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• W1996785365 abstract "Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser225, but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser225. The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser225 phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser225 region of SK1. Finally, both basal and tumor necrosis factor-α-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation. Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser225, but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser225. The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser225 phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser225 region of SK1. Finally, both basal and tumor necrosis factor-α-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation. Sphingosine kinase 1 (SK1) 2The abbreviations used are: SK1, sphingosine kinase 1; ERK1/2, extracellular signal-regulated kinases 1 and 2; PP, protein phosphatase; PP2Ac, protein phosphatase 2A catalytic subunit; HEK293, human embryonic kidney cells; TNFα, tumor necrosis factor-α; S1P, sphingosine 1-phosphate; I-2, human recombinant protein phosphatase inhibitor-2; HA, hemagglutinin; GST, glutathione S-transferase; IgGH, immunoglobulin heavy chain. has emerged as a critical regulator of cellular signaling through the generation of the bioactive phospholipid, sphingosine 1-phosphate (S1P). S1P has been implicated in a broad range of cellular processes, including cell proliferation, apoptosis, calcium homeostasis, angiogenesis, and vascular maturation (1.Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell. Biol. 2008; 9: 139-150Crossref PubMed Scopus (2461) Google Scholar). This diverse range of actions of S1P may be due in part to the ability of this lipid mediator to function as both an intracellular second messenger and a ligand for cell surface receptors (2.Spiegel S. Kolesnick R. Leukemia. 2002; 16: 1596-1602Crossref PubMed Scopus (115) Google Scholar). Cellular S1P levels are largely controlled by the actions of sphingosine kinases, which catalyze the formation of S1P from sphingosine (3.Leclercq T.M. Pitson S.M. IUBMB Life. 2006; 58: 467-472Crossref PubMed Scopus (51) Google Scholar). Although low cellular levels of S1P are present under basal conditions, growth factor and other agonist stimulation of cells can result in a rapid and transient increase in S1P levels as a direct consequence of SK1 activation (4.Pitson S.M. Moretti P.A. Zebol J.R. Xia P. Gamble J.R. Vadas M.A. D'Andrea R.J. Wattenberg B.W. J. Biol. Chem. 2000; 275: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Thus, the activation state of SK1 is critical in the control of cellular S1P levels and the cellular effects of this bioactive phospholipid. A basal level of SK activity has been proposed to play a “housekeeping” role in maintaining relatively low levels of sphingosine and ceramide in the cell (5.Wattenberg B.W. Pitson S.M. Raben D.M. J. Lipid Res. 2006; 47: 1128-1139Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In contrast, agonist stimulation can result in a rapid and transient increase in SK1 activity, as a result of an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for this phosphorylation, which occurs at Ser225 in human SK1 (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar). Interestingly, this phosphorylation results not only in increased SK1 activity, but is also crucial for agonist-induced translocation of SK1 from the cytosol to the plasma membrane (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar), and subsequent oncogenic signaling by this enzyme (7.Pitson S.M. Xia P. Leclercq T.M. Moretti P.A. Zebol J.R. Lynn H.E. Wattenberg B.W. Vadas M.A. J. Exp. Med. 2005; 201: 49-54Crossref PubMed Scopus (227) Google Scholar). Phosphorylation of cellular proteins is a reversible process and the transient activation/phosphorylation of SK1 (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar, 8.Xia P. Wang L. Gamble J.R. Vadas M.A. J. Biol. Chem. 1999; 274: 34499-34505Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) suggests that levels of SK1 phosphorylation at Ser225 are governed by the competing actions of the protein kinases and protein phosphatases that mediate SK1 phosphorylation and dephosphorylation, respectively. As noted above, we have previously identified ERK1/2 as the protein kinases responsible for phosphorylation of SK1 at Ser225 (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar). Despite its likely importance in the regulation of SK1, and SK1-mediated cell signaling, the protein phosphatase responsible for dephosphorylation of this residue has, however, remained undefined. In this study we report that protein phosphatase 2A (PP2A) deactivates SK1 through dephosphorylation of phospho-Ser225. Materials—Protein Phosphatase 1 (PP1) purified from rabbit skeletal muscle was obtained from New England Biolabs (Ipswich, MA). Protein phosphatase 2A1 (PP2A1) purified from bovine kidney, okadaic acid, human recombinant protein phosphatase inhibitor-2 (I-2), and fostriecin were obtained from Calbiochem. Monoclonal anti-FLAG (M2) antibody, anti-HA antibody and p-nitrophenyl phosphate were obtained from Sigma. Anti-His antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PP2A antibody, recognizing the PP2A catalytic subunit, was obtained from Upstate/Millipore (Temecula, CA). Mouse monoclonal antibody (DM1A) to α-tubulin was obtained from Abcam (Cambridge, UK). The phosphopeptide based on the SK1 phospho-Ser225 region (phospho-SK1 peptide) was synthesized by Auspep (Victoria, Australia) and had the sequence CGSKTPApSPVVVQQ, where pS denotes phospho-Ser. Protein G-Sepharose was obtained from Invitrogen (Carlsbad, CA), and recombinant human tumor necrosis factor-α (TNFα) was from R&D Systems (Minneapolis, MN). Generation of Expression Constructs—The generation of mammalian expression vectors for FLAG-epitope-tagged wild-type human SK1 and a truncated version of SK1 (SK1ΔCT) lacking 17 residues (368–384) at the C terminus have been previously described (9.Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (166) Google Scholar). Human PP2A catalytic subunit (PP2Ac) cDNA (GenBank™ accession number NM_002715) was amplified by Pfu DNA polymerase chain reaction from placenta cDNA with the oligonucleotide primers 5′-TAGAATTCCAATGGACGAGAAGGTGTTCAC-3′ and 5′-TAGGTACCTTACAGGAAGTAGTCTGGGGT-3′. The resultant product was cloned into pCMV(HA) (Clontech Laboratories Inc., Mountain View, CA) with EcoRI and KpnI to allow expression of N-terminal hemagglutinin (HA)-tagged PP2Ac in mammalian cells. To generate a glutathione S-transferase (GST)-PP2Ac fusion protein in bacteria, PP2Ac was also subcloned into pGEX4T2 (GE Healthcare, Piscataway, NJ) following digestion with EcoRI and NotI. Human protein phosphatase 4 catalytic subunit (PP4c) (GenBank™ accession number BC001416) was amplified by Pfu DNA polymerase chain reaction from placenta cDNA with the oligonucleotide primers 5′-TAGAATTCCCATGGCGGAGATCAGCG-3′ and 5′-TAGAATTCTCACAGGAAGTAGTCGGCC-3′. The resultant product was cloned into pCMV(HA) and pGEX4T2, with EcoRI for mammalian and bacterial expression, respectively. The HA-tagged catalytically inactive versions of PP2Ac (10.Ogris E. Mudrak I. Mak E. Gibson D. Pallas D.C. J. Virol. 1999; 73: 7390-7398Crossref PubMed Google Scholar) and PP4c (11.Hu M.C. Tang-Oxley Q. Qiu W.R. Wang Y.P. Mihindukulasuriya K.A. Afshar R. Tan T.H. J. Biol. Chem. 1998; 273: 33561-33565Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) (PP2AcR89A (human protein phosphatase 2A catalytic subunit with Arg89 → Ala mutation) and PP4cR236L (human protein phosphatase 4 catalytic subunit with Arg236 → Leu mutation), respectively) were generated by QuikChange™ mutagenesis using the primers 5′-GGAGATTATGTTGACGCCGGCTATTATTCAGTTGAA-3′, 5′-TTCAACTGAATAATAGCCGGCGTCAACATAATCTCC-3′ and 5′-CGCAGCCAATGACATCGATATGATCTGCCTTGCCCACCAACTGGT-3′, 5′-ACCAGTTGGTGGGCAAGGCAGATCATATCGATGTCATTGGCTGCG-3′, respectively. DNA sequencing verified the integrity and orientation of all cloned cDNAs. Cell Culture and Transfection—Human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (GIBCO® Invitrogen), containing 10% bovine calf serum (JRH Biosciences, Lenexa, KS), 2 mm glutamine, 0.2% (w/v) sodium bicarbonate, 1 mm HEPES, penicillin (1.2 mg/ml), and streptomycin (1.6 mg/ml). Cells were transiently transfected using Lipofectamine™ 2000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions. Transfected cells were harvested and lysed 24 h post-transfection. For immunoprecipitations, transfected cells were lysed by sonication in a Bioruptor™ (Diagenode, NY) (4 × 25 s pulses with 25 s breaks, 200 watts) in extraction buffer composed of 50 mm Tris/HCl buffer (pH 7.4) containing 150 mm NaCl, 10% glycerol, 0.05% Triton X-100, 1 mm dithiothreitol, 2 mm Na3VO4, 10 mm NaF, 10 mm β-glycerophosphate, 1 mm EDTA, and protease inhibitors (Complete™, Roche Applied Sciences). Where samples were used for coimmunoprecipitation, transfected cells were lysed in extraction buffer containing no dithiothreitol, and drawn 5 times through a 26.5-gauge needle rather than being sonicated. All lysates were clarified by centrifugation (17,000 × g, 15 min, 4 °C) prior to subsequent analyses. Lysates for use in in vitro phosphatase assays were prepared in a modified format, as described in detail below. Preparation of GST Fusion Proteins and Pulldown Analyses—GST fusion proteins were prepared and purified as described previously (12.Sutherland C.M. Moretti P.A. Hewitt N.M. Bagley C.J. Vadas M.A. Pitson S.M. J. Biol. Chem. 2006; 281: 11693-11701Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), except that after growth and induction with isopropyl 1-thio-β-d-galactopyranoside, bacteria were lysed in phosphate-buffered saline supplemented with protease inhibitors (Complete™). For pulldown analyses, 2 μg of each fusion protein immobilized on glutathione-Sepharose (GE Healthcare, Piscataway, NJ) was incubated with 1 μg of recombinant His-tagged SK1 protein (13.Pitson S.M. Moretti P.A. Zebol J.R. Zareie R. Derian C.K. Darrow A.L. Qi J. D'Andrea R.J. Bagley C.J. Vadas M.A. Wattenberg B.W. J. Biol. Chem. 2002; 277: 49545-49553Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) in extraction buffer for 1.5 h at 4 °C with constant mixing. Protein complexes were then washed three times in extraction buffer and bound SK1 resolved by SDS-PAGE and visualized by immunoblotting via its His epitope. Coimmunoprecipitation and Immunoblotting—For coimmunoprecipitation analyses, lysates from transfected cells were incubated in the presence of the appropriate antibody (anti-FLAG or anti-HA) with mixing for 1.5 h at 4 °C, prior to addition of protein G-Sepharose and further incubation with mixing (1 h, 4 °C). Immunocomplexes were washed three times in extraction buffer prior to SDS-PAGE, transfer of the proteins to nitrocellulose and immunoblotting. Following incubation with primary antibodies and horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Pierce) as appropriate, an enhanced chemiluminescence kit (ECL, Amersham Biosciences) was used for detection. In Vitro Phosphatase Assays—Phosphorylase b was 32P-labeled by phosphorylase kinase to form 32P-phosphorylase a as previously described (14.Tung H.Y. Pelech S. Fisher M.J. Pogson C.I. Cohen P. Eur. J. Biochem. 1985; 149: 305-313Crossref PubMed Scopus (57) Google Scholar). This labeled phosphorylase a was then used as a substrate to standardize the activities of the commercial PP1 and PP2A phosphatases via scintillation counting of 32P released, such that 1 unit of each protein phosphatase could be included in subsequent experiments. A unit of phosphatase activity is defined as the amount of enzyme required to release 1 nmol of 32P/min from [32P]phosphorylase a. Assays to detect endogenous phosphatase activity were performed as previously described (15.Mayer-Jaekel R.E. Ohkura H. Ferrigno P. Andjelkovic N. Shiomi K. Uemura T. Glover D.M. Hemmings B.A. J. Cell Sci. 1994; 107: 2609-2616Crossref PubMed Google Scholar). Briefly, whole cell extracts from cells overexpressing SK1 were incubated at 37 °C in phosphatase buffer (50 mm Tris/HCl (pH 7.0), 5 mm MnCl2, 5 mm MgCl2, 0.1 mm EDTA, 5 mm dithiothreitol, 0.025% Tween 20) in a 10-μl total reaction volume. Phosphatase inhibitors (1 μm I-2, 1.6 μm fostriecin) were added, as specified in individual experiments. Control reactions were carried out in the presence of the relevant vehicle solutions. Reactions were terminated by the addition of 5× Laemmli sample buffer with boiling, and then subjected to SDS-PAGE. Dephosphorylation of SK1 was quantitated by immunoblotting with antibodies recognizing phospho-Ser225 of SK1 (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar), with total SK1 levels assessed by anti-FLAG antibodies. Where overexpressed phosphatases were immunoprecipitated prior to use in in vitro phosphatase assays, transfected HEK293 cells were lysed by sonication in modified extraction buffer containing 50 mm Tris/HCl (pH 7.4), 150 mm NaCl, 10% (v/v) glycerol, 1 mm dithiothreitol, 0.05% (v/v) Triton X-100, and Complete™ protease inhibitors. Lysates were clarified by centrifugation (17,000 × g, 15 min, 4 °C), and overexpressed phosphatases were immunoprecipitated with anti-HA antibody and protein G-Sepharose. Immunoprecipitation complexes were washed three times in wash buffer (50 mm Tris/HCl (pH 7.4), 150 mm NaCl, 10% (v/v) glycerol) prior to use in in vitro phosphatase assays with either p-nitrophenyl phosphate (0.9 mg/ml) or phospho-SK1 peptide (625 μm) substrates, as per the manufacturer's protocols for Ser/Thr assay kit 1 (Upstate Biotechnology Inc., Waltham, MA) and as described previously (16.Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. Van Hooser A. Hu M.C. Brinkley B.R. Tan T.H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Sphingosine Kinase Assays—Sphingosine kinase activity was routinely determined using d-erythro-sphingosine and [γ-32P]ATP as substrates, as described previously (9.Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (166) Google Scholar). A unit of sphingosine kinase activity is defined as the amount of enzyme required to produce 1 pmol of S1P/min. siRNA Silencing—The siGENOME SMART pool (M-003598) targeting human PP2A catalytic subunit, α isoforms (NM_ 002715) was purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO). The Stealth universal negative control siRNA reagent and transfection reagents (Lipofectamine™ 2000 and Lipofectamine™ RNAiMAX) were purchased from Invitrogen. For transfection of siRNA into HEK293 cells, Lipofectamine™ RNAiMAX was used according to the manufacturer's guidelines. For cotransfection of plasmid DNA and siRNA into HEK293 cells, Lipofectamine™ 2000 was used according to the manufacturer's guidelines. siRNAs were used at a final concentration of 10 nm. Cells were harvested 72–96 h post-transfection and lysed as described above. Cell Fractionation—For analyses of soluble versus membrane localization of SK1, cells were lysed first by sonication as described above, but in extraction buffer containing no Triton X-100. These whole cell extracts were then clarified by centrifugation (17,000 × g, 15 min, 4 °C) yielding the supernatant as the soluble fraction. The cell pellets were then sonicated as above in extraction buffer containing 1% (v/v) Triton X-100, and clarified as above, yielding the supernatant as the membrane fraction. PP2A-family Phosphatases Dephosphorylate SK1—A number of growth factors and cytokines induce a rapid and transient activation of SK1 (4.Pitson S.M. Moretti P.A. Zebol J.R. Xia P. Gamble J.R. Vadas M.A. D'Andrea R.J. Wattenberg B.W. J. Biol. Chem. 2000; 275: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). In the case of TNFα, we have previously shown that this correlated with the rapid phosphorylation and subsequent rapid dephosphorylation of SK1 at Ser225 (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar). Because this dephosphorylation and deactivation of SK1 suggested the importance of a protein phosphatase in SK1 regulation, we sought to establish the identity of this enzyme. To do this, we initially exploited the observation that overexpression of SK1 in HEK293 cells results in the detectable phosphorylation of this protein at Ser225 (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar). Assay conditions were then established to follow SK1 dephosphorylation in vitro due to endogenous protein phosphatase activity. Specifically, lysates were prepared from cells overexpressing SK1 and incubated under conditions that permitted phosphatase activity. Immunoblot analysis with an antibody that specifically detects phospho-Ser225 of SK1 (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar) revealed a noticeable decrease in phospho-SK1 levels over time, while levels of total SK1 protein remained unchanged (Fig. 1A). Human Ser/Thr-specific phosphatases can be grouped into five broad classes, comprising the PP1, PP2/4/6, PP3, PP5, and PP7 subfamilies (17.Cohen P.T. Philp A. Vazquez-Martin C. FEBS Lett. 2005; 579: 3278-3286Crossref PubMed Scopus (132) Google Scholar). However, the PP1 and PP2/4/6 subclasses have been reported to contribute most of the Ser/Thr phosphatase activity in cells (18.Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). PP2A is by far the best-characterized member within the PP2/4/6 subclass, given that its catalytic, structural, and regulatory subunits have been sequenced and identified, and its susceptibility to inhibition by compounds such as okadaic acid and fostriecin is well known (19.Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1542) Google Scholar). Although the related PP4 and PP6 proteins are known to be similarly susceptible to active site inhibitors (20.Prickett T.D. Brautigan D.L. J. Biol. Chem. 2006; 281: 30503-30511Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 21.Brewis N.D. Street A.J. Prescott A.R. Cohen P.T. EMBO J. 1993; 12: 987-996Crossref PubMed Scopus (200) Google Scholar), considerably less is known about their component subunits. Using the conditions we established to follow SK1 dephosphorylation, we assessed the relative contributions to this activity from endogenous PP1- and PP2A-family phosphatases, given that these are known to constitute the majority of cellular phosphatase activity. In order to distinguish between these phosphatase subclasses, we included I-2 and fostriecin in the assay, which are selective inhibitors of the PP1- and PP2A-family phosphatases, respectively. Fostriecin displayed noticeable inhibition of SK1 dephosphorylation under these conditions, whereas I-2 showed no such effect (Fig. 1B), supporting a role for the PP2A-family phosphatases, but not PP1-family phosphatases, in regulating levels of phospho-SK1. Direct Association of PP2Ac and Recombinant SK1—The previous assays demonstrated that PP2A activity reduced phospho-SK1 levels in cell lysates, but did not demonstrate a direct interaction between the proteins. To address whether PP2A exerted direct or indirect effects on phospho-SK1 levels, we prepared the catalytic subunit of PP2A as a GST fusion protein (GST-PP2Ac) and used this in pulldown analyses with purified recombinant human SK1 generated in insect cells (13.Pitson S.M. Moretti P.A. Zebol J.R. Zareie R. Derian C.K. Darrow A.L. Qi J. D'Andrea R.J. Bagley C.J. Vadas M.A. Wattenberg B.W. J. Biol. Chem. 2002; 277: 49545-49553Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We found that this purified recombinant SK1 specifically associated with GST-PP2Ac, but not GST alone (Fig. 2), indicating a direct interaction between PP2Ac and SK1. To reinforce the role of PP2A in SK1 dephosphorylation, we examined the ability of commercial preparations of PP1 and PP2A to dephosphorylate purified recombinant SK1 (13.Pitson S.M. Moretti P.A. Zebol J.R. Zareie R. Derian C.K. Darrow A.L. Qi J. D'Andrea R.J. Bagley C.J. Vadas M.A. Wattenberg B.W. J. Biol. Chem. 2002; 277: 49545-49553Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), which is known to be phosphorylated at Ser225 (22.Stahelin R.V. Hwang J.H. Kim J.H. Park Z.Y. Johnson K.R. Obeid L.M. Cho W. J. Biol. Chem. 2005; 280: 43030-43038Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Immunoblotting to detect levels of phospho-SK1 indicated that PP2A treatment resulted in substantial SK1 dephosphorylation, while, in contrast, PP1 had no effect on phospho-SK1 levels (Fig. 3A). Furthermore, PP2A-treated recombinant SK1 demonstrated reduced catalytic activity in vitro (Fig. 3B). This is consistent with observations that phosphorylation of SK1 at Ser225 correlates with an increase in the activity of the protein (6.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (459) Google Scholar), and thus dephosphorylation of this site results in reduced SK1 activity. Coimmunoprecipitation of PP2Ac and SK1—The GST pulldown analysis above indicated that protein complexes of SK1 and PP2Ac could form in vitro. Next, we investigated whether similar protein complexes could form within cells by performing coimmunoprecipitation analysis using lysates from HEK293 cells coexpressing HA-tagged PP2Ac and FLAG-tagged SK1. We found that PP2Ac could be detected in SK1 immunoprecipitates (Fig. 4A), and additionally, SK1 was detected in equivalent PP2Ac immunoprecipitates (Fig. 4B). These results indicated that the catalytic subunit of PP2A could interact with SK1 protein not only in vitro, but also in a more physiologically relevant cellular context. Role of the Pro-rich C Terminus of SK1 in Regulation by PP2Ac—Thus far, our data indicated a direct interaction between SK1 and PP2Ac, but did not highlight the binding interface(s) between these proteins. It seemed most likely that the PP2A catalytic subunit would interact with the phospho-Ser225 motif of SK1, given that its activity was directed toward this region of the protein. However, it seemed likely that other regions of the protein might also participate in the interaction, given previous reports of the interactions of the related PP2A-family member, PP4, and its substrates. The PP4 substrate hematopoietic progenitor kinase 1, an upstream activating kinase in the pathway leading to c-Jun N-terminal kinase activation, binds PP4c through a Pro-rich region in its C terminus (23.Zhou G. Boomer J.S. Tan T.H. J. Biol. Chem. 2004; 279: 49551-49561Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Because SK1 also possesses a Pro-rich C-terminal region (13.Pitson S.M. Moretti P.A. Zebol J.R. Zareie R. Derian C.K. Darrow A.L. Qi J. D'Andrea R.J. Bagley C.J. Vadas M.A. Wattenberg B.W. J. Biol. Chem. 2002; 277: 49545-49553Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), we sought to investigate its significance for the SK1-PP2Ac interaction. For these studies, HEK293 cells were cotransfected with PP2Ac and a truncated version of SK1, lacking the 17 extreme C-terminal residues, which constitutes the Prorich region (SK1ΔCT) (Fig. 5A). We found that while PP2Ac coimmunoprecipitated with both SK1 and SK1ΔCT, the amount of PP2Ac detected in SK1ΔCT immunoprecipitates was substantially reduced (Fig. 5B). This prompted us to examine whether this reduced interaction with PP2Ac also had implications for the levels of phosphorylated SK1ΔCT in cells. We found that similar to wildtype SK1, coexpression of PP2Ac reduced levels of SK1ΔCT phosphorylated at Ser225 (Fig. 5C). Strikingly, however, in the absence of PP2A coexpression, the basal levels of Ser225 phosphorylation were found to be considerably higher for SK1ΔCT compared with wild-type SK1 (Fig. 5C). In light of the reduced amount of PP2Ac detected in SK1ΔCT immunoprecipitates (Fig. 5B), it is possible that this could result from a reduced ability of this mutant to interact with endogenous PP2A. Taken together, these results suggest that while the Pro-rich C terminus of SK1 is not essential for the interaction with PP2Ac, it may contribute to both the affinity of the PP2Ac-SK1 interaction and also the ability of PP2Ac to dephosphorylate SK1 at Ser225. Implications of PP2A Activity for Cellular Phospho-SK1 Levels—Thus far, our overexpression studies strongly suggested that PP2A regulated cellular levels of phospho-SK1. We next performed complementary experiments using a catalytically inactive form of PP2A, given that the use of such a dominant-negative is a more physiological tool than overexpression of an active phosphatase. Thus, we cotransfected HEK293 cells with SK1 and catalytically inactive forms of either PP2Ac, or the related PP4c (PP2AcR89A or PP4cR236L, respectively). Immunoblotting analysis on these cell lysates for phospho-SK1 indicated that PP2AcR89A expression resulted in hyperphosphorylation of SK1 compared with the control cells (Fig. 6A). In contrast, coexpression of PP4cR236L had no effect on cellular phospho-SK1 levels (Fig. 6A). This again supported a role for PP2A in the regulation of SK1 phosphorylation, rather than its related family members, such as PP4. To further confirm this finding, we expressed PP2Ac and the related PP4c in HEK293 cells, immunoprecipitated the active phosphatases, and assessed their activities toward a phosphopeptide substrate based on the phospho-Ser225 region of SK1 (phospho-SK1 peptide) using an in vitro assay measuring phosphate release. PP2Ac dephosphorylated the phospho-SK1 peptide, whereas PP4c had no noticeable activity toward the substrate (Fig. 6B), despite both protein phosphatases displaying activity against a generic substrate, p-nitrophenyl phosphate (Fig. 6B). Notably, even when 3.5-fold more PP4c was used in these assays, dephosphorylation of the phospho-SK1 peptide substrate remained undetectable, while activity toward the pNPP substrate increased proportionately (data not shown). Thus, it appeared that PP2Ac, but not the related PP4c, had the ability to directly dephosphorylate the phospho-SK1 peptide. These results were in keeping with the above coexpression studies, and provided further evidence supporting a role for PP2A, rather than the related PP4, in regulating cellular levels of phospho-SK1. PP2A Mediates Deactivation of SK1 following TNFα Stimulation—Although our findings indicated a role for PP2A activity in SK1 regulation, all cellular experiments thus far relied on ectopic expression of SK1 and PP2Ac. We next chose to investigate the potential for PP2A to participate in the regulation of SK1 phosphorylation in" @default.
• W1996785365 created "2016-06-24" @default.
• W1996785365 creator A5018351867 @default.
• W1996785365 creator A5032213369 @default.
• W1996785365 creator A5032903954 @default.
• W1996785365 creator A5037001168 @default.
• W1996785365 creator A5052287744 @default.
• W1996785365 date "2008-12-01" @default.
• W1996785365 modified "2023-10-17" @default.
• W1996785365 title "Deactivation of Sphingosine Kinase 1 by Protein Phosphatase 2A" @default.
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• W1996785365 cites W1595085053 @default.
• W1996785365 cites W1966452335 @default.
• W1996785365 cites W1975748957 @default.
• W1996785365 cites W1981686290 @default.
• W1996785365 cites W1983755623 @default.
• W1996785365 cites W1985430829 @default.
• W1996785365 cites W1995294841 @default.
• W1996785365 cites W1997218197 @default.
• W1996785365 cites W2005071794 @default.
• W1996785365 cites W2015379895 @default.
• W1996785365 cites W2017092095 @default.
• W1996785365 cites W2022589671 @default.
• W1996785365 cites W2023879911 @default.
• W1996785365 cites W2024106968 @default.
• W1996785365 cites W2027509121 @default.
• W1996785365 cites W2042268974 @default.
• W1996785365 cites W2051485704 @default.
• W1996785365 cites W2057221801 @default.
• W1996785365 cites W2061243113 @default.
• W1996785365 cites W2066717022 @default.
• W1996785365 cites W2068712775 @default.
• W1996785365 cites W2069853996 @default.
• W1996785365 cites W2070341850 @default.
• W1996785365 cites W2076269247 @default.
• W1996785365 cites W2081280187 @default.
• W1996785365 cites W2084815981 @default.
• W1996785365 cites W2085444732 @default.
• W1996785365 cites W2088811318 @default.
• W1996785365 cites W2090509124 @default.
• W1996785365 cites W2101400842 @default.
• W1996785365 cites W2101803513 @default.
• W1996785365 cites W2108813293 @default.
• W1996785365 cites W2115065585 @default.
• W1996785365 cites W2121559592 @default.
• W1996785365 cites W2123556335 @default.
• W1996785365 cites W2126167700 @default.
• W1996785365 cites W2129727010 @default.
• W1996785365 cites W2132805096 @default.
• W1996785365 cites W2136584723 @default.
• W1996785365 cites W2137221055 @default.
• W1996785365 cites W2151948261 @default.
• W1996785365 cites W2152009878 @default.
• W1996785365 cites W2154089780 @default.
• W1996785365 cites W2155952015 @default.
• W1996785365 cites W2156532107 @default.
• W1996785365 cites W2157033357 @default.
• W1996785365 cites W2164422031 @default.
• W1996785365 cites W2349550891 @default.
• W1996785365 cites W2902314794 @default.
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• W1996785365 doi "https://doi.org/10.1074/jbc.m804658200" @default.
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