A new model for regulation of sphingosine kinase 1 translocation to the plasma membrane in breast cancer cells

The translocation of sphingosine kinase 1 (SK1) to the plasma membrane (PM) is crucial in promoting oncogenesis. We have previously proposed that SK1 exists as both a monomer and dimer equilibrium, although it unclear whether these species translocate PM via same or different mechanisms. therefore investigated structural determinants involved better understand how might potentially be targeted for therapeutic intervention. report here monomeric WT mouse (GFP-mSK1) translocates MCF-7L cells stimulated with carbachol phorbol 12-myristate 13-acetate, whereas response sphingosine-1-phosphate; thus, equilibrium between sensitive cellular stimulus. In addition, 13-acetate induced GFP-mSK1 lamellipodia, sphingosine-1-phosphate dimeric filopodia, suggesting regulates cell biological processes dependent on dimerization. mutants designed modulate dimerization confirmed this difference localization. Regulation by C-terminal tail was using truncations. Removal last five amino acids (PPEEP) prevented enzyme PM, removal ten restored translocation. This suggests penultimate (SRRGP) function brake, which can released sequestration PPEEP sequence. propose alter arrangement N-terminal domains SK1, leading unique surfaces promote differential PM. Sphingosine-1-phosphate (S1P) bioactive lipid formed phosphorylation (Sph) kinase, there are two isoforms (sphingosine [SK1] 2). 2 encoded genes distinct subcellular localization biochemical properties (1Pyne S. Adams D.R. Pyne N.J. Sphingosine 1-phosphate kinases health disease: Recent advances.Prog. Lipid Res. 2016; 62: 93-106Crossref PubMed Scopus (117) Google Scholar). S1P degraded lyase, produce (E)-2-hexadecenal phosphoethanolamine, phosphatase, dephosphorylates form Sph from specific transporters then binds stimulates family G protein–coupled receptors, receptors (S1P1-S1P5), act intracellular targets, such histone deacetylase 1/2, induce responses An important role cancer evident studies showing high expression tumors linked poor prognosis patients (2Watson C. Long J.S. Orange Tannahill C.L. Mallon E. McGlynn L.M. Edwards J. High S1P1 S1P3, 1, extracellular signal-regulated kinase-1/2 associated development tamoxifen resistance estrogen receptor-positive breast patients.Am. Pathol. 2010; 177: 2205-2215Abstract Full Text PDF (135) Scholar, 3Ohotski Elsberger B. Watson Identification novel functional spatial associations other signaling proteins affect prognostic outcome cancer.Int. Cancer. 2013; 132: 605-616Crossref (37) 4Wang Q. Li G. Y. Xu M. Fu Prognostic significance non-small lung cancer.Tumour Biol. 2014; 35: 363-368Crossref (41) also promotes transformation, epithelial mesenchymal transition invasiveness, survival, replicative immortality, tumor neovascularization, aerobic glycolysis—the so-called hallmarks 5Pyne cancer.Nat. Rev. 10: 489-503Crossref (634) 6Maczis Milstien Spiegel cancer.Adv. Regul. 60: 160-165Crossref (33) Therefore, target intervention cancer. Indeed, oncogenic transformation NIH3T3 overexpression (7Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A. Wattenberg B.W. D'Andrea R.J. Vadas M.A. kinase.Curr. 2000; 1527-1530Abstract (354) Scholar), involves its cytoplasm process allows access substrate (Sph). importance kinase-dead G82D hSK1 mutant fails thereby demonstrating dependency catalytic activity A key step signal–regulated (ERK) Ser225 hSK1, (8Pitson Zebol Lynn H.E. Xia Activation ERK1/2-mediated phosphorylation.EMBO 2003; 22: 5491-5500Crossref (436) 9Pitson Leclercq T.M. Phosphorylation-dependent drives signaling.J. Exp. Med. 2005; 201: 49-54Crossref (223) underlined fact constitutive (9Pitson Moreover, demonstrated activation subsequent stimulation S1P2 receptor (‘inside-out’ signaling) increase transferrin (10Pham D.H. Powell J.A. Gliddon B.L. Tsykin A. Van der Hoek Kenyon R. Goodall G.J. Enhanced contributes 1.Oncogene. 33: 5559-5568Crossref (24) critically evidenced data neutralizing anti–transferrin antibody blocks oncogenesis Dynamically regulated clearly enzyme. Although basis controlled targeting remains incompletely understood at present, emergence crystal structures has begun shed some light issue recent years. adopts bidomain organization domain (CTD) hosts binding site an (NTD) ATP; center interface (as reviewed (11Adams kinases: Emerging structure-function insights.Trends Biochem. Sci. 41: 395-409Abstract (44) Scholar)). bound within J-shaped cavity (the ‘J-channel’), packing three extended lipid-binding loop against ?-sandwich core CTD. exposed hydrophobic patch exterior (LBL-1) (L194/F197/L198 human [hSK1]) been shown determinant engagement, including curvature-sensitive (12Shen H. Giordano F. Wu Chan Zhu Milosevic I. X. Yao K. Chen Baumgart T. Sieburth D. De Camilli Coupling endocytosis recruitment.Nat. Cell 16: 652-662Crossref (68) 13Pulkoski-Gross M.J. Jenkins M.L. Truman J.P. Salama M.F. Clarke C.J. Burke J.E. Hannun Y.A. Obeid intrinsic controls function.J. 2018; 59: 462-474Abstract (20) LBL-1 implicated calcium- integrin-binding protein, calcium-sensing partner protein (14Jarman K.E. Translocation mediated 1.J. Chem. 285: 483-492Abstract (113) Acidic phospholipids play critical notably phosphatidylserine (PS) phosphatidic acid (PA) (15Stahelin R.V. Hwang J.H. Kim Park Z.Y. Johnson K.R. Cho W. mechanism 280: 43030-43038Abstract (124) 16Olivera Rosenthal Effect acidic kinase.J. 1996; 529-537Crossref (91) 17Delon Manifava Wood Thompson Krugmann Ktistakis N.T. effector acid.J. 2004; 279: 44763-44774Abstract (170) Early Thr54 Asn89 interaction PS 18Hengst Guilford J.M. Fox T.E. Conroy E.J. Yun J.K. localized raft microdomain overcomes serum deprivation growth inhibition.Arch. Biophys. 2009; 492: 62-73Crossref (39) but attention focused set basic residues candidate phospholipid engagement. first identified analysis all currently available structures, assembly (19Wang Z. Min Xiao S.H. Johnstone Romanow Meininger Liu Dai Thibault Walker N. Molecular recognition catalysis.Structure. 21: 798-809Abstract (116) 20Gustin D.J. Brown Schmitt Wanska Connors Cardozo Cheng A.C. Jeffries Franks Shen Wong et al.Structure guided design series (SphK) inhibitors.Bioorg. Lett. 23: 4608-4616Crossref (69) 21Wang Knapp Elkins Crystal structure PF-543.ACS 5: 1329-1333Crossref consistent coimmunoprecipitation showed dimerize (22Lim K.G. Tonelli Lu Bittman FTY720 analogues inhibitors: Enzyme inhibition kinetics, allosterism, proteasomal degradation, actin rearrangement MCF-7 cells.J. 2011; 286: 18633-18640Abstract (105) mode generates pronounced groove aligned parallel loops protomers assembly. grooved surface exhibits positive electrostatic potential because contributions NTD tip LBL-1, topographically coordinated patches provide suitable membrane-binding mutagenesis validated engagement specifically K27, K29, R186 PA-enriched membranes (13Pulkoski-Gross ERK (conserved [mSK1]) occupies prominently solvent-exposed position regulatory ‘R-loop’) packs reverse side CTD opposite surface. underpinning not yet elucidated. However, may involve interdomain movement affects presentation relative thence presents combination elements misaligned patches; R-loop impact capacity adopt principle As added complexity, additional drivers independent S225 SK1. For example, Gq mSK1 HEK293 manner generation activated G?q subunit downstream [Ca2+]i elevation (23ter Braak Danneberg Lichte Liphardt Hla Jakobs K.H. Meyer zu Heringdorf Galpha(q)-mediated kinase-1 cross-activation receptors.Biochim. Acta. 1791: 357-370Crossref (26) proteins, necrosis factor receptor-associated (24Xia Albanese Chai interacts TRAF2 dissects factor-alpha 2002; 277: 7996-8003Abstract (251) Scholar) phosphatase (25Barr R.K. Khew-Goodall Deactivation 2A.J. 2008; 283: 34994-35002Abstract (45) suggested regulating truncation immediately preceding Gly364 remove (21 residues) PS-containing microdomains (26Hengst Enhancement deletion 21 COOH-terminus.Arch. 494: 23-31Crossref (13) phosphorylation. existence phosphorylation-dependent phosphorylation-independent mechanisms raises question any mechanistic intersection differing modes level structure. occur, if destabilizes autoinhibitory folding so correctly present regions required phospholipids. would represent unified localization, promoted either alteration tail. structural/functional facilitate mSK1. includes defining forms localize aspects enzyme’s pleiotropic functions, Such inform evolutionary conserved structural–functional enable identification possible deregulated controlling preclinical murine disease models. Confirmation Western blot anti-GFP (Fig. 1A). treatment (5 ?M, 10 min), (PMA) (1 (100 min) endogenous (detected anti-SK1 antibody) Carbachol M1- Gq-coupled M3 muscarinic (27Fiszman G.L. Middonno M.C. de la Torre Farina Español A.J. Sales M.E. cholinergic induces proliferation angiogenesis stimulating nitric oxide synthase activity.Cancer Ther. 2007; 6: 1106-1113Crossref (35) Scholar); bind S1P3 (28Long Tovey Mair K.M. Schiff Natarajan V. tolerance epidermal prevents formation migratory phenotype 1- phosphate cells.Mol. Cell. 30: 3827-3841Crossref (85) S1P, PMA, activate phospholipase D (PLD) PA (29Brizuela Rábano Gangoiti Narbona Macarulla Trueba Gómez- Muñoz aldosterone secretion through involving PI3K/PKB MEK/ERK 1/2 pathways.J. 48: 2264-2274Abstract 30Kiss Crilly K.S. Anderson W.H. Phorbol ester phosphatidylcholine synthesis requires C-alpha D.Biochim. 1998; 1392: 109-118Crossref (7) 31Schmidt Hüwe Fasselt Homann Rümenapp U. Sandmann Mechanisms m3 acetylcholine receptors. Evidence involvement tyrosine phosphorylation.Eur. 1994; 225: 667-675Crossref (88) quantified measuring immunoreactivity GFP intensity area under curve (AUC) described Experimental procedures. Both exhibited significant increases AUC after ligands B C). Results were percentage containing PM-associated transiently overexpressing cells, appeared very little majority perinuclear region. Treatment GFP-mSK1, These observed redistribution cytoplasm/perinuclear GFP-transfected carbachol, did show S1). To confirm identity treated siRNA, reduced immunostaining 42-kDa detected lysates decreased immunofluorescence ligand 1D).Figure 1Effect MEK-1 inhibitor, PD98059, cells. expressing pretreated without PD98059 (50 h) siRNA (200 nM, 24 before ?M) PMA ?M,) min. Cells processed (see procedures) mounted DAPI stain DNA (blue). A, photomicrographs 40× oil magnification FITC-conjugated secondary immunofluorescence, respectively). Inset probed P-ERK antibodies ERK-1/2 PD98059. Reprobing total GAPDH used similar loading. Representative results experiments. C, measurements made individual stitched together (n = 5) (B) (C). B, bar graph represents 5); ??p < 0.01 alone versus PD98059; +p 0.05, ++p 0.01, +++p 0.001 stimulus control (two-way ANOVA Tukey's post hoc test). left transfected 5). right translocated 3); ????p 0.0001 ++++p D, effect expression. upper 3) scrambled ???p lower scrambled, ?p 0.001, AUC, curve; ERK, kinase; mSK1, SK1; 13-acetate; 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure