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• W2023879911 abstract "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. 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. Sphingosine kinases are important signaling enzymes because of their role in the synthesis of the bioactive lipid sphingosine 1-phosphate (S1P). 3The abbreviations used are: S1P, sphingosine 1-phosphate; CaM, calmodulin; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; hSK1, human sphingosine kinase 1; hSK2, human sphingosine kinase 2; PCB, putative calmodulin binding region; PA, phosphatidic acid; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; TNFα, tumor necrosis factor-α. 3The abbreviations used are: S1P, sphingosine 1-phosphate; CaM, calmodulin; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; hSK1, human sphingosine kinase 1; hSK2, human sphingosine kinase 2; PCB, putative calmodulin binding region; PA, phosphatidic acid; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; TNFα, tumor necrosis factor-α. Many studies have shown that S1P can affect a diverse array of biological processes, including calcium mobilization, mitogenesis, apoptosis, atherosclerosis, inflammatory responses, cell motility, and angiogenesis (1Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (659) Google Scholar, 2Spiegel S. English D. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 3Watterson K. Sankala H. Milstein S. Spiegel S. Prog. Lipid Res. 2003; 42: 344-357Crossref PubMed Scopus (94) Google Scholar, 4Baumruker T. Bornancin F. Billich A. Immunol. Lett. 2005; 96: 175-185Crossref PubMed Scopus (79) Google Scholar). Although some of these varied effects of S1P result from its action as a ligand for S1P-specific cell-surface G-protein-coupled receptors (5Hla T. Lee M.-J. Ancellin N. Paik J.H. Kluk M.J. Science. 2001; 294: 1875-1878Crossref PubMed Scopus (477) Google Scholar), significant evidence now exists that indicates S1P can also function intracellularly as a second messenger, particularly in the regulation of cell proliferation and apoptosis (6Maceyka M. Payne S.G. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1585: 193-201Crossref PubMed Scopus (493) Google Scholar).Two sphingosine kinases exist in humans (hSK1 and hSK2), with most studies to date focusing on hSK1. Although these two enzymes originate from different genes and differ in size, tissue distribution, developmental expression, substrate specificity, specific activity, and possibly in their cellular roles (7Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 8Liu H. Toman R.E. Goparaju S.K. Maceyka M. Nava V.E. Sankala H. Payne S.G. Bektas M. Ishii I. Chun J. Milstein S. Spiegel S. J. Biol. Chem. 2003; 278: 40330-40336Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 9Igarashi N. Okada T. Hayashi S. Fujita T. Jahangeer S. Nakamura S. J. Biol. Chem. 2003; 278: 46832-46839Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 10Roberts J.L. Moretti P.A.B. Darrow A.L. Derian C.K. Vadas M.A. Pitson S.M. Anal. Biochem. 2004; 331: 122-129Crossref PubMed Scopus (37) Google Scholar), their polypeptide sequences possess a high degree of similarity. In fact, almost all of the hSK1 sequence aligns with regions of the larger hSK2 sequence with 80% overall similarity (45% identity) (7Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). However, hSK2 also possesses two additional polypeptide regions at its N terminus and within the central region of its sequence that are quite distinct from hSK1.Although hSK1 has considerable intrinsic catalytic activity (11Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A.B. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (165) Google Scholar), its activity can be enhanced by exposure of cells to various growth factors and other agonists (6Maceyka M. Payne S.G. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1585: 193-201Crossref PubMed Scopus (493) Google Scholar). This agonist-induced activation of hSK1 results in an increase in cellular levels of S1P, which appears critical in mediating the cellular effects attributed to S1P (12Pitson S.M. Moretti P.A.B. 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). We have recently identified that activation of hSK1 occurs directly via its phosphorylation at Ser225 by ERK1/2 (13Pitson S.M. Moretti P.A.B. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (453) Google Scholar). We have also observed that this phosphorylation and, in particular, the subsequent phosphorylation-dependent translocation of hSK1 to the plasma membrane are critical steps for the pro-proliferative, anti-apoptotic effects of this enzyme in the cell (14Pitson S.M. Xia P. Leclercq T.M. Moretti P.A.B. Zebol J.R. Lynn H.E. Wattenberg B.W. Vadas M.A. J. Exp. Med. 2005; 201: 49-54Crossref PubMed Scopus (227) Google Scholar). Thus, considerable insight into the regulation of hSK1 has been recently gained. However, much is still not known regarding the exact molecular mechanisms controlling the activation and cellular localization of this enzyme.Numerous studies have shown that both human and mouse SK1 associate with calmodulin (CaM) in a Ca2+-dependent manner (11Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A.B. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (165) Google Scholar, 15Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 16Pitson S.M. Moretti P.A.B. Zebol J.R. Vadas M.A. D'Andrea R.J. Wattenberg B.W. FEBS Lett. 2001; 509: 169-173Crossref PubMed Scopus (16) Google Scholar, 17Pitson S.M. Moretti P.A.B. 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, 18Yokota S. Taniguchi Y. Kihara A. Mitsutake S. Igarashi Y. FEBS Lett. 2004; 578: 106-110Crossref PubMed Scopus (40) Google Scholar). Because CaM is a common regulatory protein controlling the activity and function of many signaling enzymes (19Chin D. Means A.R. Trends Cell Biol. 2000; 10: 322-328Abstract Full Text Full Text PDF PubMed Scopus (1113) Google Scholar), its potential role in the regulation of hSK1 has long been speculated (11Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A.B. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (165) Google Scholar, 20Olivera A. Spiegel S. Prostaglandins. 2001; 64: 123-134Crossref PubMed Scopus (145) Google Scholar, 21Liu H. Chakravarty D. Maceyka M. Milstien S. Spiegel S. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 493-511Crossref PubMed Google Scholar). Although we have shown previously that neither Ca2+-bound nor Ca2+-free CaM (Ca2+/CaM and apoCaM, respectively) had an effect on hSK1 activity in vitro (11Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A.B. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (165) Google Scholar), other studies have suggested it may play a role in Ca2+-dependent translocation of hSK1 to the plasma membrane because this process is inhibited by the Ca2+/CaM antagonist W-7 (22Young K.W. Willets J.M. Parkinson M.J. Bartlett P. Spiegel S. Nahorski S.R. Challiss R.A.J. Cell Calcium. 2003; 33: 119-128Crossref PubMed Scopus (54) Google Scholar). Further elucidation of the direct role of CaM in the regulation of hSK1 has, however, been limited by the lack of molecular tools to specifically ablate CaM binding to hSK1. In this study we have identified the CaM-binding sites of hSK1 and hSK2. This has enabled the generation of CaM-binding-deficient versions of the proteins and allowed further molecular examination of the role of CaM in sphingosine kinase regulation.EXPERIMENTAL PROCEDURESCell Culture and Transfection—Human embryonic kidney cells (HEK293T) were cultured in Dulbecco's modified Eagle's medium (JRH Biosciences, Lenexa, KS) containing 10% bovine calf serum (JRH Biosciences), 2 mm glutamine, 0.2% (w/v) sodium bicarbonate, penicillin (1.2 mg/ml), and streptomycin (1.6 mg/ml). Cells were transiently transfected using the calcium phosphate precipitation method, harvested, and lysed by sonication as described previously (12Pitson S.M. Moretti P.A.B. 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). Protein concentrations in cell homogenates were determined with Coomassie Brilliant Blue reagent (Sigma) using bovine serum albumin as standard. Assays for colony formation in soft agar were performed as detailed previously (23Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A.B. D'Andrea R.J. Wattenberg B.W. Vadas M.A. Curr. Biol. 2000; 19: 1527-1530Abstract Full Text Full Text PDF Scopus (358) Google Scholar).Construction of Sphingosine Kinase Mutants—hSK1 cDNA (GenBank™ accession number AF200328) was FLAG epitope tagged at the 3′ end and subcloned into pALTER site-directed mutagenesis vector (Promega Corp., Annandale, Australia), as described previously (12Pitson S.M. Moretti P.A.B. 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). Single-stranded DNA was prepared and used as template for oligonucleotide-directed mutagenesis as detailed in the manufacturer's protocol. The mutagenic oligonucleotides used to generate the mutant constructs were as follows: hSK1L134Q, 5′-ATGAAGACCAATTGACCAACT-3′; hSK1L147Q, 5′-GCCGGCTGCAGTCACCCAT-3′; hSK1L-153Q, 5′-TGAACCTGCAAAGCTTGCACACGG-3′; hSK1R185A/R186A, 5′-GAGAGTGAGAAGTATGCGGCCCTAGGGGAGATGCGCTTC-3′; hSK1L187Q, 5′-GAAGTATCGTCGACAGGGGGAGAT-3′; hSK1L194Q, 5′-AGATGCGCTTCACTCAGGGTACCTTCCTGCGTCTGGCA-3′; hSK1F197A, 5′-GCGCTTCACTCTGGGTACCGCCCTGCGTCTGGCAGC-3′; hSK1L198Q,5′-CTCTGGGCACTTTCCAGCGCTTGGCAGCCTTGCGCA-3′; hSK1F197A/L198Q, 5′-GCGCTTCACTCTGGGTACCGCCCAGCGCTTGGCAGC-3′; hSK1L200Q, 5′-GCACTTTCCTGCGTCAGGCAGCCTTACGCACTTACCGCGGC-3′; hSK1L194Q/L200Q,5′-AGATGCGCTTCACTCAGGGTACCTTCCTGCGTCAGGCAGCCTTACGCACTTACCGCGGC-3′; hSK1V290N, 5′-CTGTTCTACAACCGGGCCGGCGTGTCTCGT-3′; and hSK1F303H, 5′-CTGCTGCGCCTCCAGCTGGCCATGGAG-3′. The mutants were sequenced to verify incorporation of the desired modification, and the cDNA was subsequently subcloned into pcDNA3 (Invitrogen) for transient transfection into HEK293T cells.The hSK1F197A/L198Q was tagged at the N terminus with enhanced green fluorescent protein (EGFP) by using methods described previously for wild type hSK1 (13Pitson S.M. Moretti P.A.B. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (453) Google Scholar). hSK2V327A/L328Q was generated from hSK2 cDNA in pcDNA3 (10Roberts J.L. Moretti P.A.B. Darrow A.L. Derian C.K. Vadas M.A. Pitson S.M. Anal. Biochem. 2004; 331: 122-129Crossref PubMed Scopus (37) Google Scholar) by QuikChange mutagenesis (Stratagene) using the primers 5′-TTCACACTGGGCACGGCGCAAGGCCTCGCCACACTG-3′ and 5′-CAGTGTGGCGAGGCCTTGCGCCGTGCCCAGTGTGAA-3′ and sequenced to verify incorporation of the desired modifications. hSK2 was also tagged at the N terminus with EGFP. Briefly, EGFP was PCR-amplified from pEGFP-1 (Clontech) with the primers 5′-TAGGATCCGCCACCATGGTGAGCAAGG-3′ and 5′-TAGAATTCCGGCTTGTACAGCTCGTCCATGC-3′, and the resultant product was then cloned into pcDNA3 following digestion with BamHI and EcoRI. Wild type hSK2 and hSK2V327A/L328Q were amplified with 5′-TAGAATTCATGGCCCCGCCCCCA-3′ and 5′-TAGAATTCAGGGCTCCCGCCCCG-3′ primers, digested with EcoRI, and cloned into pcDNA3-EGFP. The orientation was determined by restriction analysis, with sequencing verifying the integrity of the EGFP-hSK2 cDNA sequences. hSK1hSK2-CBS and hSK2hSK1-CBS were generated from hSK1 and hSK2 cDNAs in pcDNA3 by QuikChange mutagenesis using the primers 5′-AGTGAGAAGTATCGGGCCCTGGGATCCGCGCGCTTCACTCTGGG-3′, 5′-CCCAGAGTGAAGCGCGCGGATCCCAGGGCCCGATACTTCTCACT-3′,5′-TTCACTCTGGGCACTGTCCTAGGTCTGGCAACCTTGCACACTTACCGCGGCCG-3′, and 5′-CGGCCGCGGTAAGTGTGCAAGGTTGCCAGACCTAGGACAGTGCCCAGAGTGAA-3′ for hSK1hSK2-CBS and 5′-GAGCGAGCGCTTCAGACGTCTGGGCGAGATGCGCTTCACACTGGGC-3′, 5′-GCCCAGTGTGAAGCGCATCTCGCCCAGACGTCTGAAGCGCTCGCTC-3′, 5′-TTCACACTGGGCACGTTTCTGCGACTCGCCGCACTACGTACCTACCGCGGACGC-3′, and 5′-GCGTCCGCGGTAGGTACGTAGTGCGGCGAGTCGCAGAAACGTGCCCAGTGTGAA-3′ for hSK2hSK1-CBS.Sphingosine Kinase Assays—Sphingosine kinase activity was routinely determined using d-erythro-sphingosine (Biomol, Plymouth Meeting, PA) and [γ-32P]ATP as substrates as described previously (10Roberts J.L. Moretti P.A.B. Darrow A.L. Derian C.K. Vadas M.A. Pitson S.M. Anal. Biochem. 2004; 331: 122-129Crossref PubMed Scopus (37) Google Scholar). A unit of sphingosine kinase activity is defined as the amount of enzyme required to produce 1 pmol of S1P/min.Mass Measurement of S1P—Measurement of cellular S1P levels were performed using the enzymatic method described by Edsall et al. (24Edsall S. Vann L. Milstein S. Spiegel S. Methods Enzymol. 2000; 312: 9-16Crossref PubMed Google Scholar).Calmodulin Binding Assays—Assays to assess CaM binding of sphingosine kinase were performed as detailed previously (17Pitson S.M. Moretti P.A.B. 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). Briefly, HEK293T cells overexpressing wild type or mutant hSK1 or hSK2 were harvested and lysed as described above. The cell lysates were then centrifuged (13,000 × g, 15 min at 4 °C) to remove cell debris. Aliquots of the supernatants were added to tubes containing CaM-Sepharose 4B (Amersham Biosciences) pre-equilibrated with binding buffer composed of 50 mm Tris/HCl, pH 7.4, 100 mm NaCl, 10% (w/v) glycerol, 0.05% (w/v) Triton X-100, 1 mm dithiothreitol, 1.5 mm Na3VO4, 7.5 mm NaF, and protease inhibitors (Complete™, Roche Applied Science) and incubated with either 5 mm CaCl2 or5mm EGTA for 30 min at 4 °C with continuous mixing. The CaM-Sepharose 4B beads were then pelleted by centrifugation (5000 × g, 5 min at 4 °C) and washed twice with binding buffer. Bound hSK1 or hSK2 was then resolved by SDS-PAGE and visualized by Western blotting via the FLAG epitope. Sepharose CL-4B (Amersham Biosciences) was used as a control for nonspecific binding to CaM-Sepharose 4B.For studies to elucidate the role of hSK1 phosphorylation on its association with CaM, purified recombinant hSK1 generated in E. coli was phosphorylated in vitro at Ser225 by ERK2 as described previously (13Pitson S.M. Moretti P.A.B. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (453) Google Scholar).Generation of GST Peptides and Pull-down Analyses—The sequences encoding peptides containing the putative CaM binding (PCB) regions of hSK1 were PCR-amplified from the hSK1 cDNA with the following primers: PCB1/2, 5′-TAGGATCCCCGCCGGTCACCAATGAAGACCTCCT-3′ and 5′-TAGAATTCAAGCCGTGTGCAGAGACAGC-3′; PCB3, 5′-TAGGATCCCCGCCGCTAGAGAGTGAGAAGTATCGG-3′ and 5′-TAGAATTCAGCCGCGGTAAGTGCGCAA-3′; and PCB4, 5′-TAGGATCCCCGCCGGCTGGCGTCATGCATCTGT-3′ and 5′-TAGAATTCAATGCCTGCCCTTCTCCATG-3′. The products were subsequently digested with EcoRI and cloned into pGEX2T. Cultures of Escherichia coli JM109 transformed with these pGEX2T-PCB plasmids were grown overnight in Luria broth containing 100 mg/liter ampicillin at 37 °C with shaking. The cultures were then diluted 1 in 10 into the same medium and grown at 37 °C for 1 h with shaking until reaching an A600 of ∼0.6. Expression of the GST-PCB peptides was induced by the addition of isopropyl 1-thio-β-d-galactopyranoside to a final concentration of 1 mm, and the cultures were incubated for an additional 3 h. The cells were harvested by centrifugation at 6000 × g for 15 min at 4 °C and lysed by sonication (3 times, 30-s pulses of 5 watts) in 50 mm Tris/HCl, pH 7.4, containing 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, and protease inhibitors. Following clarification of the lysate by centrifugation at 20,000 × g for 30 min at 4 °C, glutathione-Sepharose (Amersham Biosciences) was added, and the mixture was incubated at 4 °C for 1 h with constant agitation. After this time the glutathione-Sepharose was washed three times with cold phosphate-buffered saline, and the GST peptides were eluted with 20 mm glutathione in 50 mm Tris/HCl, pH 8.5, for 10 min at 4 °C. Pull-down analyses with CaM-Sepharose and the GST peptide fusion proteins was performed as described above using ∼1 μg of each purified GST peptide or GST alone. Peptide binding to CaM-Sepharose was detected using an anti-GST antibody.Limited Proteolysis and N-terminal Sequencing—Recombinant hSK1 was generated and purified from Sf9 cells as described previously (17Pitson S.M. Moretti P.A.B. 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). Limited proteolysis of this hSK1 (1.5 μg in 15 μl) was performed in the presence or absence of a 3-fold molar excess of purified bovine CaM (Sigma) by the addition of 2 or 5 ng of trypsin (Roche Applied Science) in 100 mm Tris/HCl, pH 8.5. The mixture was then incubated at 37 °C for 60 min, stopped by the addition of 1.5 μl of 100 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride (Roche Applied Science), and incubated for an additional 5 min at 37 °C. Tryptic cleavage products were then resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Following Coomassie staining of the membrane, bands that were protected in the presence of CaM were excised, and their N-terminal sequences were determined by 6 cycles of automated Edman degradation using an Applied Biosystems 494 Procise Protein Sequencing System at the Australian Proteome Analysis Facility.Western Blotting—SDS-PAGE was performed on cell lysates using 12% acrylamide gels. Proteins were transferred to nitrocellulose, and the membranes were blocked overnight at 4 °C in phosphate-buffered saline containing 5% skim milk powder and 0.1% (w/v) Triton X-100. hSK1 expression levels in cell lysates were quantitated over a dilution series of the lysates with the monoclonal M2 anti-FLAG antibody (Sigma), with the immunocomplexes detected with horseradish peroxidase anti-mouse (Pierce) IgG using an enhanced chemiluminescence kit (ECL, Amersham Biosciences).Fluorescence Microscopy and Subcellular Fractionation—HEK293T cells expressing EGFP fusion proteins were plated onto poly-l-lysine-coated (Sigma) 8-well glass chamber slides and incubated for 24 h. Following incubation for 30 min with or without PMA, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 10 min. Epifluorescence microscopy was then performed on an Olympus BX-51 microscope equipped with a fluorescein excitation filter (494 nm), acquired to a Cool Snap FX charge-coupled device camera (Photometrics). COS-7 cells expressing EGFP fusion proteins were analyzed in a similar manner, except instead of PMA treatment, the effect of cell density on hSK2 cellular localization was determined by plating the cells at a wide range of cell densities. Cell lysates were fractionated into cytosol and membrane fractions as described previously (13Pitson S.M. Moretti P.A.B. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (453) Google Scholar).RESULTSMutagenesis of Predicted CaM-binding Sites in hSK1—hSK1 and its murine orthologue have been shown to bind CaM in a Ca2+-dependent manner (11Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A.B. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (165) Google Scholar, 15Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 16Pitson S.M. Moretti P.A.B. Zebol J.R. Vadas M.A. D'Andrea R.J. Wattenberg B.W. FEBS Lett. 2001; 509: 169-173Crossref PubMed Scopus (16) Google Scholar, 17Pitson S.M. Moretti P.A.B. 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, 18Yokota S. Taniguchi Y. Kihara A. Mitsutake S. Igarashi Y. FEBS Lett. 2004; 578: 106-110Crossref PubMed Scopus (40) Google Scholar). Previous analysis of the hSK1 polypeptide sequence indicated the presence of three potential Ca2+/CaM binding regions (11Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A.B. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (165) Google Scholar) as follows: two that overlap between residues 134 and 153 (termed here PCB1 and PCB2), and one spanning residues 290-303 (PCB4) (Fig. 1). All three of these putative Ca2+/CaM-binding sites are conserved in the mouse SK1 sequence (27Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Further analysis of the hSK1 polypeptide sequence using the CaM target data base (26Yap K.L. Kim J. Truong K. Sherman M. Yuan T. Ikura M. J. Struct. Funct. Genomics. 2000; 1: 8-14Crossref PubMed Scopus (454) Google Scholar) also revealed another putative Ca2+/CaM-binding site spanning residues 191-206 (PCB3) (Fig. 1). Again, this putative Ca2+/CaM binding region is also conserved in murine SK1.To examine the potential role of the putative Ca2+/CaM-binding sites in the interaction of hSK1 with CaM, we performed site-directed mutagenesis of hSK1 in these regions. In particular, this mutagenesis concentrated on the conserved hydrophobic residues within these motifs that are generally critical in Ca2+/CaM binding (28Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (689) Google Scholar, 29Vetter S.W. Leclerc E. Eur. J. Biochem. 2003; 270: 404-414Crossref PubMed Scopus (281) Google Scholar, 30Bhattacharya S. Bunick C.G. Chazin W.J. Biochim. Biophys. Acta. 2004; 1742: 69-79Crossref PubMed Scopus (209) Google Scholar), changing them to structurally conservative hydrophilic residues. PCB1 and PCB2 were targeted by a mutations of Leu147 → Gln (hSK1L147Q) and Leu153 → Gln (hSK1L153Q), respectively. PCB3 was targeted by mutations of Leu187 → Gln (hSK1L187Q) and Leu200 → Gln (hSK1L200Q), and PCB4 was targeted by a Phe303 → His mutation (hSK1F303H). These versions of hSK1 were then expressed in HEK293T cells and analyzed for both their ability to bind CaM-Sepharose and their catalytic activity, as a measure of retained gross protein folding. Although these hSK1 mutants all retained at least some catalytic activity (Fig. 2B), somewhat surprisingly, all five bound Ca2+/CaM with similar efficiency to wild type hSK1 (Fig. 2A). This suggested that these predicted Ca2+/CaM binding regions of hSK1 may not be involved in CaM binding. Unfortunately, further mutagenesis of other conserved hydrophobic residues within these putative Ca2+/CaM binding regions (i.e. Leu134 → Gln and Val290 → Asn) yielded catalytically inactive hSK1 proteins (Fig. 2B) that were therefore not further analyzed because of the likelihood that the mutations caused disruption to the gross folding of these proteins.FIGURE 2Site-directed mutagenesis of predicted CaM binding regions of hSK1. A, selective binding of the hSK1 mutants to CaM-Sepharose (CaM) was examined using extracts from HEK293T cells expressing the various hSK1 mutants (Load). Bound hSK1 proteins were visualized by Western blotting via their FLAG epitope. Binding to Sepharose CL-4B (CL4B) was used as a control to account for any nonspecific binding to the CaM-Sepharose beads. B, relative catalytic activity of the hSK1 mutants. Data are mean (±S.D.) from three independent experiments. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Direct Identification of the CaM-binding Site in hSK1—Because our mutagenesis experiments suggested that the Ca2+/CaM-binding sites of hSK1 predicted from sequence analysis were not responsible for CaM binding, we performed further experiments to directly identify the CaM binding region in hSK1. This was initially performed using limited proteolysis of purified recombinant hSK1 and identifying cleavage sites in hSK1 that were protected by the presence of Ca2+/CaM. Such an approach has been used previously to identify the CaM-binding sites of other proteins (31Moore C.P. Rodney G. Zhang J. Santacruz-Toloza L. Strasburg G. Hamilton S.L. Biochemistry. 1999; 38: 8532-8537Crossref PubMed Scopus (119) Google Scholar, 32Zhang H. Zhang J. Danila C.I. Hamilton S.L. J. Biol. Chem. 2003; 278: 8348-8355Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Limited proteolysis of purified recombinant hSK1 with trypsin generated several detectable cleavage products ranging in size from ∼9 to 32 kDa (Fig. 3A). Inclusion of Ca2+/CaM during this limited proteolysis, however, resulted in the loss of a number of hSK1-derived products in the 17-22-kDa range and the accumulation of larger hSK1-derived polypeptides (Fig. 3A). The two most notable polypeptides not generated during limited proteolysis in the presence of Ca2+/CaM had approximate molecular masses of 21 and 22 kDa (Fig. 3A). These polypeptides represented C-terminal fragments of hSK1 because they retained the His tag that resides at this end of the intact recombinant hSK1 (Fig. 3B), and they were presumably not generated because of the presence of bound Ca2+/CaM in close proximity to at least two tryptic cleavage sites within the central region of hSK1." @default.
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• W2023879911 title "The Calmodulin-binding Site of Sphingosine Kinase and Its Role in Agonist-dependent Translocation of Sphingosine Kinase 1 to the Plasma Membrane" @default.
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