Protein Kinase D-mediated Phosphorylation and Nuclear Export of Sphingosine Kinase 2

Abstract

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling. Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling. Sphingosine kinases (SPHKs) 3The abbreviations used are:SPHKsphingosine kinaseP-SPHK2phosphorylated SPHK2PKDprotein kinase DNLSnuclear localization signalNESnuclear export signalGFPgreen fluorescent proteinHAinfluenza hemagglutininsiRNAsmall interfering RNACRM1chromosomal region maintenance 1FRAPfluorescence recovery after photobleachingPMAphorbol 12-myristate 13-acetateDAPI4′,6-diamidino-2-phenylindoleDICdifferential interference contrast catalyze the formation of sphingosine 1-phosphate, a bioactive lipid that regulates a diverse range of cellular processes, including cell growth, survival, differentiation, motility, and cytoskeletal organization (1Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (661) Google Scholar, 2Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Some of these cellular processes are mediated by five sphingosine 1-phosphate-specific G protein-coupled receptors, whereas others appear to be controlled by intracellular sphingosine 1-phosphate through as yet unidentified intracellular targets (2Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 3Watterson K. Sankala H. Milstien S. Spiegel S. Prog. Lipid Res. 2003; 42: 344-357Crossref PubMed Scopus (94) Google Scholar). sphingosine kinase phosphorylated SPHK2 protein kinase D nuclear localization signal nuclear export signal green fluorescent protein influenza hemagglutinin small interfering RNA chromosomal region maintenance 1 fluorescence recovery after photobleaching phorbol 12-myristate 13-acetate 4′,6-diamidino-2-phenylindole differential interference contrast Two distinct SPHK isoforms, SPHK1 and SPHK2, have been cloned and characterized (4Kohama 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 (473) Google Scholar, 5Liu 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 (563) Google Scholar). Diverse external stimuli, particularly growth and survival factors, stimulate SPHK1, and intracellularly generated sphingosine 1-phosphate has been implicated in their mitogenic and anti-apoptotic effects (6Olivera A. Kohama T. Edsall L. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol. 1999; 147: 545-558Crossref PubMed Scopus (462) Google Scholar, 7Edsall L.C. Cuvillier O. Twitty S. Spiegel S. Milstien S. J. Neurochem. 2001; 76: 1573-1584Crossref PubMed Scopus (170) Google Scholar, 8Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A.B. Wattenberg B.W. D'Andrea R.J. Vadas M.A. Curr. Biol. 2000; 10: 1527-1530Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 9Nava V.E. Hobson J.P. Murthy S. Milstien S. Spiegel S. Exp. Cell Res. 2002; 281: 115-127Crossref PubMed Scopus (200) Google Scholar, 10Shu X. Wu W. Mosteller R.D. Broek D. Mol. Cell. Biol. 2002; 22: 7758-7768Crossref PubMed Scopus (245) Google Scholar, 11Xia P. Wang L. Moretti P.A. Albanese N. Chai F. 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Cell Biol. 1999; 147: 545-558Crossref PubMed Scopus (462) Google Scholar, 8Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A.B. Wattenberg B.W. D'Andrea R.J. Vadas M.A. Curr. Biol. 2000; 10: 1527-1530Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 16Olivera A. Rosenfeldt H.M. Bektas M. Wang F. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 46452-46460Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and induced tumor formation in mice (8Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A.B. Wattenberg B.W. D'Andrea R.J. Vadas M.A. Curr. Biol. 2000; 10: 1527-1530Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 9Nava V.E. Hobson J.P. Murthy S. Milstien S. Spiegel S. Exp. Cell Res. 2002; 281: 115-127Crossref PubMed Scopus (200) Google Scholar). In contrast to SPHK1 much less is known about SPHK2. Although highly similar in amino acid sequence and possessing five evolutionarily conserved domains found in all SPHKs (17Liu H. Chakravarty D. Maceyka M. Milstien S. Spiegel S. Prog. Nucleic Acids Res. 2002; 71: 493-511Crossref PubMed Google Scholar), SPHK2 diverges in its N terminus and central regions. These two isoforms have different kinetic properties and differ in developmental and tissue expression (5Liu 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 (563) Google Scholar) implying that they may have distinct physiological functions. In fact, studies from our laboratory have demonstrated that, in contrast to cytosolic distribution of SPHK1, SPHK2 enters nuclei and inhibits DNA synthesis or induces apoptosis under stressful conditions such as serum deprivation (18Igarashi 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 (346) Google Scholar, 19Okada T. Ding G. Sonoda H. Kajimoto T. Haga Y. Khosrowbeygi A. Gao S. Miwa N. Jahangeer S. Nakamura S. J. Biol. Chem. 2005; 280: 36318-36325Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Similarly, Liu et al. have reported that SPHK2 induces apoptosis through its putative BH3 domain (20Liu H. Toman R.E. Goparaju S.K. Maceyka M. Nava V.E. Sankala H. Payne S.G. Bektas M. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 40330-40336Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). More recently, SPHK2 has been shown to phosphorylate an immunosuppressant drug FTY720 (21Billich A. Bornancin F. Devay P. Mechtcheriakova D. Urtz N. Baumruker T. J. Biol. Chem. 2003; 278: 47408-47415Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 22Paugh S.W. Payne S.G. Barbour S.E. Milstien S. Spiegel S. FEBS Lett. 2003; 554: 189-193Crossref PubMed Scopus (269) Google Scholar) and, further, to associate with interleukin-12 receptor in T cells (23Yoshimoto T. Furuhata M. Kamiya S. Hisada M. Miyaji H. Magami Y. Yamamoto K. Fujiwara H. Mizuguchi J. J. Immunol. 2003; 171: 1352-1359Crossref PubMed Scopus (60) Google Scholar), suggesting a potential immunomodulatory action. However, the physiological role of SPHK2 remains largely unknown. Nucleo/cytoplasmic shuttling of protein and RNA molecules plays an important role in eukaryotic cell function (24Weis K. Trends Biochem. Sci. 1998; 23: 185-189Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). A related family of shuttling transport factors, importins and exportins, recognize nuclear localization signal (NLS)-containing or nuclear export signal (NES)-containing proteins. The coordination of trafficking between the nucleus and the cytoplasm is determined by the balance of nuclear import and export activity (25Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (918) Google Scholar, 26Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1009) Google Scholar, 27Hood J.K. Silver P.A. Curr. Opin. Cell Biol. 1999; 11: 241-247Crossref PubMed Scopus (114) Google Scholar), which is specifically regulated by various physiological or environmental determinants. Modification of proteins regulates their function, location, and stability. The present studies were undertaken to investigate the post-translational phosphorylation of SPHK2 and its involvement in the regulation of nucleo/cytoplasmic shuttling of this protein. Here we report, for the first time, that SPHK2 can be phosphorylated in an agonist-dependent manner. have identified a in SPHK2, function is regulated by protein we have identified the kinase for the phosphorylation of the serine residue in the as protein kinase D and that the phosphorylation of SPHK2 by PKD is for its nuclear export under physiological influenza hemagglutinin was as T. Ding G. Sonoda H. Kajimoto T. Haga Y. Khosrowbeygi A. Gao S. Miwa N. Jahangeer S. Nakamura S. J. Biol. Chem. 2005; 280: 36318-36325Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and cloned and were from a and or by and for and for and for to was the to various The used were as putative and and phosphorylation and active and and and active and and and active and small interfering RNA and were by the PKD cells were with active or after cells were in and in the at 1 1 and PKD was for at with the and for at with in kinase 1 by phosphorylation by PKD was determined as J.L. J. J. EMBO J. PubMed Scopus Google Scholar). or the was phosphorylated by PKD in the of and at the was by and the with the was determined by Cell and cells were in and in a at for and and and and the and were at HeLa cells were both with and various expression of an has been (18Igarashi 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 (346) Google Scholar). SPHK2 was in a the region with were by through a with the can be used for and for the of cell of were with of with for at In this SPHK2 was to the with an recovery of The were by and to followed by or were and to the localization studies were as S. Okada T. N. Fujita T. Jahangeer S. Nakamura S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). used were and The fluorescence of and was under a with at a and with at a after was with the as G. J. G. 2004; PubMed Scopus Google Scholar). HeLa cells with fluorescent protein were or with for and nuclear or region of was by for with an at the of fluorescence in the nuclei after nuclear or in fluorescence in the nuclei after was by fluorescence with at the after all of the the were by RNA was from HeLa cells to the synthesis and were as T. Ding G. Sonoda H. Kajimoto T. Haga Y. Khosrowbeygi A. Gao S. Miwa N. Jahangeer S. Nakamura S. J. Biol. Chem. 2005; 280: 36318-36325Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The and were as and and and and The expression of was to the of phosphorylation of proteins is an important protein function, and stability. SPHK2 can be HeLa cells were with a tumor phorbol 12-myristate 13-acetate which is known to phosphorylation of various cellular proteins Y. PubMed Scopus Google Scholar). of cells with for to phosphorylation of SPHK2 as by the of suggesting that SPHK2 can be phosphorylated by of of SPHK2 in the have reported that distribution of SPHK2 cell conditions such as (18Igarashi 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 (346) Google Scholar) or serum T. Ding G. Sonoda H. Kajimoto T. Haga Y. Khosrowbeygi A. Gao S. Miwa N. Jahangeer S. Nakamura S. J. Biol. Chem. 2005; 280: 36318-36325Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) resulted in the accumulation of SPHK2 in the nucleus. have identified an that is important for its nuclear import and localization (18Igarashi 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 (346) Google Scholar). Because protein can the function and localization of and SPHK2 was phosphorylated by we studied the of the localization of SPHK2, which under basal conditions is in the nucleus of HeLa that, in HeLa cells with under conditions phosphorylation is a of SPHK2 from the nucleus to the cytoplasm and the of accumulation of SPHK2 be to an in the nuclear export or an of nuclear import which of these was important for the the shuttling of SPHK2 was characterized by HeLa cells were for the of the fluorescent protein after the fluorescent protein in the nucleus was recovery of nuclear fluorescence was between and cells A and the other fluorescence in the region was the of in fluorescence in the nuclear region was enhanced by These results indicate that an in the nuclear export of SPHK2, an of nuclear import which results in the accumulation of SPHK2 in the we found that cells were with to an accumulation of SPHK2 in the nucleus. can to a residue in the nuclear export receptor chromosomal region maintenance 1 K. 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Because the is under basal conditions and two potential phosphorylation after its we that or both of the serine and in the may be the of phosphorylation that is important for the regulation of the nuclear shuttling of this protein. this we an in the sequence and and its localization with SPHK2 after expression in HeLa The the activity or the expression of the SPHK2 in contrast to SPHK2 the its to from the nucleus in to and suggesting that nucleo/cytoplasmic shuttling of SPHK2 may be regulated by phosphorylation of the in the the of phosphorylation of SPHK2 its nuclear we generated an that specifically the phosphorylated which has two serine at and that can be the of this phosphorylated SPHK2 SPHK2 was phosphorylated in a in by PKD and to the protein was in the A and the the protein HeLa cells SPHK2 were with in cells recognize of In this SPHK2 cells were with phosphorylation of SPHK2 was by SPHK2 in HeLa cells in cells that phosphorylation of in the of SPHK2 the nuclear export of this that phosphorylation of SPHK2 in its sequence and its subsequent nuclear export to protein the localization of SPHK2 was studied in SPHK2 was in the nucleus with a nucleo/cytoplasmic under basal conditions with SPHK2 with a in nuclear SPHK2, which resulted in a nucleo/cytoplasmic The of as by an in was in the cytoplasm of cells as with that in These results a physiological of phosphorylation of SPHK2 in the regulation of nucleo/cytoplasmic shuttling of this protein. of SPHK2 at the a of was to a protein kinase for the phosphorylation of in the of SPHK2. the sequence the with the sequence for phosphorylation by PKD K. A. L.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). PKD was for phosphorylation of SPHK2 under in phosphorylation of the by PKD was active phosphorylated the of an PKD was used as a SPHK2 phosphorylation by PKD was in an cell HeLa cells were both with and an or a active and by or was was in the nucleus with and In and active were in HeLa SPHK2 was in the with SPHK2 in the of cells the active PKD in cells with an was the protein was by of active PKD expression and in the nucleus. These results indicate that PKD has the to phosphorylate SPHK2 both in and in PKD as a for the of is important to PKD is a physiologically relevant protein kinase that SPHK2 for the regulation of nucleo/cytoplasmic shuttling under various including this phosphorylation of SPHK2 in the domain was determined under conditions the expression of PKD was by PKD HeLa cells and with the expression of with with or the HeLa cells were both with a and various cells were to followed by with the whereas and a and a of phosphorylation of SPHK2, with the of the expression of and a of all the PKD by specific resulted in an of phosphorylation of SPHK2, suggesting a function for PKD in the phosphorylation of SPHK2. expression of a active in HeLa cells resulted in phosphorylation of SPHK2 and its from the nucleus under basal conditions with our that SPHK2 phosphorylation in to and from the of HeLa cells PKD was by all PKD that in contrast to cells phosphorylation of SPHK2 and its subsequent from the nucleus were in cells and of and similar results Expression of an in the cells the phosphorylation of serine in the of SPHK2 and its subsequent from the nucleus cells with cells to the involvement of PKD in SPHK2 phosphorylation under physiological conditions we distribution of SPHK2 in cells was by PKD this enzyme was in the in these cells under basal conditions down-regulation of PKD expression by resulted in an in the cell localization of SPHK2 in the nucleus this in distribution of SPHK2 by PKD down-regulation with phosphorylation by These results indicate that PKD is a physiologically relevant protein kinase for the phosphorylation of serine in the of SPHK2, which is important for its nuclear export both under basal as as have identified and characterized for the first the in SPHK2 in its nuclear The is under basal conditions and Upon stimulation with SPHK2 phosphorylation of serine in the and from the nucleus studies for the first that SPHK2 is by a novel of PKD expression and as as phosphorylation of SPHK2 by in and in vivo demonstrated that PKD is a physiologically relevant protein kinase for the phosphorylation of serine in the of SPHK2 in to various stimuli, including A serine and was to both phosphorylation and nuclear and A in the both serine to acid was to the activity the other a of a serine to was from the nucleus in to the protein However, a at a serine of the activity nuclear as in the and these was used as a the present In to the sequence acid in the present studies we identified other in SPHK2. of was in the conserved region amino acid putative may be active under basal conditions a of this putative sequence and its SPHK2 was phosphorylated at serine in the acid the nucleus after stimulation suggesting that phosphorylation of SPHK2 may the nucleus. was by the that SPHK2 was phosphorylated at of the in nuclei from HeLa cells the putative In has been reported that PKD nucleus stimulation of cells by O. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar) of SPHK2 is under basal conditions and may be to that phosphorylation of SPHK2 by PKD may the PKD has with SPHK2 as by have reported that distribution of SPHK2 cell and cellular growth Upon SPHK2 was in the nucleus in HeLa whereas the enzyme was in the cytoplasm of and cells (18Igarashi 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 (346) Google Scholar). our that SPHK2 phosphorylation and from the nucleus to SPHK2 in cells be phosphorylated highly under basal conditions that in HeLa In fact, SPHK2 in cells was in the cytoplasm and was phosphorylated that in HeLa cells as determined by down-regulation of PKDs through RNA interference resulted in the accumulation of SPHK2 in the nucleus under basal conditions with a in the phosphorylation the the physiological of PKD in this phosphorylation of SPHK2 and its subsequent export from the nucleus both under basal as as have shown that SPHK2 in the nucleus under stressful conditions such as or serum in apoptosis or the of cell proliferation (18Igarashi 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 (346) Google Scholar, 19Okada T. Ding G. Sonoda H. Kajimoto T. Haga Y. Khosrowbeygi A. Gao S. Miwa N. Jahangeer S. Nakamura S. J. Biol. Chem. 2005; 280: 36318-36325Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). PKD may such stressful by SPHK2. PKD activation under such conditions remains to be The other isoform, SPHK1, is a cytosolic protein with an of A for SPHK1 has been identified A protein of this may the nucleus. two in SPHK1 have been identified Y. A. S. Y. Biochem. Res. 2003; PubMed Scopus Google Scholar). These may be under basal acid the phosphorylation of the in SPHK2 are highly conserved from to suggesting that the the nucleo/cytoplasmic shuttling of SPHK2 may be to and be implicated in the regulation of cell functions. sequence SPHK1 has phosphorylation sequence for SPHK1 and SPHK2 similar these two are regulated in suggesting distinct physiological functions. The PKD have been implicated in a of cellular including organization and and cell proliferation O. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). the present the activation of PKD results in SPHK2 phosphorylation, which its nuclear export and may such as cell proliferation and The role of PKD to be in of cellular mediated by SPHK2. N. and Y. for the with