Interferon-α-induced Expression of Phospholipid Scramblase 1 through STAT1 Requires the Sequential Activation of Protein Kinase Cδ and JNK

Abstract

Phospholipid scramblase 1 (PLSCR1), a calcium-binding protein that either inserts into the plasma membrane or binds to genomic DNA in the nucleus, has been shown to contribute to the cell proliferation, differentiation, and apoptosis as well as antiviral activity of interferon (IFN). The expression of PLSCR1 protein is also known to be markedly increased in response to IFN and to some differentiation inducing agents such as all-trans retinoic acid, but the precise mechanisms of this response remain to be investigated. In this study, we show that the protein kinase Cδ (PKCδ)-specific inhibitor rottlerin and the dominant negative mutant of PKCδ significantly antagonized IFN-induced PLSCR1 expression. The influence of PKCδ on IFN-mediated induction of PLSCR1 was dependent upon the phosphorylation of STAT1 at Ser-727. Furthermore, PKCδ-mediated activation of STAT1 required the activation of JNK, as the inhibition of JNK activity by its specific inhibitor or transfection of its dominant negative mutant suppressed both serine phosphorylation of STAT1 and PLSCR1 expression but not the activation of PKCδ. In conclusion, our results suggest that the induction of PLSCR1 transcription through STAT1 depends upon sequential activation of PKCδ and JNK. Phospholipid scramblase 1 (PLSCR1), a calcium-binding protein that either inserts into the plasma membrane or binds to genomic DNA in the nucleus, has been shown to contribute to the cell proliferation, differentiation, and apoptosis as well as antiviral activity of interferon (IFN). The expression of PLSCR1 protein is also known to be markedly increased in response to IFN and to some differentiation inducing agents such as all-trans retinoic acid, but the precise mechanisms of this response remain to be investigated. In this study, we show that the protein kinase Cδ (PKCδ)-specific inhibitor rottlerin and the dominant negative mutant of PKCδ significantly antagonized IFN-induced PLSCR1 expression. The influence of PKCδ on IFN-mediated induction of PLSCR1 was dependent upon the phosphorylation of STAT1 at Ser-727. Furthermore, PKCδ-mediated activation of STAT1 required the activation of JNK, as the inhibition of JNK activity by its specific inhibitor or transfection of its dominant negative mutant suppressed both serine phosphorylation of STAT1 and PLSCR1 expression but not the activation of PKCδ. In conclusion, our results suggest that the induction of PLSCR1 transcription through STAT1 depends upon sequential activation of PKCδ and JNK. Human phospholipid scramblase 1 (PLSCR1) 3The abbreviations used are: PLSCR1phospholipid scramblase 1ATRAall-trans-retinoic acidDNdominant negativeMAPKmitogen-activated protein kinasePKCδprotein kinase CδPMAphorbol 12-myristate 13-acetateSTAT1signal transducer and activator of transcription-1JNKc-Jun N-terminal kinaseERKextracellular signal-regulated kinaseIFNinterferonRTreverse transcription.3The abbreviations used are: PLSCR1phospholipid scramblase 1ATRAall-trans-retinoic acidDNdominant negativeMAPKmitogen-activated protein kinasePKCδprotein kinase CδPMAphorbol 12-myristate 13-acetateSTAT1signal transducer and activator of transcription-1JNKc-Jun N-terminal kinaseERKextracellular signal-regulated kinaseIFNinterferonRTreverse transcription. is a multiply palmitoylated endofacial plasma membrane protein with a proline-rich cytoplasmic domain containing several Src homology 3 and WW domain binding motifs (1Sims P.J. Wiedmer T. Thromb. Haemostasis. 2001; 86: 266-275Crossref PubMed Scopus (187) Google Scholar, 2Zhao J. Zhou Q. Wiedmer T. Sims P.J. Biochemistry. 1998; 37: 6361-6366Crossref PubMed Scopus (39) Google Scholar). PLSCR1 protein is also phosphorylated by several tyrosine kinases such as c-Abl and c-Src, which participate in multiple growth factor receptor signaling pathways (3Sun J. Zhao J. Schwartz M.A. Wang J.Y. Wiedmer T. Sims P.J. J. Biol. Chem. 2001; 276: 28984-28990Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4Nanjundan M. Sun J. Zhao J. Zhou Q. Sims P.J. Wiedmer T. J. Biol. Chem. 2003; 278: 37413-37418Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 5Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). As an example, tyrosine phosphorylation of PLSCR1 by c-Src occurs in response to epidermal growth factor, resulting in the association of phosphorylated PLSCR1 with Shc and the activated epidermal growth factor receptor complex (6Sun J. Nanjundan M. Pike L.J. Wiedmer T. Sims P.J. Biochemistry. 2002; 41: 6338-6345Crossref PubMed Scopus (75) Google Scholar). PLSCR1 was also reported to be a substrate of protein kinase Cδ (PKCδ), although this could not be confirmed in our recent experiments with recombinant PKCδ and apoptosis induction by Fas ligation in Jurkat cells (7Zhao K.W. Li X. Zhao Q. Huang Y. Li D. Peng Z.G. Shen W.Z. Zhao J. Zhou Q. Chen Z. Sims P.J. Wiedmer T. Chen G.Q. Blood. 2004; 104: 3731-3738Crossref PubMed Scopus (89) Google Scholar). When it fails to be palmitoylated, PLSCR1 can be imported into the nucleus where it binds to genomic DNA, suggesting a potential role for this protein in gene transcription (8Chen M.H. Ben-Efraim I. Mitrousis G. Walker-Kopp N. Sims P.J. Cingolani G. J. Biol. Chem. 2005; 280: 10599-10606Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 9Ben-Efraim I. Zhou Q. Wiedmer T. Gerace L. Sims P.J. Biochemistry. 2004; 43: 3518-3526Crossref PubMed Scopus (56) Google Scholar). phospholipid scramblase 1 all-trans-retinoic acid dominant negative mitogen-activated protein kinase protein kinase Cδ phorbol 12-myristate 13-acetate signal transducer and activator of transcription-1 c-Jun N-terminal kinase extracellular signal-regulated kinase interferon reverse transcription. phospholipid scramblase 1 all-trans-retinoic acid dominant negative mitogen-activated protein kinase protein kinase Cδ phorbol 12-myristate 13-acetate signal transducer and activator of transcription-1 c-Jun N-terminal kinase extracellular signal-regulated kinase interferon reverse transcription. These biochemical studies strongly indicate that PLSCR1 exerts wide biological effects. Whereas the role of PLSCR1 in remodeling plasma membrane phospholipids and in cell surface exposure of phosphatidylserine, which was originally identified (10Basse F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 11Zhou Q. Zhao J. Stout J.G. Luhm R.A. Wiedmer T. Sims P.J. J. Biol. Chem. 1997; 272: 18240-18244Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar), remains a topic of great controversy, there is increasing evidence to suggest a direct role of this protein in regulating cell proliferation and terminal differentiation. As an example, proliferation and terminal differentiation of myeloid precursor cells in response to selective growth factors are impaired in PLSCR1–/– mice (12Zhou Q. Zhao J. Wiedmer T. Sims P.J. Blood. 2002; 99: 4030-4038Crossref PubMed Scopus (192) Google Scholar). Furthermore, the suppression of PLSCR1 expression by small interfering RNA and antisense RNA inhibits the all-trans-retinoic acid (ATRA) and/or phorbol 12-myristate 13-acetate (PMA)-induced leukemic cell differentiation (7Zhao K.W. Li X. Zhao Q. Huang Y. Li D. Peng Z.G. Shen W.Z. Zhao J. Zhou Q. Chen Z. Sims P.J. Wiedmer T. Chen G.Q. Blood. 2004; 104: 3731-3738Crossref PubMed Scopus (89) Google Scholar, 13Nakamaki T. Okabe-Kado J. Yamamoto-Yamaguchi Y. Hino K. Tomoyasu S. Honma Y. Kasukabe T. Exp. Hematol. 2002; 30: 421-429Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, the mechanisms of regulation of PLSCR1 expression remain largely unknown thus far. Recently, we reported that PKCδ mediates ATRA and PMA-induced PLSCR1 expression (7Zhao K.W. Li X. Zhao Q. Huang Y. Li D. Peng Z.G. Shen W.Z. Zhao J. Zhou Q. Chen Z. Sims P.J. Wiedmer T. Chen G.Q. Blood. 2004; 104: 3731-3738Crossref PubMed Scopus (89) Google Scholar). PLSCR1 has also been shown to be among the most potently activated of the interferon (IFNs)-stimulated genes, suggesting that it possibly participates in cellular response to this important cytokine (14Zhou Q. Zhao J. Al-Zoghaibi F. Zhou A. Wiedmer T. Silverman R.H. Sims P.J. Blood. 2000; 95: 2593-2599Crossref PubMed Google Scholar, 15Der S.D. Zhou A. Williams B.R. Silverman R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15623-15628Crossref PubMed Scopus (1509) Google Scholar). As recently reported, gene deletion of PLSCR1 or suppression of PLSCR1 expression by small interfering RNA in wild type cells was found to markedly attenuate the antiviral activity of IFNs, which appeared to be related to diminished expression of a subset of IFN-stimulated genes with known antiviral activities in cells failing to up-regulate PLSCR1 (16Dong B. Zhou Q. Zhao J. Zhou A. Harty R.N. Bose S. Banerjee A. Slee R. Guenther J. Williams B.R. Wiedmer T. Sims P.J. Silverman R.H. J. Virol. 2004; 78: 8983-8993Crossref PubMed Scopus (88) Google Scholar). However, the mechanism by which IFN induces PLSCR1 expression also remains unresolved. Because IFNs was also shown to activate PKCδ (17Uddin S. Sassano A. Deb D.K. Verma A. Majchrzak B. Rahman A. Malik A.B. Fish E.N. Platanias L.C. J. Biol. Chem. 2002; 277: 14408-14416Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), in the present study we investigated whether and how activated PKCδ contributes to the transcriptional activation of PLSCR1 by IFN. Reagents and Cell Lines—IFNα2a was purchased from Roche Applied Science. PMA and all specific inhibitors, including Gö6976, SB203580, U0126, and SP600125, were from Calbiochem, except for rottlerin (from Biomol, Plymouth, PA). Rabbit polyclonal antibodies for p38, ERK, c-Jun, JNK, and their phosphoylated forms as well as phospho-PKCδ (Ser-643), phospho-STAT1 (Ser-727 and Tyr-701), and horseradish peroxidase-linked secondary antibodies were purchased from Cell Signaling (Beverly, MA), except for anti-phospho STAT1 (Ser-727) used in the experiments shown in Fig. 7 (Upstate Biotechnology, Inc., Waltham, MA). Rabbit polyclonal anti-PKCδ antibody and mouse monoclonal anti-STAT1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), but anti-STAT1 antibody to detect transfection of mutant STAT1-S727A was purchased from BD Biosciences. Mouse monoclonal anti-β-actin antibody (Ab-5) was from NeoMarkers (Fremont, CA). Human monocytic leukemic cell line U937 (from Cell Bank of Shanghai Institutes for Biological Sciences, Shanghai, China) was cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT). The human fibrosarcoma cell line HT1080 (also called 2fTGH) and its STAT1–/– cell line U3A (18McKendry R. John J. Flavell D. Muller M. Kerr I.M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11455-11459Crossref PubMed Scopus (228) Google Scholar) (kindly provided by Dr. George Stark, The Lerner Research Institute, Cleveland, OH) were each cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum. All cells were maintained in 5% CO2-95% air at 37 °C. During experiments, cell viability was kept at least 90% by trypan blue exclusion assay. Plasmids and Transfection —Plasmid pcDNA3-PKC-DNδ carrying the dominant negative (DN) fragment of PKCδ and its empty vector pcDNA3 were generous gifts from Dr. Jae-Won Soh (Inha University, Incheon, Korea) (19Soh J.W. Weinstein I.B. J. Biol. Chem. 2003; 278: 34709-34716Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). pcDNA3-DN-JNK1 was kindly provided by Dr. Young-Mi Ham (Seoul National University, Seoul, Korea) (20Ham Y.M. Choi J.S. Chun K.H. Joo S.H. Lee S.K. J. Biol. Chem. 2003; 278: 50330-50337Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). pRc/CMV-STAT1 and pRc/CMV-STAT1-S727A were generously provided by Dr. James Darnell (Rockefeller University, New York) (21Zhang J.J. Zhao Y. Chait B.T. Lathem W.W. Ritzi M. Knippers R. Darnell Jr., J.E. EMBO J. 1998; 17: 6963-6971Crossref PubMed Scopus (188) Google Scholar). These plasmids were transfected into H1080 and U3A cells using Polyfect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Expression of transfected cDNAs was confirmed by Western blotting. Real-time Quantitative RT-PCR for PLSCR1 mRNA—Total RNA was isolated by Trizol kit (Invitrogen) and treated with DNase (Promega, Madison, WI). Complementary DNA was synthesized by using the cDNA synthesis kit according to manufacturer's instructions (Applied CA), and RT-PCR was as we (7Zhao K.W. Li X. Zhao Q. Huang Y. Li D. Peng Z.G. Shen W.Z. Zhao J. Zhou Q. Chen Z. Sims P.J. Wiedmer T. Chen G.Q. Blood. 2004; 104: 3731-3738Crossref PubMed Scopus (89) Google Scholar) with specific for PLSCR1 antisense and for antisense The is shown as S.D. in with the vector and human PLSCR1 which was a vector with a DNA fragment of the and the of the of the human PLSCR1 gene as (14Zhou Q. Zhao J. Al-Zoghaibi F. Zhou A. Wiedmer T. Silverman R.H. Sims P.J. Blood. 2000; 95: 2593-2599Crossref PubMed Google Scholar), were transfected into HT1080 cells with or dominant negative or dominant negative PKCδ using the Polyfect reagent the manufacturer's were treated with in the or of rottlerin or for activity was using a the manufacturer's The activity was activity for each and activity was as empty vector activity by PKCδ cells were in phosphorylation 1 10% and inhibitor Cell were with anti-PKCδ and were with phosphorylation and with kinase 1 of phosphatidylserine, and and in of kinase containing of as an to which of was The was for at and by the of were by and phosphorylated was by S. Li Y. Verma A. Sassano A. Majchrzak B. Deb D.K. S. N. Rahman A. S. S. M.S. Fish E.N. Platanias L.C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Western were and with phosphorylation as and an of Cell were on 10% and to membrane with 5% in the were with monoclonal PLSCR1 antibody (14Zhou Q. Zhao J. Al-Zoghaibi F. Zhou A. Wiedmer T. Silverman R.H. Sims P.J. Blood. 2000; 95: 2593-2599Crossref PubMed Google Scholar) and antibodies by horseradish peroxidase-linked secondary was by kit according to the manufacturer's instructions were and with mouse monoclonal anti-β-actin antibody to of As the signal of were the using a from Shanghai, and the was with was used to the of was to be As reported (14Zhou Q. Zhao J. Al-Zoghaibi F. Zhou A. Wiedmer T. Silverman R.H. Sims P.J. Blood. 2000; 95: 2593-2599Crossref PubMed Google Scholar, 15Der S.D. Zhou A. Williams B.R. Silverman R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15623-15628Crossref PubMed Scopus (1509) Google Scholar), PLSCR1 and protein in leukemic U937 cells as by RT-PCR and Western to PLSCR1 and in an in PKCδ phosphorylation in As shown in Fig. increased phosphorylated PKCδ to at IFN and the of PKCδ in IFN induction of leukemic U937 cells were treated with in the and of the PKCδ with rottlerin for 1 IFN activation of PKCδ as by PKCδ phosphorylation and the of PKCδ to was that in to PKCδ several including p38, ERK, and could not be in such the for in kinase of PKCδ. with rottlerin IFN induction of PLSCR1 and protein In Gö6976, an inhibitor of was to IFN-induced PLSCR1 expression As reported rottlerin from phosphorylation and thus cellular that could of J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar). the rottlerin has also been shown to kinases M. K. M. 2001; PubMed Scopus (36) Google Scholar, M. J. 2000; PubMed Scopus Google Scholar). the from using rottlerin be we also the of a dominant negative mutant of PKCδ which of PKCδ (19Soh J.W. Weinstein I.B. J. Biol. Chem. 2003; 278: 34709-34716Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). the of the human fibrosarcoma cell line HT1080 was a containing the PLSCR1 to cDNA or the empty vector was transfected into HT1080 As in Fig. IFN in an in the which was significantly suppressed by rottlerin but not by not In with the of transfection of IFN induction of PLSCR1 and the PLSCR1 All of indicate that IFN activation of PKCδ to and contributes to the induction of rottlerin and also the of protein and its suggesting that activity of PKCδ possibly contributes to the expression of Because PKCδ is known to STAT1 on in response to and PLSCR1 induction were in HT1080 in with its the U3A cell line (18McKendry R. John J. Flavell D. Muller M. Kerr I.M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11455-11459Crossref PubMed Scopus (228) Google Scholar). In response to STAT1 phosphorylation increased at of Fig. which appeared activation of PKCδ the induction of PLSCR1 and protein was not 3 and of results were in U937 cells not with our (14Zhou Q. Zhao J. Al-Zoghaibi F. Zhou A. Wiedmer T. Silverman R.H. Sims P.J. Blood. 2000; 95: 2593-2599Crossref PubMed Google Scholar), PLSCR1 was not by IFN in the U3A cells the role of STAT1 phosphorylation in IFN-induced PLSCR1 expression. this wild type STAT1 and mutant STAT1-S727A were transfected into U3A Western the of transfection of wild type STAT1 and mutant the but not the phosphorylated on The results that expression of STAT1 but not STAT1-S727A increased PLSCR1 expression in U3A cells In the of we our to the potential PKCδ and STAT1 in the induction of this HT1080 cells were treated with in with either rottlerin or transfection The results that both rottlerin and transfection phosphorylation on STAT1 at suggesting that PKCδ or to STAT1 studies L.C. 2003; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar) that IFN to activation of several kinases including mitogen-activated protein kinases although the is several of the including and JNK, as well as the JNK substrate transcription factor c-Jun, were but phosphorylated upon we to whether of contribute to PLSCR1 expression in response to U937 cells were for 1 with J.M. J. 1998; Google Scholar), S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and A. S. Y. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). The results that SP600125, which phosphorylation of c-Jun by JNK, of STAT1 phosphorylation and PLSCR1 expression but and to These diminished phosphorylation of and p38, and These results indicate that JNK, but not or p38, contributes to STAT1 phosphorylation and PLSCR1 expression by with this a dominant negative mutant JNK, also phosphorylation of STAT1 by and expression of both the PLSCR1 and PLSCR1 in HT1080 cells negative JNK inhibits phosphorylation of STAT1 and PLSCR1 transcription. HT1080 cells were transfected with the dominant negative mutant of JNK or empty was for an cell were used in Western for and The the The of or cells is shown as S.D. from with with with HT1080 cells were transfected with either plasmids or vector with vector and was for an The cells were for the of activities and The are as S.D. of activities of from an The experiments were at least and results were each with with with we to whether the of PKCδ and JNK on STAT1 phosphorylation are or in the signaling we found that inhibition of PKCδ by rottlerin activation of JNK in U937 as by phosphorylation of However, inhibition of JNK with on the IFN of PKCδ activation In with the of inhibitors, transfection of could the phosphorylation of JNK and c-Jun, to the activation of PKCδ in HT1080 cells These results indicate that PKCδ is of and required for JNK activation and that both kinases are in IFN signaling to the PLSCR1 of PKCδ could JNK activation by IFN. HT1080 cells were transfected with a dominant negative mutant of PKCδ or dominant negative mutant of JNK was for an cell were used in Western for phosphorylation of JNK, and c-Jun and JNK, and The of or cells is shown as S.D. from with with with 1 we reported that the activation of PKCδ by phosphorylation mediates ATRA and PMA-induced PLSCR1 expression in leukemic cells (7Zhao K.W. Li X. Zhao Q. Huang Y. Li D. Peng Z.G. Shen W.Z. Zhao J. Zhou Q. Chen Z. Sims P.J. Wiedmer T. Chen G.Q. Blood. 2004; 104: 3731-3738Crossref PubMed Scopus (89) Google Scholar). In this we to show that the inhibition of PKCδ activation by the inhibitor rottlerin and transfection of the dominant negative mutant of PKCδ significantly expression of PLSCR1 and such inhibition was not for Gö6976, an inhibitor of the These with our that transfection of the fragment of PKCδ PLSCR1 expression (7Zhao K.W. Li X. Zhao Q. Huang Y. Li D. Peng Z.G. Shen W.Z. Zhao J. Zhou Q. Chen Z. Sims P.J. Wiedmer T. Chen G.Q. Blood. 2004; 104: 3731-3738Crossref PubMed Scopus (89) Google Scholar), strongly suggest that PKCδ is a for PLSCR1 expression by or PLSCR1 expression has been shown to the antiviral activity of IFNs (16Dong B. Zhou Q. Zhao J. Zhou A. Harty R.N. Bose S. Banerjee A. Slee R. Guenther J. Williams B.R. Wiedmer T. Sims P.J. Silverman R.H. J. Virol. 2004; 78: 8983-8993Crossref PubMed Scopus (88) Google Scholar). studies also that expression of PLSCR1 contributes to leukemic cell differentiation (7Zhao K.W. Li X. Zhao Q. Huang Y. Li D. Peng Z.G. Shen W.Z. Zhao J. Zhou Q. Chen Z. Sims P.J. Wiedmer T. Chen G.Q. Blood. 2004; 104: 3731-3738Crossref PubMed Scopus (89) Google Scholar). Furthermore, and ATRA in the induction of cell differentiation and growth inhibition S. Li Y. Verma A. Sassano A. Majchrzak B. Deb D.K. S. N. Rahman A. S. S. M.S. Fish E.N. Platanias L.C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). it is of that and ATRA both up-regulate PLSCR1 expression through PKCδ. is well known that binding to the type IFN receptor a tyrosine phosphorylation kinases and resulting in the of STAT1 and These phosphorylated are to the nucleus in a complex with to IFN-stimulated response and activate the transcription of genes Jr., J.E. Science. 1997; 277: PubMed Scopus Google Scholar, L.C. Fish E.N. Exp. Hematol. Full Text Full Text PDF PubMed Scopus Google Scholar, G.R. Kerr I.M. Williams B.R. Silverman R.H. 1998; PubMed Scopus Google Scholar). However, an phosphorylation on STAT1 is required for a IFN-stimulated transcriptional response Z. Z. Darnell Jr., J.E. Full Text PDF PubMed Scopus Google Scholar). was reported to activate expression of the human PLSCR1 gene at the transcriptional through a IFN-stimulated response in the (14Zhou Q. Zhao J. Al-Zoghaibi F. Zhou A. Wiedmer T. Silverman R.H. Sims P.J. Blood. 2000; 95: 2593-2599Crossref PubMed Google Scholar). to PLSCR1 in U3A we that with wild type but not STAT1 mutant PLSCR1 expression. These an role for of STAT1 in PLSCR1 expression. studies that PKCδ can STAT1 on that a role for PKCδ in PLSCR1 expression is S. S. J. Li Y. Sassano A. Majchrzak B. S. M.S. Fish E.N. Platanias L.C. Exp. Hematol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). has been that PKCδ is activated of the IFN receptor and that such an activation of PKCδ for phosphorylation of STAT1 on (17Uddin S. Sassano A. Deb D.K. Verma A. Majchrzak B. Rahman A. Malik A.B. Fish E.N. Platanias L.C. J. Biol. Chem. 2002; 277: 14408-14416Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) also that PKCδ STAT1 through phosphorylation of STAT1 on in shown that the PKCδ inhibitor rottlerin or a dominant negative mutant of PKCδ significantly antagonized STAT1 phosphorylation on Ser-727. our a role for PKCδ in the phosphorylation of STAT1 required for IFN induction of PKCδ activation to phosphorylation of STAT1 is unresolved. results in phosphorylation of signaling including receptor substrate S. Fish E.N. D.A. Platanias L.C. J. 1997; Google Scholar, S. Fish E.N. D. M. P.M. Platanias L.C. Blood. 1997; Google Scholar), E.N. S. M. Majchrzak B. Platanias L.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and S. Majchrzak B. J. Y. R. Fish E.N. Platanias L.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. F. N. Majchrzak B. I. Fish E.N. Platanias L.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). recent that agents such as JNK activation is by with expression of a PKCδ and of PKCδ by small interfering that PKCδ is in for induction of JNK K. Y. D. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). that a JNK but not of and p38, both STAT1 phosphorylation and PLSCR1 expression IFN transfection of a dominant negative mutant of JNK also phosphorylation of STAT1 and PLSCR1 expression. results that JNK contributes to both STAT1 phosphorylation and an IFN transcriptional response as by expression of However, inhibition of JNK not phosphorylation and activation of PKCδ. JNK activation is of both of kinases are required for STAT1 In PKCδ inhibition suppressed JNK STAT1 and expression of induction of PLSCR1 through STAT1 that is by is dependent upon sequential activation of both PKCδ and JNK Jae-Won Young-Mi and James Darnell for generously the are also to Dr. George Stark, kindly provided with HT1080 and U3A cell