substrate from the ceramide de novo synthesis pathway. However plasma membrane-restricted sphingosine kinase cannot access the pool of dihydrosphingosine. Therefore whereas sphingosine kinase localization does not affect downstream metabolism of sphingosine-1-phosphate, localization has an important effect on the pools of substrate to which this key signaling enzyme has access. 相似文献
6.
The calmodulin-binding site of sphingosine kinase and its role in agonist-dependent translocation of sphingosine kinase 1 to the plasma membrane 总被引:2,自引:0,他引:2
Sutherland CM Moretti PA Hewitt NM Bagley CJ Vadas MA Pitson SM 《The Journal of biological chemistry》2006,281(17):11693-11701
Sphingosine kinases catalyze the formation of sphingosine 1-phosphate, a bioactive lipid involved in many aspects of cellular regulation, including the fundamental biological processes of cell growth and survival. A diverse range of cell agonists induce activation of human sphingosine kinase 1 (hSK1) and, commonly, its translocation to the plasma membrane. Although the activation of hSK1 in response to at least some agonists occurs directly via its phosphorylation at Ser225 by ERK1/2, many aspects governing the regulation of this phosphorylation and subsequent translocation remain unknown. Here, in an attempt to understand some of these processes, we have examined the known interaction of hSK1 with calmodulin (CaM). By using a combination of limited proteolysis, peptide interaction analysis, and site-directed mutagenesis, we have identified that the CaM-binding site of hSK1 resides in the region spanned by residues 191-206. Specifically, Phe197 and Leu198 are critically involved in the interaction because a version of hSK1 incorporating mutations of both Phe197 --> Ala and Leu198 --> Gln failed to bind CaM. We have also shown for the first time that human sphingosine kinase 2 (hSK2) binds CaM, and does so via a CaM binding region that is conserved with hSK1 because comparable mutations in hSK2 also ablate CaM binding to this protein. By using the CaM-binding-deficient version of hSK1, we have begun to elucidate the role of CaM in hSK1 regulation by demonstrating that disruption of the CaM-binding site ablates agonist-induced translocation of hSK1 from the cytoplasm to the plasma membrane, while having no effect on hSK1 phosphorylation and catalytic activation. 相似文献
7.
Wattenberg BW 《World journal of biological chemistry》2010,1(12):362-368
The sphingosine kinases, SK1 and SK2, produce the potent signaling lipid sphingosine-1-phosphate (S1P). These enzymes have garnered increasing interest for their roles in tumorigenesis, inflammation, vascular diseases, and immunity, as well as other functions. The sphingosine kinases are considered signaling enzymes by producing S1P, and their activity is acutely regulated by a variety of agonists. However, these enzymes are also key players in the control of sphingolipid metabolism. A variety of sphingolipids, such as sphingosine and the ceramides, are potent signaling molecules in their own right. The role of sphingosine kinases in regulating sphingolipid metabolism is potentially a critical aspect of their signaling function. A central aspect of signaling lipids is that their hydrophobic nature constrains them to membranes. Most enzymes of sphingolipid metabolism, including the enzymes that degrade S1P, are membrane enzymes. Therefore the localization of the sphingosine kinases and S1P is likely to be important in S1P signaling. Sphingosine kinase localization affects sphingolipid signaling in several ways. Translocation of SK1 to the plasma membrane promotes extracellular secretion of S1P. SK1 and SK2 localization to specific sites appears to direct S1P to intracellular protein effectors. SK localization also determines the access of these enzymes to their substrates. This may be an important mechanism for the regulation of ceramide biosynthesis by diverting dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, from the de novo production of ceramide. 相似文献
8.
The localization and activity of sphingosine kinase 1 are coordinately regulated with actin cytoskeletal dynamics in macrophages 总被引:1,自引:0,他引:1
Kusner DJ Thompson CR Melrose NA Pitson SM Obeid LM Iyer SS 《The Journal of biological chemistry》2007,282(32):23147-23162
The physiologic and pathologic functions of sphingosine kinase (SK) require translocation to specific membrane compartments. We tested the hypothesis that interactions with actin filaments regulate the localization of SK1 to membrane surfaces, including the plasma membrane and phagosome. Macrophage activation is accompanied by a marked increase in association of SK1 with actin filaments. Catalytically-inactive (CI)- and phosphorylation-defective (PD)-SK1 mutants exhibited reductions in plasma membrane translocation, colocalization with cortical actin filaments, membrane ruffling, and lamellipodia formation, compared with wild-type (WT)-SK1. However, translocation of CI- and PD-SK1 to phagosomes were equivalent to WT-SK1. SK1 exhibited constitutive- and stimulus-enhanced association with actin filaments and F-actin-enriched membrane fractions in both intact macrophages and a novel in vitro assay. In contrast, SK1 bound G-actin only under stimulated conditions. Actin inhibitors disrupted SK1 localization and modulated its activity. Conversely, reduction of SK1 levels or activity via RNA interference or specific chemical inhibition resulted in dysregulation of actin filaments. Thus, the localization and activity of SK1 are coordinately regulated with actin dynamics during macrophage activation. 相似文献
9.
Roberts JL Moretti PA Darrow AL Derian CK Vadas MA Pitson SM 《Analytical biochemistry》2004,331(1):122-129
Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro. 相似文献
10.
CR Gault ST Eblen CA Neumann YA Hannun LM Obeid 《The Journal of biological chemistry》2012,287(38):31794-31803
Sphingosine kinase 1 (SK1) is an important enzyme involved in the production of the bioactive lipid sphingosine 1-phosphate (S1P). SK1 is overexpressed in many forms of cancer, however, the contribution of SK1 to cancer progression is still unclear. One of the best characterized mutations found in several forms of human cancer is an activating point mutation in the Ras oncogene, which disrupts its GTPase activity and leads to stimulation of the MEK/ERK pathway. Because SK1 activity and subcellular localization have been shown to be regulated by ERK, we wished to investigate the effect of oncogenic Ras, a potent activator of the Raf/MEK/ERK pathway, on the activity of SK1 and sphingolipid metabolism. Using HEK293T cells transiently transfected with the K-RasG12V oncogene and both wild type and Sphk1(-/-) mouse embryonic fibroblasts stably infected with retroviral K-RasG12V, we found that K-RasG12V increases the production of S1P and decreases the production of ceramide in a SK1-dependent manner. In addition, we found that expression of the K-RasG12V oncogene leads to plasma membrane localization of SK1 and a reduction in cytosolic levels of SK1. This effect is likely mediated by the Raf/MEK/ERK pathway as constitutively active B-Raf or MEK1 are able to activate SK1, but constitutively active Akt1 is not. We believe this research has important implications for how sphingolipids may be contributing to oncogenic transformation and provide some of the first evidence for oncogenes inducing specific changes in sphingolipid metabolism through SK1 regulation. 相似文献
11.
Bharath Ananthanarayanan Sudipto Das Sue Goo Rhee Diana Murray Wonhwa Cho 《The Journal of biological chemistry》2002,277(5):3568-3575
The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-delta isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-delta1, -delta3, and -delta4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-delta1-C2 is driven by nonspecific electrostatic interactions between the Ca(2+)-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca(2+), Asn(647), and phosphatidylserine (PS). The PS selectivity of PLC-delta1-C2 governs its specific Ca(2+)-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-delta3-C2 also involves Ca(2+)-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-delta1-C2 and PLC-delta3-C2, PLC-delta4-C2 has significant Ca(2+)-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca(2+)-binding loops and the substitution of Ser for the Ca(2+)-coordinating Asp in position 717. Consequently, PLC-delta4-C2 exhibits unique pre-localization to the plasma membrane prior to Ca(2+) import and non-selective Ca(2+)-mediated targeting to various cellular membranes, suggesting that PLC-delta4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-delta isoforms as Ca(2+)-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors. 相似文献
12.
The sphingosine kinases, SK1 and SK2, catalyse the formation of the bioactive signalling lipid, sphingosine 1-phosphate (S1P), from sphingosine. SK1 and SK2 differ in their subcellular localisation, trafficking and regulation, but the isoforms are also distinct in their selectivity toward naturally occurring and synthetic ligands as substrates and inhibitors. To date, only the structure of SK1 has been determined, and a structural basis for selectivity differences in substrate handling by SK2 has yet to be established. Here we present a structural rationale, based on homology modelling and ligand docking, to account for the capacity of SK2, but not SK1, to efficiently process the pharmacologically active substances, fingolimod (FTY720) and safingol, as substrates. We propose that two key residue differences in hSK2 (Ser305/Thr584 in place of Ala175/Ala339 in hSK1) facilitate conformational switching in the lipid head group anchor residue, Asp308 (corresponding to Asp178 in hSK1), to accommodate substrate diversity for SK2. Our analysis accounts for the contrasting behaviour of fingolimod and safingol as non-turnover inhibitors of SK1, but substrates for SK2, and the observed stereoselectivity for phosphorylation of the pro-S hydroxymethyl group of fingolimod to generate (S)-FTY720-P in vivo. We also rationalise why methylation of the pro-R hydroxymethyl of FTY720 switches the behaviour of the resulting compound, (R)-FTY720 methyl ether (ROMe), to SK2-selective inhibition. Whilst the pharmacological significance of (S)-FTY720-P is firmly established, as the active principle of fingolimod in treating relapsing-remitting multiple sclerosis, the potential importance of SK-mediated phosphorylation of other substrates, such as safingol and non-canonical naturally occuring substrates such as (4E,nZ)-sphingadienes, is less widely appreciated. Thus, the contribution of SK2-derived safingol 1-phosphate to the anti-cancer activity of safingol should be considered. Similarly, the biological role of sphingadiene 1-phosphates derived from plant-based dietary sphingadienes, which we also show here are substrates for both SK1 and SK2, merits investigation. 相似文献
13.
The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A(2) (cPLA(2)) and protein kinase C-alpha (PKC-alpha) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-alpha C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn(189) plays a key role in this specificity. In contrast, the cPLA(2) C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA(2) C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties. 相似文献
14.
El-Shewy HM Abdel-Samie SA Al Qalam AM Lee MH Kitatani K Anelli V Jaffa AA Obeid LM Luttrell LM 《Molecular endocrinology (Baltimore, Md.)》2011,25(12):2144-2156
We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1. 相似文献
15.
Asp177 in C4 domain of mouse sphingosine kinase 1a is important for the sphingosine recognition 总被引:1,自引:0,他引:1
Sphingosine kinase (SK) is the enzyme that catalyzes the formation of sphingosine 1-phosphate (S1P). Although diverse biological functions have been reported for SK, its recognition site for its substrate sphingosine (Sph) is still unclear. We constructed various mutants of mouse sphingosine kinase 1a (mSK1a), carrying mutations in the C4 domain, which we had expected to encompass the Sph-binding site. We analyzed the influence of these mutations on the SK activity and substrate kinetics. One mutation, Asp177-->Asn177, caused a dramatic decrease in SK activity (to approximately 6% of wild type) and an increase in the Km value for Sph (10.1-->108 microM), with no change in the affinity for ATP. This result suggests that the C4 domain, especially the Asp177, is involved in the specific recognition of Sph. In this report, we are able, for the first time, to provide an account of the Sph-binding site of SK. 相似文献
16.
Delon C Manifava M Wood E Thompson D Krugmann S Pyne S Ktistakis NT 《The Journal of biological chemistry》2004,279(43):44763-44774
Sphingosine kinase 1 (SK1) phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK1 is of considerable interest. We report that SK1, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK1 with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK1-PA interaction maps to the C terminus of SK1 and is independent of catalytic activity or of the diacylglycerol kinase-like domain of the enzyme. Thus SK1 constitutes a novel, physiologically relevant PA effector. 相似文献
17.
《Journal of lipid research》2023,64(2):100322
The role of ceramide in biological functions is typically based on the elevation of cellular ceramide, measured by LC-MS in the total cell lysate. However, it has become increasingly appreciated that ceramide in different subcellular organelles regulates specific functions. In the plasma membrane, changes in ceramide levels might represent a small percentage of the total cellular ceramide, evading MS detection but playing a critical role in cell signaling. Importantly, there are currently no efficient techniques to quantify ceramide in the plasma membrane. Here, we developed a method to measure the mass of ceramide in the plasma membrane using a short protocol that is based on the hydrolysis of plasma membrane ceramide into sphingosine by the action of exogenously applied bacterial recombinant neutral ceramidase. Plasma membrane ceramide content can then be determined by measuring the newly generated sphingosine at a stoichiometry of 1:1. A key step of this protocol is the chemical fixation of cells to block cellular sphingolipid metabolism, especially of sphingosine to sphingosine 1-phosphate. We confirmed that chemical fixation does not disrupt the lipid composition at the plasma membrane, which remains intact during the time of the assay. We illustrate the power of the approach by applying this protocol to interrogate the effects of the chemotherapeutic compound doxorubicin. Here we distinguished two pools of ceramide, depending on the doxorubicin concentration, consolidating different reports. In summary, we have developed the first approach to quantify ceramide in the plasma membrane, allowing the study of new avenues in sphingolipid compartmentalization and function. 相似文献
18.
Deanna L. Siow Charles D. Anderson Evgeny V. Berdyshev Anastasia Skobeleva Stuart M. Pitson Binks W. Wattenberg 《Journal of lipid research》2010,51(9):2546-2559
Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.51: 2546–2559. 相似文献
19.
Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase. 相似文献
20.
Kate E. Jarman Paul A. B. Moretti Julia R. Zebol Stuart M. Pitson 《The Journal of biological chemistry》2010,285(1):483-492
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 Ca2+-dependent manner at the previously identified “calmodulin-binding site” of SK1. We also demonstrate that CIB1 functions as a Ca2+-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. 相似文献