Importance of C1B Domain for Lipid Messenger-induced Targeting of Protein Kinase C
2002; Elsevier BV; Volume: 277; Issue: 20 Linguagem: Inglês
10.1074/jbc.m111761200
ISSN1083-351X
AutoresKaori Kashiwagi, Yasuhito Shirai, Masamitsu Kuriyama, Norio Sakai, Naoaki Saito,
Tópico(s)Cellular transport and secretion
ResumoThe molecular mechanisms by which arachidonic acid (AA) and ceramide elicit translocation of protein kinase C (PKC) were investigated. Ceramide translocated εPKC from the cytoplasm to the Golgi complex, but with a mechanism distinct from that utilized by AA. Using fluorescence recovery after photobleaching, we showed that, upon treatment with AA, εPKC was tightly associated with the Golgi complex; ceramide elicited an accumulation of εPKC which was exchangeable with the cytoplasm. Stimulation with ceramide after AA converted the AA-induced Golgi complex staining to one elicited by ceramide alone; AA had no effect on the ceramide-stimulated localization. Using point mutants and deletions of εPKC, we determined that the εC1B domain was responsible for the ceramide- and AA-induced translocation. Switch chimeras, containing the C1B from εPKC in the context of δPKC (δ(εC1B)) and vice versa (ε(δC1B)), were generated and tested for their translocation in response to ceramide and AA. δ(εC1B) translocated upon treatment with both ceramide and AA; ε(δC1B) responded only to ceramide. Thus, through the C1B domain, AA and ceramide induce different patterns of εPKC translocation and the C1B domain defines the subtype specific sensitivity of PKCs to lipid second messengers. The molecular mechanisms by which arachidonic acid (AA) and ceramide elicit translocation of protein kinase C (PKC) were investigated. Ceramide translocated εPKC from the cytoplasm to the Golgi complex, but with a mechanism distinct from that utilized by AA. Using fluorescence recovery after photobleaching, we showed that, upon treatment with AA, εPKC was tightly associated with the Golgi complex; ceramide elicited an accumulation of εPKC which was exchangeable with the cytoplasm. Stimulation with ceramide after AA converted the AA-induced Golgi complex staining to one elicited by ceramide alone; AA had no effect on the ceramide-stimulated localization. Using point mutants and deletions of εPKC, we determined that the εC1B domain was responsible for the ceramide- and AA-induced translocation. Switch chimeras, containing the C1B from εPKC in the context of δPKC (δ(εC1B)) and vice versa (ε(δC1B)), were generated and tested for their translocation in response to ceramide and AA. δ(εC1B) translocated upon treatment with both ceramide and AA; ε(δC1B) responded only to ceramide. Thus, through the C1B domain, AA and ceramide induce different patterns of εPKC translocation and the C1B domain defines the subtype specific sensitivity of PKCs to lipid second messengers. The PKC 1The abbreviations used are: PKCprotein kinase CAAarachidonic acidFRAPfluorescent recovery after photobleachingGFPgreen fluorescent proteinεPKCε subtype of protein kinase CδPKCδ subtype of protein kinase CTPA12-O-tetradecanoylphorbol-13-acetateWGAwheat germ agglutininDGdiacylglycerolPSpseudosubstrateC1conserved region 1GFPgreen fluorescent proteinCHOChinese hamster ovaryPBSphosphate-buffered salinePDBuphorbol 12,13-dibutyrate 1The abbreviations used are: PKCprotein kinase CAAarachidonic acidFRAPfluorescent recovery after photobleachingGFPgreen fluorescent proteinεPKCε subtype of protein kinase CδPKCδ subtype of protein kinase CTPA12-O-tetradecanoylphorbol-13-acetateWGAwheat germ agglutininDGdiacylglycerolPSpseudosubstrateC1conserved region 1GFPgreen fluorescent proteinCHOChinese hamster ovaryPBSphosphate-buffered salinePDBuphorbol 12,13-dibutyrate family of serine/threonine protein kinase contains at least 10 subtypes. They are divided into three subgroups based on structural differences and requirement for activators (1Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5740) Google Scholar, 2Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3523) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar, 4Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar). The conventional PKCs (cPKC; α, βI, βII, and γ) are Ca2+-dependent and activated by diacylglycerol or phorbol esters. The novel PKCs (nPKC; δ, ε, η, and θ) are activated by diacylglycerol (DG) or phorbol esters, but are Ca2+-independent (5Ono Y. Fujii T. Igarashi K. Kikkawa U. Ogita K. Nishizuka Y. Nucleic Acids Res. 1988; 16: 5199-5200Crossref PubMed Scopus (58) Google Scholar, 6Osada S. Mizuno K. Saido T.C. Suzuki K. Kuroki T. Ohno S. Mol. Cell. Biol. 1992; 12: 3930-3938Crossref PubMed Google Scholar, 7Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1216) Google Scholar). The atypical PKCs (aPKC; ζ and λ/ι) are insensitive to DG/phorbol ester, and are Ca2+-independent (8Ono Y. Fujii T. Igarashi K. Kuno T. Tanaka C. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4868-4871Crossref PubMed Scopus (391) Google Scholar, 9Akimoto K. Mizuno K. Osada S. Hirai S. Tanuma S. Suzuki K. Ohno S. J. Biol. Chem. 1994; 269: 12677-12683Abstract Full Text PDF PubMed Google Scholar, 10Selbie L.A. Schmitz Peiffer C. Sheng Y. Biden T.J. J. Biol. Chem. 1993; 268: 24296-24302Abstract Full Text PDF PubMed Google Scholar).All PKCs possess an amino-terminal regulatory domain and a catalytic domain in the carboxyl terminus. The regulatory domain of the PKCs contains a variable region 1 (V1), a pseudosubstrate motif (PS), and a conserved region 1 (C1). The V1 of εPKC has been reported to be a selective inhibitor of εPKC translocation (11Johnson J.A. Gray M.O. Chen C.H. Mochly Rosen D. J. Biol. Chem. 1996; 271: 24962-24966Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 12Hundle B. McMahon T. Dadgar J. Chen C.H. Mochly Rosen D. Messing R.O. J. Biol. Chem. 1997; 272: 15028-15035Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In the resting state, the PS is bound in the active site of the catalytic domain, keeping the enzyme inactive by blocking the catalytic site. The binding of activators to the regulatory domain causes a conformational change which releases the PS from the active site and activates the enzyme (13Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (845) Google Scholar). DG and phorbol ester binding have been localized to the C1 domain (2Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3523) Google Scholar, 8Ono Y. Fujii T. Igarashi K. Kuno T. Tanaka C. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4868-4871Crossref PubMed Scopus (391) Google Scholar, 14Newton A.C. Curr. Biol. 1995; 5: 973-976Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 15Quest A.F. Bell R.M. J. Biol. Chem. 1994; 269: 20000-20012Abstract Full Text PDF PubMed Google Scholar). Additionally, the C1 domain mediates protein-protein interactions: that of εPKC binds actin (16Prekeris R. Hernandez R.M. Mayhew M.W. White M.K. Terrian D.M. J. Biol. Chem. 1998; 273: 26790-26798Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 17Matto Yelin M. Aitken A. Ravid S. Mol. Biol. Cell. 1997; 8: 1889-1899Crossref PubMed Scopus (25) Google Scholar, 18Yao L. Suzuki H. Ozawa K. Deng J. Lehel C. Fukamachi H. Anderson W.B. Kawakami Y. Kawakami T. J. Biol. Chem. 1997; 272: 13033-13039Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar).The C1 domain of cPKCs and nPKCs have two cysteine-rich loops (C1A and C1B), each consisting of ∼50-amino acids including six cysteine and two histidine residues arranged in a zinc finger motif. The C1B of cPKCs and nPKCs showed strong phorbol esters binding, but all C1A except for γPKC showed very weak affinity for phorbol esters (19Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (140) Google Scholar). GFP-tagged C1A-C1B or C1A translocated to the plasma membrane in response to receptor or phorbol esters stimuli, whether significant plasma membrane translocation of C1B was only observed in phorbol esters stimulation (20Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (290) Google Scholar). In addition, distinct roles for the C1A and C1B domains in the activation of the enzyme have been shown (21Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2001; 276: 4218-4226Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). These results suggest that the C1A and C1B domains of PKCs are functionally distinct.The activity of PKC can be regulated not only by DG and phorbor ester but also by other lipids such as arachidonic acid (AA) (22Kasahara K. Kikkawa U. J. Biochem. (Tokyo). 1995; 117: 648-653Crossref PubMed Scopus (26) Google Scholar, 23Shinomura T. Asaoka Y. Oka M. Yoshida K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5149-5153Crossref PubMed Scopus (304) Google Scholar) and ceramide (24Huwiler A. Fabbro D. Pfeilschifter J. Biochemistry. 1998; 37: 14556-14562Crossref PubMed Scopus (106) Google Scholar, 25Macdonald N.J. Perez Polo J.R. Bennett A.D. Taglialatela G. J. Neurosci. Res. 1999; 57: 219-226Crossref PubMed Scopus (51) Google Scholar). Like DG/phorbol esters, these lipid second messengers also induce translocation of PKCs. Immunoblot analysis and immunocytochemistry in fixed cells have shown that AA induces translocation of εPKC (26Huang X.P., Pi, Y. Lokuta A.J. Greaser M.L. Walker J.W. J. Cell Sci. 1997; 110: 1625-1634PubMed Google Scholar) and ceramide translocates εPKC and δPKC from the plasma membrane to the cytoplasm (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Using green fluorescent protein (GFP)-tagged PKCs and live cell imaging, we have shown that AA translocates εPKC, but not δPKC, from the cytoplasm to the Golgi complex (28Shirai Y. Kashiwagi K. Yagi K. Sakai N. Saito N. J. Cell Biol. 1998; 143: 511-521Crossref PubMed Scopus (122) Google Scholar, 29Shirai Y. Kashiwagi K. Sakai N. Saito N. J. Cell Sci. 2000; 113: 1335-1343PubMed Google Scholar) and that ceramide translocates δPKC from the cytoplasm to the Golgi complex (30Kajimoto T. Ohmori S. Shirai Y. Sakai N. Saito N. Mol. Cell. Biol. 2001; 21: 1769-1783Crossref PubMed Scopus (84) Google Scholar). However, little is known about the mechanism underlying these translocations. Here we identified the intramolecular domains of ε- and δPKC that respond to ceramide and AA to clarify the molecular mechanisms responsible for the lipids-dependent translocation of nPKCs.DISCUSSIONThe importance of ceramide and AA as lipid messengers has only recently begun to be appreciated. Ceramide is involved in such processes as cell differentiation (34Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar), outgrowth of neurons (35Brann A.B. Scott R. Neuberger Y. Abulafia D. Boldin S. Fainzilber M. Futerman A.H. J. Neurosci. 1999; 19: 8199-8206Crossref PubMed Google Scholar), apoptosis (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 36Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1597) Google Scholar, 37Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar), and long-term depression of synaptic transmission (38Yang S.N. Neuroscience. 2000; 96: 253-258Crossref PubMed Scopus (44) Google Scholar). AA has been shown to act as a retrograde transmitter (39Herrero I. Miras Portugal M.T. Sanchez Prieto J. Nature. 1992; 360: 163-166Crossref PubMed Scopus (329) Google Scholar) in the generation of long-term potentiation, as a regulator of ion channels (40Nojima H. Sasaki T. Kimura I. Brain. Res. 2000; 852: 233-238Crossref PubMed Scopus (11) Google Scholar), a mediator of cell death (25Macdonald N.J. Perez Polo J.R. Bennett A.D. Taglialatela G. J. Neurosci. Res. 1999; 57: 219-226Crossref PubMed Scopus (51) Google Scholar, 41Scorrano L. Penzo D. Petronilli V. Pagano F. Bernardi P. J. Biol. Chem. 2001; 276: 12035-12040Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar) and is known to be necessary for superoxide generation (42Shiose A. Sumimoto H. J. Biol. Chem. 2000; 275: 13793-13801Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Both ceramide and AA can regulate the activity and/or translocation of PKC. AA activates εPKC but not δPKC in vitro (22Kasahara K. Kikkawa U. J. Biochem. (Tokyo). 1995; 117: 648-653Crossref PubMed Scopus (26) Google Scholar), and inhibits the ceramide-induced activation of ζPKC (25Macdonald N.J. Perez Polo J.R. Bennett A.D. Taglialatela G. J. Neurosci. Res. 1999; 57: 219-226Crossref PubMed Scopus (51) Google Scholar). O'Flaherty (43O'Flaherty J.T. Chadwell B.A. Kearns M.W. Sergeant S. Daniel L.W. J. Biol. Chem. 2001; 276: 24743-24750Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) demonstrated that αPKC, βPKC, and δPKC can be translocated by low concentrations of AA. In contrast, Oancea et al. (20Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (290) Google Scholar) reported that AA inhibits translocation of the C1A domain of γPKC. Ceramide translocates δPKC and εPKC from the plasma membrane to the cytoplasm (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), and αPKC from the cytoplasm to the membrane (24Huwiler A. Fabbro D. Pfeilschifter J. Biochemistry. 1998; 37: 14556-14562Crossref PubMed Scopus (106) Google Scholar). Taken together, these reports suggest that both ceramide and AA play important roles in signal transduction and implicate their involvement in the regulation of subtype-specific activation or translocation of PKCs.In this study, we showed that ceramide translocates εPKC from the cytoplasm to the perinuclear region and identified this region as the Golgi complex by WGA staining (Fig. 1). We have previously shown that AA also induces the translocation of εPKC to the Golgi complex (Fig. 1B) (28Shirai Y. Kashiwagi K. Yagi K. Sakai N. Saito N. J. Cell Biol. 1998; 143: 511-521Crossref PubMed Scopus (122) Google Scholar). In those studies, 10 μm ceramide and 100 μm AA were used to detect translocation of PKCs clearly and constantly, although the translocation to the Golgi complex could be detected even at 25 μm AA and 1 μmceramide. The concentrations of these lipids might be relatively higher than that of physiological condition. It, however, is noteworthy that εPKC is translocated to the Golgi complex when ceramide is generated by receptor stimuli with tumor necrosis factor-α as seen in the case of exogenous ceramide stimulation (data not shown), and that εPKC accumulates in the Golgi complex in the brain (data not shown). These findings suggest that translocation of εPKC to the Golgi complex occurs under physiological conditions.Although both ceramide and AA translocated εPKC from the cytoplasm to the Golgi complex, the pattern of localization was subtly, but distinctly, different. First, in ceramide-treated cells εPKC was concentrated in the well defined Golgi complex with uniform distribution in the cytosol. In contrast, upon AA treatment εPKC accumulated in a diffuse pattern around the nucleus with heterogeneous fluorescence in the cytosol. Second, TPA application after ceramide or AA revealed differences in the dissociation of εPKC from the Golgi complex. The ceramide-stimulated interaction of εPKC with the Golgi complex was transient, as shown by the TPA-induced relocalization from the Golgi complex to the plasma membrane. On the other hand, interaction of AA-stimulated εPKC was strong enough to resist being translocated by TPA stimuli. Third, FRAP analysis also revealed distinct interaction of εPKC with the Golgi complex. The fact that, in ceramide-treated cells, fluorescence recovery in the Golgi complex was coincident with decreased cytosolic fluorescence suggests the εPKC exchanges with the cytosolic pool. As staining in the unbleached regions of the Golgi complex did not change, it is unlikely that there is significant movement of εPKC in the Golgi complex in response to ceramide. On the other hand, after AA treatment, the recovery came from unbleached regions of the Golgi complex rather than the cytosol. This suggests that AA mediates a tight association of εPKC with the Golgi complex resulting in low exchange with cytosolic εPKC pools. Finally, AA-translocated εPKC was sensitive to redistribution by ceramide but AA did not alter the ceramide-translocated εPKC. This indicates that AA-mobilized εPKC is responsive to ceramide, but the ceramide-treated εPKC cannot be further translocated by AA. Taken together, these differences imply that distinct mechanisms are involved in the translocation of εPKC mediated by ceramide and AA. Similarly, different effects of ceramide and AA on PKC translocation have been reported for γPKC-C1A (20Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (290) Google Scholar). In those studies, pretreatment with AA reduced the DG-induced translocation of γPKC-C1A to the plasma membrane but pretreatment with ceramide had no effect on the translocation.To identify the domains of εPKC necessary for AA- and ceramide-induced εPKC translocation, we constructed a series of deletion mutants and studied their translocation characteristics. Loss of the V1, PS, and/or C1A domains did not alter translocation in response to ceramide or AA as compared with εPKC. However, deletion of the C1B domain rendered the mutants insensitive to both ceramide and AA. These results indicate that the C1B domain is necessary for the translocation induced by both ceramide and AA. Although the mechanisms causing the distinct translocation are unknown, they may include differences in phosphorylation, interaction partners, and/or specific conformation changes.The fact that the C1B, but not the C1A domain, is involved in the AA- and ceramide-induced translocation suggests that C1A and C1B have different roles in translocation. Even in the case of TPA, difference between C1A and C1B was observed. Unlike ceramide and AA, TPA induced translocation of both ΔC1A and ΔC1B but the mutants lacking both C1A and C1B (ΔV1-PS-C1A-C1B and ΔC1A-C1B) were insensitive to TPA, indicating that either C1A or C1B can mediate TPA-induced translocation. However, the translocation of ΔC1B was weaker than that of ΔC1A (Fig. 6). This is consistent with the report that the C1B domain of εPKC has higher affinity for phorbol esters than the C1A domain (19Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (140) Google Scholar). In addition, several reports suggest distinct contributions of C1A and C1B domain in the regulation of PKC. For example, Shieh et al. (44Shieh H.L. Hansen H. Zhu J. Riedel H. Mol. Carcinog. 1995; 12: 166-176Crossref PubMed Scopus (33) Google Scholar, 45Shieh H.L. Hansen H. Zhu J. Riedel H. Cancer Detect. Prev. 1996; 20: 576-589PubMed Google Scholar) used mutants of αPKC lacking either C1A or C1B and showed no differences in TPA stimulated activity, suggesting that TPA regulates αPKC activity via either C1A or C1B. In contrast, mezerein regulation occurs predominantly via the C1A. Second, Bogi et al. (46Bogi K. Lorenzo P.S. Acs P. Szallasi Z. Wagner G.S. Blumberg P.M. FEBS Lett. 1999; 456: 27-30Crossref PubMed Scopus (31) Google Scholar) reported that translocation of δPKC by PMA requires the C1B domain but not C1A, although C1A and C1B domains of αPKC have equivalent roles for the PMA-induced translocation. Finally, the γPKC-C1A fragment was preferentially translocated to the plasma membrane compared with the γPKC-C1B or γPKC-C1AC1B fragment upon treatment of rat basophilic leukemia cells with IgE or ligands of PAF receptor. (20Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (290) Google Scholar). Thus, there is a considerable body of literature consistent with our findings that C1A and C1B domains differentially regulate PKC translocation.We used point mutations in the C1A (P180G and C186G) and C1B (P253G and C259G) domains to confirm that the C1B domain is responsible for the AA- or ceramide-induced translocation, and that either the C1A or the C1B domain is sufficient for the TPA-induced translocation. The proline mutants have a decreased affinity for PDBu, and the cysteine to glycine mutation eliminates PDBu binding (33Kazanietz M.G. Wang S. Milne G.W. Lewin N.E. Liu H.L. Blumberg P.M. J. Biol. Chem. 1995; 270: 21852-21859Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Ceramide and AA translocate the C1A mutants, but not the C1B mutants; TPA translocates all. Collectively, these results provide strong evidence that the εC1B domain is required for ceramide- and AA-stimulated translocation, while TPA has a less stringent requirement, needing only one cysteine-rich loop of C1 domain, in either C1A or C1B, for membrane localization.Like εPKC, ceramide induces the translocation of δPKC from the cytosol to the Golgi complex (30Kajimoto T. Ohmori S. Shirai Y. Sakai N. Saito N. Mol. Cell. Biol. 2001; 21: 1769-1783Crossref PubMed Scopus (84) Google Scholar). Unlike εPKC, δPKC is not sensitive to AA (29Shirai Y. Kashiwagi K. Sakai N. Saito N. J. Cell Sci. 2000; 113: 1335-1343PubMed Google Scholar). What then, are the differences between εPKC and δPKC? Our results show that the εC1B is necessary for AA-induced translocation of εPKC. Can the differences between the C1B domains of εPKC and δPKC account for their differential sensitivity to AA? If so, the chimera of δPKC having the C1B domain of εPKC, (δ(εC1B)), should translocate in response to AA, but the chimera of εPKC having the C1B domain of δPKC, (ε(δC1B)), should not. δ(εC1B), but not ε(δC1B), translocated in response to AA. This difference is not due to a general nonresponsiveness of the ε(δC1B) as it does translocate in response to ceramide (Fig. 8). Instead the data suggests inherent differences between the δC1B and εC1B. Specifically, our data demonstrates that εC1B domain responds to both ceramide and AA, but δC1B is sensitive only to ceramide. These differences in the C1B domain may determine the subtype-specific responses of ε- and δPKC to lipid second messengers.How the differences between εPKC and δPKC to the lipid second messengers contribute their physiological roles? For example, AA is thought to be one of retrograde transmitters (39Herrero I. Miras Portugal M.T. Sanchez Prieto J. Nature. 1992; 360: 163-166Crossref PubMed Scopus (329) Google Scholar), and AA indeed facilitates long-term potentiation by the enhancement of synaptic transmission in the hippocampus (47Williams J.H. Errington M.L. Lynch M.A. Bliss T.V. Nature. 1989; 341: 739-742Crossref PubMed Scopus (496) Google Scholar, 48Drapeau C. Pellerin L. Wolfe L.S. Avoli M. Neurosci. Lett. 1990; 115: 286-292Crossref PubMed Scopus (55) Google Scholar, 49Nomura T. Nishizaki T. Enomoto T. Itoh H. Life Sci. 2001; 68: 2885-2891Crossref PubMed Scopus (26) Google Scholar). Enzymologically, εPKC is activated with AA even in the absence of DG, although δPKC is not activated with AA at all (22Kasahara K. Kikkawa U. J. Biochem. (Tokyo). 1995; 117: 648-653Crossref PubMed Scopus (26) Google Scholar). εPKC is enriched in hippocampus and cerebral cortex and is localized mainly in the presynaptic terminals (50Saito N. Itouji A. Totani Y. Osawa I. Koide H. Fujisawa N. Ogita K. Tanaka C. Brain Res. 1993; 607: 241-248Crossref PubMed Scopus (115) Google Scholar). Taken together, AA-induced activation of εPKC, but not δPKC might be in involved in the expression of hippocampal long-term potentiation. On one hand, receptor stmulations with γ-interferon, tumor necrosis factor, and vitamin D3 result in the production of ceramide, leading to apoptosis (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 36Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1597) Google Scholar, 37Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar) or cell differentiation (34Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar). It is possible that these physiological phenomena via ceramide are mediated by δ- or εPKC. In fact, Sawai et al. (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) reported that ceramide translocated δ- and εPKC, not α, β2, γ, nor ζPKC, to the cytoplasm, resulting in apoptosis in human leukemia cells (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). These results indicate that not only DG but also ceramide and AA regulate the activity and distribution of each PKC subtype, contributing to the subtype-specific physiological roles in long-term potentiation or apoptosis. In other words, even though several PKC isoforms are expressed in the same cell, each subtype of PKC can be regulated by specific activators and play a subtype-specific role in various signal transduction.In conclusion, ceramide and AA translocate εPKC to the Golgi complex by distinct mechanisms involving the C1B domain. In contrast, TPA requires only C1A or C1B domain for translocation. The subtle differences in the C1B domains of εPKC and δPKC apparently account for their differential sensitivity to AA. These results indicate that different domains of PKC mediates translocation in response to different second messengers and the distinct characteristics of the domain determine the subtype-specific translocation, thereby contributing to the subtype-specific function. The PKC 1The abbreviations used are: PKCprotein kinase CAAarachidonic acidFRAPfluorescent recovery after photobleachingGFPgreen fluorescent proteinεPKCε subtype of protein kinase CδPKCδ subtype of protein kinase CTPA12-O-tetradecanoylphorbol-13-acetateWGAwheat germ agglutininDGdiacylglycerolPSpseudosubstrateC1conserved region 1GFPgreen fluorescent proteinCHOChinese hamster ovaryPBSphosphate-buffered salinePDBuphorbol 12,13-dibutyrate 1The abbreviations used are: PKCprotein kinase CAAarachidonic acidFRAPfluorescent recovery after photobleachingGFPgreen fluorescent proteinεPKCε subtype of protein kinase CδPKCδ subtype of protein kinase CTPA12-O-tetradecanoylphorbol-13-acetateWGAwheat germ agglutininDGdiacylglycerolPSpseudosubstrateC1conserved region 1GFPgreen fluorescent proteinCHOChinese hamster ovaryPBSphosphate-buffered salinePDBuphorbol 12,13-dibutyrate family of serine/threonine protein kinase contains at least 10 subtypes. They are divided into three subgroups based on structural differences and requirement for activators (1Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5740) Google Scholar, 2Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3523) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar, 4Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar). The conventional PKCs (cPKC; α, βI, βII, and γ) are Ca2+-dependent and activated by diacylglycerol or phorbol esters. The novel PKCs (nPKC; δ, ε, η, and θ) are activated by diacylglycerol (DG) or phorbol esters, but are Ca2+-independent (5Ono Y. Fujii T. Igarashi K. Kikkawa U. Ogita K. Nishizuka Y. Nucleic Acids Res. 1988; 16: 5199-5200Crossref PubMed Scopus (58) Google Scholar, 6Osada S. Mizuno K. Saido T.C. Suzuki K. Kuroki T. Ohno S. Mol. Cell. Biol. 1992; 12: 3930-3938Crossref PubMed Google Scholar, 7Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1216) Google Scholar). The atypical PKCs (aPKC; ζ and λ/ι) are insensitive to DG/phorbol ester, and are Ca2+-independent (8Ono Y. Fujii T. Igarashi K. Kuno T. Tanaka C. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4868-4871Crossref PubMed Scopus (391) Google Scholar, 9Akimoto K. Mizuno K. Osada S. Hirai S. Tanuma S. Suzuki K. Ohno S. J. Biol. Chem. 1994; 269: 12677-12683Abstract Full Text PDF PubMed Google Scholar, 10Selbie L.A. Schmitz Peiffer C. Sheng Y. Biden T.J. J. Biol. Chem. 1993; 268: 24296-24302Abstract Full Text PDF PubMed Google Scholar). protein kinase C arachidonic acid fluorescent recovery after photobleaching green fluorescent protein ε subtype of protein kinase C δ subtype of protein kinase C 12-O-tetradecanoylphorbol-13-acetate wheat germ agglutinin diacylglycerol pseudosubstrate conserved region 1 green fluorescent protein Chinese hamster ovary phosphate-buffered saline phorbol 12,13-dibutyrate protein kinase C arachidonic acid fluorescent recovery after photobleaching green fluorescent protein ε subtype of protein kinase C δ subtype of protein kinase C 12-O-tetradecanoylphorbol-13-acetate wheat germ agglutinin diacylglycerol pseudosubstrate conserved region 1 green fluorescent protein Chinese hamster ovary phosphate-buffered saline phorbol 12,13-dibutyrate All PKCs possess an amino-terminal regulatory domain and a catalytic domain in the carboxyl terminus. The regulatory domain of the PKCs contains a variable region 1 (V1), a pseudosubstrate motif (PS), an
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