Calmodulin Is Involved in the Ca2+-dependent Activation of Ceramide Kinase as a Calcium Sensor

We recently demonstrated that the activation of ceramide kinase (CERK) and the formation of its product, ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca2+ (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca2+ as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca2+-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca2+. The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM·CERK complexes. An in vitro CaM binding assay also demonstrated Ca2+-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca2+ sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca2+-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca2+-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein kinase II, which had been assumed to be the main target of CaM in mast cells. We recently demonstrated that the activation of ceramide kinase (CERK) and the formation of its product, ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca2+ (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca2+ as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca2+-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca2+. The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM·CERK complexes. An in vitro CaM binding assay also demonstrated Ca2+-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca2+ sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca2+-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca2+-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein kinase II, which had been assumed to be the main target of CaM in mast cells. Sphingolipids and their metabolites have emerged as a new class of lipid mediator of various cell functions (1Hakomori S. Igarashi Y. Adv. Lipid. Res. 1993; 25: 147-162PubMed Google Scholar, 2Spiegel S. Merrill A.H. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (636) Google Scholar, 3Igarashi Y. J. Biochem. (Tokyo). 1997; 122: 1080-1087Crossref PubMed Scopus (131) Google Scholar). Ceramide (N-acylsphingosine; Cer), 2The abbreviations used are: CerceramideCERKceramide kinaseC1Pceramide 1-phosphateS1Psphingosine 1-phosphateCaMcalmodulinCaMKIIcalmodulin-dependent protein kinase IIRBLrat basophilic leukemiaCHOChinese hamster ovaryPIPES1,4-piperazinediethanesulfonic acidFLfull-length.2The abbreviations used are: CerceramideCERKceramide kinaseC1Pceramide 1-phosphateS1Psphingosine 1-phosphateCaMcalmodulinCaMKIIcalmodulin-dependent protein kinase IIRBLrat basophilic leukemiaCHOChinese hamster ovaryPIPES1,4-piperazinediethanesulfonic acidFLfull-length. the precursor for all sphingolipids, functions as a lipid second messenger in a variety of cellular events (4Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (293) Google Scholar). Many stimuli, such as tumor necrosis factor α, Fas ligand, γ-irradiation, anti-tumor reagents, and heat shock, cause an elevation in intracellular Cer content following the hydrolysis of sphingomyelin by endogenous sphingomyelinase. These changes can result in cell cycle arrest, cell differentiation, and apoptosis (5Obeid L.M. Linardic C.M. Hannun Y.A. Science. 1993; 256: 1769-1771Crossref Scopus (1585) Google Scholar, 6Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 3125-3128Abstract Full Text PDF Google Scholar). Cer is converted to sphingosine through the action of ceramidase, and in turn the sphingosine is metabolized to sphingosine 1-phosphate (S1P) by sphingosine kinase. S1P has been found to regulate cell growth (7Oliver A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (807) Google Scholar) and motility (8Kupperman E. An S. Osborne N. Waldron S. Stainier D.Y. Nature. 2000; 406: 192-195Crossref PubMed Scopus (339) Google Scholar, 9Kohno T. Matsuyuki H. Inagaki Y. Igarashi Y. Genes Cells. 2003; 8: 685-697Crossref PubMed Scopus (37) Google Scholar). Interestingly, S1P inhibits apoptosis induced by Cer and Fas ligand (10Cuvillier O. Pirianow G. Kleuser B. Venek P.G. Coso O.A. Gutkind J.S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1327) Google Scholar), indicating that the balance of Cer/sphingosine/S1P can affect cell phenotype. The Cer/sphingosine/S1P pathway had been considered to be the major metabolic pathway of Cer. However, in 2002, the enzyme ceramide kinase (CERK), which metabolizes Cer to ceramide 1-phosphate (C1P), was cloned (11Sugiura M. Kono K. Liu H. Shimizugawa T. Minekura H. Spiegel. S. Kohama, T. J. Biol. Chem. 2002; 277: 23294-23300Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), revealing a new pathway for Cer metabolism. CERK activity was initially described as a Ca2+-stimulated lipid kinase activity that was co-purified with brain synaptic vesicles (12Bajjalieh S.M. Martin T.F. Floor E. J. Biol. Chem. 1989; 264: 14354-14360Abstract Full Text PDF PubMed Google Scholar); it has since been reported in HL60 cells (13Dressler K.A. Kolesnick R.N. J. Biol. Chem. 1990; 265: 14917-14921Abstract Full Text PDF PubMed Google Scholar) and neutrophils (14Rile G. Yatomi Y. Takafuta T. Ozaki Y. Acta Haematol. 2003; 109: 76-83Crossref PubMed Scopus (36) Google Scholar). Additionally, CERK is thought to be involved in phagolysosome formation in polymorphonuclear leukocytes and to promote liposome fusion (15Hinkovska-Galcheva V.T. Boxer L.A. Mansfield P.J. Harsh D. Blackwood A. Shayman J.A. J. Biol. Chem. 1998; 273: 33203-33209Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). ceramide ceramide kinase ceramide 1-phosphate sphingosine 1-phosphate calmodulin calmodulin-dependent protein kinase II rat basophilic leukemia Chinese hamster ovary 1,4-piperazinediethanesulfonic acid full-length. ceramide ceramide kinase ceramide 1-phosphate sphingosine 1-phosphate calmodulin calmodulin-dependent protein kinase II rat basophilic leukemia Chinese hamster ovary 1,4-piperazinediethanesulfonic acid full-length. The product of CERK activity, C1P, has been reported to have mitogenic effects (16Gijsbers S. Mannaerts G.P. Himpens B. Veldhoven P.P. FEBS Lett. 1999; 453: 269-272Crossref PubMed Scopus (25) Google Scholar), although exogenously added C1P is rapidly hydrolyzed by a phosphatase (17Boudker O. Futerman A.H. J. Biol. Chem. 1993; 268: 22150-22155Abstract Full Text PDF PubMed Google Scholar). C1P was found to be a direct activator of cytosolic phospholipase A2 and to be involved in arachidonic acid release (18Pettus B.J. Bielawska A. Spiegel S. Roddy P. Hannun A.H. Chalfant C.E. J. Biol. Chem. 2003; 278: 38206-38213Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 19Pettus B.J. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. Evans J.H. Freiberg J. Roddy P. Hannun Y.A. Chalfant C.E. J. Biol. Chem. 2004; 279: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Additionally, our previous report revealed that CERK was a mediator of Ca2+-dependent degranulation in mast cells (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In both arachidonic acid release and mast cell degranulation, the intracellular elevation of Ca2+ is a crucial event that acts as a regulatory mechanism of CERK activity. However, there are no apparent Ca2+ binding domains in the primary structure of CERK (such as an EF-hand or C2 domain), and the Ca2+ regulatory mechanism has not been well understood. Calmodulin (CaM) has been recognized as a calcium sensor that interacts with and regulates multiple protein targets (21Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (731) Google Scholar). When intracellular Ca2+ levels rise, four Ca2+ ions bind to CaM and the Ca2+·CaM complex binds to the target protein, initiating various signaling cascades. In this manner, CaM is known to regulate ion channels, cell cycle, and cytoskeletal organization and to influence development (22Heuman R.G. Arkinson R.C. Andruss B.F. Bolduc C. Kovalick G.E. Backingham K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2420-2425Crossref PubMed Scopus (49) Google Scholar, 23Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (680) Google Scholar). In the work presented here, we investigated the mechanism of the Ca2+-dependent activation of CERK and found that CaM was in fact involved. Utilizing point mutation analysis, we also identified the CaM binding site in CERK. Materials—Ceramide (C18:0, d18:1), cardiolipin, p-nitrophenyl N-acetyl β-d-glucosaminide, A23187, and an anti-FLAG monoclonal antibody (M2) were all purchased from Sigma. [32P]ATP and [3H]sphingosine were from PerkinElmer and American Radiolabeled Chemicals (St. Louis, MO), respectively. The calmodulin antagonist W-7 and the calmodulin-dependent kinase II inhibitor KN-93 were from Seikagaku Corp. (Tokyo, Japan). An imidoester cross-linker, dimethyl 3,3-dithiobispropionimidate/2HCl, and an anti-CaM antibody were from Pierce and Zymed Laboratories Inc.(South San respectively. were of the CERK kinase activity of CERK was as described (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), with cells were in a and inhibitor was for at in a cardiolipin, and Cer (C18:0, of and CaM were added to this In were with an W-7 or at the concentration for and the enzyme activity was were and on using as the to C1P were using an and basophilic leukemia cells and the CERK and (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) were as in with and Chinese hamster ovary cells were in with and cells were purchased from and to the were with the using for cells and for respectively. metabolic cells were with of acid in of for at the the cells were and with and W-7 was added to the and cells were for the cells were at with A23187, a calcium for were by of were by of The was and to a to as described (13Dressler K.A. Kolesnick R.N. J. Biol. Chem. 1990; 265: 14917-14921Abstract Full Text PDF PubMed Google Scholar). were by the of and J. Biochem. PubMed Scopus Google Scholar). was on and as for the CERK CERK was cloned a as described (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The following were used to deletion mutants of for and for and and for and The was with by using CERK as a the and with the were and with in the of kinase as described Y. Y. T. M. Res. 25: Scopus Google Scholar). were using a to the The following were used to point for and for and for and and and for and The mutant was used as the in for and and CERK was used for the mutant was used as a cells were for at in and inhibitor at for primary were added to the and were at were by for at with of protein in to their in CERK the were and in CERK the were with to and in the were in of and the were by to the of Nature. PubMed Scopus Google Scholar). were a to the described by H. T. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a with in the was with at a with the was for with with the were using an as by the and were using CaM cells CERK or CERK mutants were and with and inhibitor the were with of and were for at to The were added to of that had been with binding and or and the was for at were by with binding to the were by as described The was with an antibody that had been (21Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (731) Google Scholar). or cells were and with a in the or of the concentration of W-7 or the cells were at with for were at for and the activity was in both the and cell using p-nitrophenyl N-acetyl as a T. T. K. S. J. Biochem. (Tokyo). PubMed Scopus Google Scholar). was as a of the activity the CaM in the Ca2+-dependent of CERK and the Ca2+-dependent of reported that CERK is involved in Ca2+-dependent degranulation in mast cells and that C1P elevation following CERK activation is for the elevation of intracellular Ca2+ (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). This that the activation of CERK by Ca2+ is for (11Sugiura M. Kono K. Liu H. Shimizugawa T. Minekura H. Spiegel. S. Kohama, T. J. Biol. Chem. 2002; 277: 23294-23300Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar) reported that concentrations of Ca2+ were to CERK in However, in our previous CERK concentrations of Ca2+ for The major was the of the used cell from we used the These the of a for CERK that its Ca2+ a that is lost the CaM is a well known Ca2+ sensor that the activity of a of metabolic (21Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (731) Google Scholar). We investigated CaM be involved in the Ca2+ activation of initially by the effects of the CaM antagonist W-7 on CERK activity in We used a cell that CERK and an activity that of its cells (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). were from the cells as cell or CERK. in W-7 had no on the activity of the it decreased the CERK activity in the in a W-7 is known to bind to CaM and to enzyme activity. is that the cause the of CaM with the in which the W-7 have no on the activity. However, in the enzyme of the cell W-7 the binding of Ca2+ from in a in the Ca2+-dependent activation of CERK. in the of with the of CERK and Ca2+. The activity in the of of the CERK was that of the but it activation by with the that endogenous Ca2+·CaM had the CERK in the to the of CaM in the activation of CaM from brain was added to of the and CERK were Ca2+ concentrations were using a as by the In the of CaM and the CERK activity and This was completely by W-7 indicating that the activation was dependent on In the of CaM and W-7 no The of activation in the the added CaM was to W-7 and that CaM in complex with CERK the and as a Ca2+ we the intracellular C1P formation be by In a previous we that C1P formation in cells could be induced by the calcium (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). cells were with acid for and for with W-7 decreased the Ca2+-dependent formation of in a This is with the that CaM acts as a calcium sensor for CERK. Ca2+-dependent of CERK with the CaM is to were using from cells CERK were with in the of or CERK was with in the of Ca2+ This result the that CaM binds to CERK and that this binding is we endogenous CaM is also to CERK in CERK was in and the cells were with the cross-linker, dimethyl CaM was using an anti-CaM antibody, the was with and the was for CERK was co-immunoprecipitated with CaM the anti-CaM antibody was added the CERK levels were the in both These that as well as endogenous CaM binds to CERK in a Ca2+-dependent of the CaM in the CaM binding domain in we a of mutants of CERK as in mutant was in and cell were with in the of or CERK and the mutant both CaM in the of The was also to bind results that CaM binding to CERK is to the region CaM targets of of a motif or of the Ca2+-dependent or or its (21Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (731) Google Scholar). and are by amphipathic with a in which at or to using the Calmodulin there are for a CaM binding site present in CERK. which were also present in of a type 1-8-14B CaM binding The motif to 1-8-14B is of the with a of to The amino acids and to the and amino and the of this region is in of CERK are in and in their were involved in CaM we or in the of CERK with to the These mutants were and CaM binding were type and similar Ca2+-dependent CaM binding the mutant had binding activity to CaM by or amino acids are apparent in helical of The helical of of CERK is in The the The of the and amino In amino acids and are located in the of the The region from in CERK an amphipathic that the of a CaM binding the results of the binding the of and to be for CaM amino acids and on the to a by the the amino acid in the of the to be for the formation of the domain recognized by However, the mutation for and CaM binding activity of indicating that although amino acids not be for CaM are for the helical structure of the CaM binding We also the which had the on the CaM binding of the CERK The CaM binding activity of this mutant was with that of type CERK Furthermore, the of and were in to that of the type CERK binding of CaM to This the of CaM binding although the binding was with the that of the binding in the region from amino acids to this region can be considered the CaM binding site in CERK. the of this CaM binding domain in we the CaM binding domain could Ca2+-dependent formation of in the cells were with and calcium intracellular C1P by of These results with our previous report (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and that CERK was by intracellular Ca2+. However, the cells were with and which contained the CaM binding domain calcium to intracellular C1P that could saturate the CaM and hence block activation of CERK. These results that the CaM binding domain of CERK interacts with and that this is for the Ca2+-dependent of CERK. CERK in a CaM-dependent previous the of CERK in mast cell degranulation (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In the present study, we have that is involved in the activation of CERK. we to the activation of CERK and degranulation of mast cells. of cells with W-7 decreased Ca2+-dependent degranulation indicating that is involved in the degranulation This is in with D.S. Kim J. 2000; PubMed Scopus Google Scholar, M. T. M. Biol. 2003; PubMed Scopus Google Scholar). In the degranulation of and have been to be the target of CaM M. T. M. Biol. 2003; PubMed Scopus Google Scholar). report has the of a but pathway in mast cell degranulation D.S. Kim J. 2000; PubMed Scopus Google Scholar). the CaM-dependent and CERK in Ca2+-dependent degranulation in the of the inhibitor KN-93 was In this we used and that we had (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). degranulation was decreased by KN-93 in a with the effects a at Interestingly, the cells to KN-93 and even at the of CERK has been to the of degranulation (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), the present suggest that CERK to the In our previous study, the of CERK activity a in Ca2+-dependent degranulation and the of CERK cells was to cause a degranulation (20Mitsutake S. Kim T.-J. Inagaki Y. Kato M. Yamashita T. Igarashi Y. J. Biol. Chem. 2004; 279: 17570-17577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). results that CERK acts as a target of to degranulation in mast and that the pathway involved be from that of which was recognized as of the intracellular targets of However, the of CERK and CaM-dependent are not from our and be of a CaM can bind with to a region of amino an amphipathic in The binding mechanism CaM and its target is as the CaM binding site in the CERK protein, we to the helical structure and the of its amphipathic These mutants lost their CaM binding to the CaM binding The of the CaM binding also activity, we were to the the CaM binding and the CERK activity in However, the cells were with that contained the CaM binding Ca2+-dependent formation of C1P was that could saturate the CaM and hence block activation of CERK. results that CaM is involved in Ca2+-dependent CERK In the of Ca2+ the activity of the CERK was not by W-7 indicating that activation of CaM can also of the the of intracellular concentrations of Ca2+ to intracellular CaM can Ca2+-dependent CERK activation in to in Ca2+. Ca2+ C1P formation was decreased with W-7 indicating that the of CaM is was from the in vitro CERK was cloned on to sphingosine kinase (11Sugiura M. Kono K. Liu H. Shimizugawa T. Minekura H. Spiegel. S. Kohama, T. J. Biol. Chem. 2002; 277: 23294-23300Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). of was reported to be dependent on CaM P. Spiegel S. 2003; PubMed Scopus Google Scholar) However, the activity of was not by W-7 indicating that CaM was not as an activator of this The present the of an enzyme in the signaling pathway using CaM as a Ca2+ In to cell and stimuli, the intracellular content Cer is by the action of and functions as a second messenger in a variety of cellular apoptosis and cell kinase phospholipase protein and all of which as in various are by Cer (4Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (293) Google Scholar, A. D. J. 1998; PubMed Scopus Google Scholar, T. T. K. M. Y. T. H. N. T. 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