Phosphorylation of Serine 147 of tis21/BTG2/pc3 by p-Erk1/2 Induces Pin-1 Binding in Cytoplasm and Cell Death

Treatment of U937 cells with epidermal growth factor (EGF) induces phosphorylation of tis21 and subsequent interaction of tis21 with Pin-1, resulting in the increased cell death with mitochondrial depolarization. Ser147 and Ser149 residues of tis21 were strongly phosphorylated by p-Erk1/2 and p-p38MAPK, respectively, but not by JNK. To investigate the significance of phosphorylation of the Ser147 residue, Pin-1, one of the mitotic regulators that binds to the Ser(P)/Thr(P)-Pro region, was employed. Wild type tis21 phosphorylated by p-Erk1/2 clearly increased its binding to Pin-1, but not the P148A mutant, indicating that Pin-1 was bound to the Ser(P)147-Pro148 region of tis21. Transfection of tis21 significantly enhanced EGF-induced Pin-1 diffusion to cytoplasm, compared with that in the vector-transfected cells. Knockdown of tis21 expression by using shRNAi significantly inhibited EGF-induced Pin-1 diffusion, and analysis by flow cytometry after JC-1 stain and confocal microscope revealed that EGF aggravated tis21-induced mitochondrial depolarization and cell death. Furthermore, tis21 was bound to cyclin B1 and Cdc2 and inhibited its activity in vivo and in vitro. In summary, treatment of U937 cells with EGF activates Erk1/2, which in turn phosphorylates Ser147 of tis21 and induces tis21 and Pin-1 binding and mitochondrial depolarization. These data suggest, for the first time, a mechanism of how EGF can be antiproliferative in human tumor cells: binding of tis21/BTG2/pc3 to Pin-1 or cyclin B1-Cdc2 complex and induction of mitochondrial depolarization. Treatment of U937 cells with epidermal growth factor (EGF) induces phosphorylation of tis21 and subsequent interaction of tis21 with Pin-1, resulting in the increased cell death with mitochondrial depolarization. Ser147 and Ser149 residues of tis21 were strongly phosphorylated by p-Erk1/2 and p-p38MAPK, respectively, but not by JNK. To investigate the significance of phosphorylation of the Ser147 residue, Pin-1, one of the mitotic regulators that binds to the Ser(P)/Thr(P)-Pro region, was employed. Wild type tis21 phosphorylated by p-Erk1/2 clearly increased its binding to Pin-1, but not the P148A mutant, indicating that Pin-1 was bound to the Ser(P)147-Pro148 region of tis21. Transfection of tis21 significantly enhanced EGF-induced Pin-1 diffusion to cytoplasm, compared with that in the vector-transfected cells. Knockdown of tis21 expression by using shRNAi significantly inhibited EGF-induced Pin-1 diffusion, and analysis by flow cytometry after JC-1 stain and confocal microscope revealed that EGF aggravated tis21-induced mitochondrial depolarization and cell death. Furthermore, tis21 was bound to cyclin B1 and Cdc2 and inhibited its activity in vivo and in vitro. In summary, treatment of U937 cells with EGF activates Erk1/2, which in turn phosphorylates Ser147 of tis21 and induces tis21 and Pin-1 binding and mitochondrial depolarization. These data suggest, for the first time, a mechanism of how EGF can be antiproliferative in human tumor cells: binding of tis21/BTG2/pc3 to Pin-1 or cyclin B1-Cdc2 complex and induction of mitochondrial depolarization. tis21 (TPA-inducible sequences 21), antiproliferative gene, has been known as one of the early growth response genes (1Sukhatme V.P. Kartha S. Toback F.G. Taub R. Hoover R.G. Tsai-Morris C.H. Oncogene Res. 1987; 1: 343-355PubMed Google Scholar) and isolated from SW3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) 1The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; JNK, c-Jun N-terminal kinase; Cdc2, cell division cycle 2 kinase; Pin, protein-interacting NIMA; shRNAi, short hairpin RNA interference; EGF, epidermal growth factor; ORF, open reading frame; PBS, phosphate-buffered saline; GST, glutathione S-transferase; WT, wild type; FACS, fluorescence-activated cell sorting; MAPK, mitogen-activated protein kinase; IP, immunoprecipitation. (2Fletcher B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.A. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Abstract Full Text PDF PubMed Google Scholar). tis21 mRNA is very rapidly and transiently induced by growth factors and tumor promoter and superinduced by inhibition of protein biosynthesis (3Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (946) Google Scholar). pc3 found in rat PC12 cells (4Bradbury A. Possenti R. Shooter E.M. Tirone F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3353-3357Crossref PubMed Scopus (177) Google Scholar) and BTG2 (B-cell translocation gene 2) in humans (5Rouault J.P. Falette N. Guehenneux F. Guillot C. Rimokh R. Wang Q. Berthet C. Moyret-Lalle C. Savatier P. Pain B. Shaw P. Berger R. Samarut J. Magaud J.P. Ozturk M. Samarut C. Puisieux A. Nat. Genet. 1996; 14: 482-486Crossref PubMed Scopus (353) Google Scholar) are tis21-homologous genes. BTG2 has been cloned from chromosomal segment 1q32 and contains a wild type p53 response element in the 5′-flanking region (–74 to –122) (6Duriez C. Falette N. Audoynaud C. Moyret-Lalle C. Bensaad K. Courtois S. Wang Q. Soussi T. Puisieux A. Gene (Amst.). 2002; 282: 207-214Crossref PubMed Scopus (57) Google Scholar), thus confirming the observation that BTG2 expression is dependent on wild type p53 function (5Rouault J.P. Falette N. Guehenneux F. Guillot C. Rimokh R. Wang Q. Berthet C. Moyret-Lalle C. Savatier P. Pain B. Shaw P. Berger R. Samarut J. Magaud J.P. Ozturk M. Samarut C. Puisieux A. Nat. Genet. 1996; 14: 482-486Crossref PubMed Scopus (353) Google Scholar). TIS21/BTG2/Pc3 belongs to the APRO (antiproliferative) protein family (7Matsuda S. Rouault J. Magaud J. Berthet C. FEBS Lett. 2001; 497: 67-72Crossref PubMed Scopus (220) Google Scholar), which contains the APRO homology domain: Box-A (Tyr49–Asn71) and Box-B (Leu96–Glu115). Overexpression of pc3 inhibits G1-S progression through transcriptional regulation of cyclin D1 in the presence of pRB (8Guardavaccaro D. Corrente G. Covone F. Micheli L. D'Agnano I. Starace G. Caruso M. Tirone F. Mol. Cell. Biol. 2000; 20: 1797-1815Crossref PubMed Scopus (206) Google Scholar), whereas tis21 down-regulates expressions of cyclin E and cyclin-dependent kinase 4 in the absence of pRB function (9Lim I.K. Lee M.S. Ryu M.S. Park T.J. Fujiki H. Eguchi H. Paik W.K. Mol. Carcinog. 1998; 23: 25-35Crossref PubMed Scopus (66) Google Scholar). It has been known that tis21/BTG2/pc3 interacts with transcription factor caf1 (10Rouault J.P. Prévôt D. Berthet C. Birot A.M. Billaud M. Magaud J.P. Corbo L. J. Biol. Chem. 1998; 273: 22563-22569Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), protein arginine methyltransferase PRMT1 (11Lin W.J. Gary J.D. Yang M.C. Clarke S. Herschman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar), and homeogene Hoxb9 (12Prevot D. Voeltzel T. Birot A.M. Morel A.P. Rostan M.C. Magaud J.P. Corbo L. J. Biol. Chem. 2000; 275: 147-153Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), which can regulate cell cycle and differentiation. Interestingly, tis21 is a substrate of PRMT1, suggesting the existence of a feedback regulatory pathway between the two genes (13Lim I.K. Park T.J. Kim S. Lee H.W. Paik W.K. Biochem. Mol. Biol. Int. 1998; 45: 871-878PubMed Google Scholar). According to the published sequences of tis21, there is only one serine residue (147SPSK150) that can potentially be phosphorylated by proline-directed kinases such as Erk1/2, p38MAPK, JNK, and Cdc2 (cell division cycle 2 kinase) (14Shah O.J. Ghosh S. Hunter T. J. Biol. Chem. 2003; 278: 16433-16442Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), suggesting a possibility of TIS21 as one of the PIN-1-interacting proteins. Pin-1 (protein-interacting NIMA), NIMA-interacting protein, is a nuclear peptidyl-prolyl cis/trans-isomerase essential for regulation of mitosis (15Lu K.P. Hanes S.D. Hunter T. Nature. 1996; 380: 544-547Crossref PubMed Scopus (808) Google Scholar), which contains group IV WW domain and binds to Ser(P)/Thr(P)-Pro in mitotic phospho-proteins (16Yaffe M.B. Schutkowski M. Shen M. Zhou X.Z. Stukenberg P.T. Rahfeld J.U. Xu J. Kuang J. Kirschner M.W. Fischer G. Cantley L.C. Lu K.P. Science. 1997; 278: 1957-1960Crossref PubMed Scopus (682) Google Scholar). It has been suggested to be required for both normal mitotic progression (17Rippmann J.F. Hobbie S. Daiber C. Guilliard B. Bauer M. Birk J. Nar H. Garin-Chesa P. Rettig W.J. Schnapp A. Cell Growth & Differ. 2000; 11: 409-416PubMed Google Scholar) and reentry into the cell cycle from quiescence (18Joseph J.D. Yeh E.S. Swenson K.I. Means A.R. Winkler K.E. Prog. Cell Cycle Res. 2003; 5: 477-487PubMed Google Scholar). Recently, increasing interest has been focused on the role of Pin-1 and its binding proteins in cell cycle regulation and on the value of Pin-1 as a potential therapeutic target in breast cancer (19Wulf G. Ryo A. Liou Y.C. Lu K.P. Breast Cancer Res. 2003; 5: 76-82Crossref PubMed Scopus (66) Google Scholar), prostate carcinoma (20Ayala G. Wang D. Wulf G. Frolov A. Li R. Sowadski J. Wheeler T.M. Lu K.P. Bao L. Cancer Res. 2003; 63: 6244-6251PubMed Google Scholar), oral squamous cell carcinoma (21Miyashita H. Uchida T. Mori S. Echigo S. Motegi K. Oncol. Rep. 2003; 10: 1045-1048PubMed Google Scholar, 22Miyashita H. Mori S. Motegi K. Fukumoto M. Uchida T. Oncol. Rep. 2003; 10: 455-461PubMed Google Scholar), and hepatocellular carcinoma (23Pang R. Yuen J. Yuen M.F. Lai C.L. Lee T.K. Man K. Poon R.T. Fan S.T. Wong C.M. Ng I.O. Kwong Y.L. Tse E. Oncogene. 2004; 23: 4182-4186Crossref PubMed Scopus (102) Google Scholar). Indeed, Pin-1 has been shown to play an important role in oncogenesis (24Ryo A. Liou Y.C. Lu K.P. Wulf G. J. Cell Sci. 2003; 116: 773-783Crossref PubMed Scopus (171) Google Scholar) through several different mechanisms, such as the expression of cyclin D1 by cooperating with Ras signaling (25Wulf G.M. Ryo A. Wulf G.G. Lee S.W. Niu T. Petkova V. Lu K.P. EMBO J. 2001; 20: 3459-3472Crossref PubMed Scopus (486) Google Scholar, 26Ryo A. Liou Y.C. Wulf G. Nakamura M. Lee S.W. Lu K.P. Mol. Cell. Biol. 2002; 22: 5281-5295Crossref PubMed Scopus (227) Google Scholar), direct stabilization of cyclin D1 protein (27Liou Y.C. Ryo A. Huang H.K. Lu P.J. Bronson R. Fujimori F. Uchida T. Hunter T. Lu K.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1335-1340Crossref PubMed Scopus (295) Google Scholar), and inhibition of β-catenin interaction with the tumor A. Nakamura M. Wulf G. Liou Y.C. Lu K.P. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar). Furthermore, Pin-1 is an target gene essential for the of cells A. Liou Y.C. Wulf G. Nakamura M. Lee S.W. Lu K.P. Mol. Cell. Biol. 2002; 22: 5281-5295Crossref PubMed Scopus (227) Google Scholar) as as a of the tumor p53 and in response to P. M. Uchida T. C. F. S. G. C. G. Nature. 2002; PubMed Scopus Google Scholar, G.M. Liou Y.C. Ryo A. Lee S.W. Lu K.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). These strongly that several regulators of and such as Cdc2, and cyclin be the potential proteins to with a of on the of Pin-1 binding to proteins are the that of human are or M. K. M.S. Soussi T. R. T. E. B. R. Res. 22: Google Scholar), is not to regulation of the cell division cycle by the interaction of tis21/BTG2/pc3 with Pin-1 in human to the that tis21/BTG2/pc3 is a response gene induced by or growth factors (1Sukhatme V.P. Kartha S. Toback F.G. Taub R. Hoover R.G. Tsai-Morris C.H. Oncogene Res. 1987; 1: 343-355PubMed Google Scholar, B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.A. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Abstract Full Text PDF PubMed Google Scholar, H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (946) Google Scholar), cell are to BTG2 expression in the tumor cells to the in normal K. P. R. K. M. J. H. Cancer Res. 2004; PubMed Scopus Google Scholar). It be that of normal tis21 strongly tumor function of tis21 in and that of BTG2 is a in cancer of tumor by tis21/BTG2/pc3 in not been in the that tis21 protein interacts with Pin-1 after phosphorylation of its Ser147 residue by which was induced by EGF and inhibition of cyclin Cdc2 treatment of U937 cells with EGF clearly enhanced Pin-1 diffusion from to cytoplasm, by of tis21 expression with shRNAi hairpin RNA mitochondrial depolarization and increased cell death found a mechanism of how EGF can be through Pin-1 binding to resulting in increased mitochondrial depolarization. Cell U937 cells were in with in To and p38MAPK, cells were treated with epidermal growth factor (EGF) for and with for cells were by treatment with for tis21 was in A. Liou Y.C. Wulf G. Nakamura M. Lee S.W. Lu K.P. Mol. Cell. Biol. 2002; 22: 5281-5295Crossref PubMed Scopus (227) Google Scholar), and reading was by with the and the were into after with and To P148A and tis21 several were and was first was with as a and the which is to region of was with the first and as a and the of one was to of the tis21 gene which of the using and as and with tis21 as a were into after with and were by of TIS21 focused on the potential in a of glutathione proteins was induced in cells with and the cells were in and and by of by 4 the proteins in the were with to the proteins were using tis21 proteins were in after from the by for 4 tis21 protein that has of were with the of tis21 not homology with family genes and to be was with an of for the first and with for subsequent were by protein and using a protein expression of cell were to on a proteins were to a of used were as and from Cell and from Oncogene. were with with or and the proteins were by of the enhanced were with and for 4 cell were with protein for 4 p-p38MAPK, cyclin and Pin-1 were by cell 4 with p-p38MAPK, cyclin and Pin-1 were with the To investigate tis21 can be phosphorylated and which kinase potentially phosphorylates tis21 protein, in vivo and in kinase were using the and cyclin B1 were with mitogen-activated protein kinase and Cdc2 kinase was used for in kinase were by substrate proteins and in a of for the were by and by mitotic proteins with a was and proteins were to and with of the U937 cells in the 4 for were with the and the bound proteins were by using cyclin B1 and Pin-1 from U937 cell of were 4 for with tis21 or tis21 P148A proteins which been phosphorylated with or p-Erk1/2 were with the and the bound proteins were with using To the of tis21 on Cdc2 Cdc2 were from U937 cells with tis21, or with B1 were with kinase and the was by and in a of was for and by the of the substrate protein was by after To in the inhibition of Cdc2 activity by tis21, protein and was to the and Cdc2 kinase activity was by the U937 cells were with EGF after with or the and after and of EGF To investigate the of Pin-1, the cells were in for with in PBS, and on for with was 4 and the cells were with in with with for To was to the and the cells were with in for were by confocal microscope To tis21 tis21 mRNA was using the shRNAi to the the as and of tis21 mRNA sequences and of were sequences can hairpin region, and to of and of tis21 mRNA promoter with of the or the were in by and in transcription was for and the were through the with and In to of tis21 expression by shRNAi analysis was U937 cells were with tis21 in with or using of for in cells were after and tis21 expression was by using tis21 and with of to to expression was for mRNA with and with of to to In to investigate the of EGF to tis21-induced cell of mitochondrial potential were by JC-1 on the cell in a of JC-1 and the of JC-1 S. A. C. A. FEBS Lett. 1997; PubMed Scopus Google Scholar). U937 cells with tis21 or its were with EGF for and with cell was with JC-1 for and and was by fluorescence-activated cell after the cells with p-Erk1/2 and tis21 but Erk1/2, JNK, and with Cdc2 are proline-directed kinases S. J. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), tis21 residues were as potential phosphorylation by the kinase were U937 cells were with EGF for to strongly the p-Erk1/2 an in kinase was and only tis21 and not the be by indicating that Ser147 residue in tis21 protein be the of phosphorylation by of protein was to activity of the in In to p38MAPK, U937 cells were with and was from the cells after with the tis21, and proteins were to the in kinase strongly phosphorylated tis21, but not and indicating that the Ser149 residue in tis21 is the potential for To phosphorylates tis21 protein, an in kinase was not tis21 as to the phosphorylation of which as a for the not Pin-1 and B1 with tis21 on the observation that the Ser147 residue in tis21 protein can be phosphorylated by p-Erk1/2 and the residue can be by Pin-1 protein P.J. Zhou X.Z. Liou Y.C. J.P. Lu K.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), that the Ser147 residue of in the tis21 with To Pin-1 the tis21 protein that been phosphorylated with p-Erk1/2 was bound to Pin-1 the proteins were by analysis with To the of the binding between Pin-1 and tis21, the tis21 P148A was and phosphorylated with there was tis21 binding to Pin-1 thus indicating that Ser(P)147-Pro148 residues of tis21 a binding for the Pin-1 interaction was by a not only Cdc2 but cyclin B1 was found to to both the tis21 and but not to that the Ser147 residue is very for interaction but not for the B1 and the of tis21 was for binding of tis21 protein to the cyclin B1-Cdc2 of Cdc2 by tis21 in and in of the interaction of tis21 with Cdc2, in vivo and in of the Cdc2 activity by tis21. the of Cdc2 was by cyclin B1 in U937 cells. the cell of the tis21, and were with B1 and to kinase with and as a phosphorylation by Cdc2 was found to be significantly inhibited only in the tis21 and not in the and cells of Cdc2 activity by tis21 was by in or proteins were to the cyclin kinase activity was inhibited by but not by the of tis21 be essential to Cdc2 kinase tis21 Pin-1 into after EGF significance of Pin-1 binding to tis21, the or U937 cells were with EGF, and of of Pin-1 were shown in Pin-1 was in of the vector-transfected EGF treatment for significantly increased Pin-1 in the of both and cells. the translocation was in Pin-1 translocation into the in the cells in the vector-transfected In EGF significantly induced and of in the compared with the vector-transfected cells which was by of tis21 and its shRNAi the were a confocal the Pin-1 translocation was significantly increased in the tis21 to the and the cells of tis21 expression by shRNAi was by analysis These strongly that treatment of U937 cells with EGF rapidly induces phosphorylation of the Ser147 residue in tis21 protein by and Pin-1 binding and its diffusion into with nuclear of U937 cells. EGF tis21-induced and Cell in U937 on M.S. Lee M.S. E. Lim I.K. Cell Res. 2004; PubMed Scopus Google Scholar), EGF treatment the of tis21 on of U937 cells. the cells were with and the cell cycle was by shown in EGF significantly in the U937 cells after the from the of cells in and was to nuclear a microscope and tis21 was found to significantly cell death and EGF treatment enhanced the tis21 EGF tis21-induced of U937 the of EGF on tis21-induced cell of mitochondrial potential were by analysis with JC-1 stain of the cells. shown in a revealed the presence of cells with increased compared with that of cells. the cells in the and region were EGF treatment was found to cells in the region, whereas of the of cells was in the region These data clearly that EGF mitochondrial an early of cell cells the tis21 gene, thus resulting in cell death with by Cdc2 In the a mechanism of activity of tis21, through its direct binding to Pin-1 and Cdc2 both of which are very important regulators of of Pin-1 to tis21 required phosphorylation of Ser147 by and residue in tis21 protein residues of tis21 were required for inhibition of Cdc2 activity were not for interaction with cyclin B1 and Cdc2 Treatment of U937 cells with EGF clearly induced Pin-1 in and increased cell death in Interestingly, Pin-1 translocation was in the tis21 the vector-transfected cells that expression of in U937 cells induces cell and inhibition of cyclin B1 binding to Cdc2 M.S. Lee M.S. E. Lim I.K. Cell Res. 2004; PubMed Scopus Google Scholar). on the can that EGF-induced tis21 phosphorylation Ser147 residue Pin-1 to of tis21 protein, which in turn inhibited Cdc2 activity through the inhibition of cyclin B1-Cdc2 complex These in increased cell death through mitochondrial depolarization mitotic Cdc2 been of which are phosphorylated only Cdc2 but been in the proline-directed kinases S. J. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the proline-directed family has an of has its substrate be to between or different for in the that Ser147 and Ser149 residues be phosphorylated by p-Erk1/2 and p-p38MAPK, and p-Erk1/2 as the tis21 Ser147 It that there be between the kinase complex and p-Erk1/2 signaling which can the phosphorylation of tis21. possibility is in with that of pathway inhibits Cdc2 to of in T.M. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar), and that the of Cdc2 interacts with of Erk1/2, and inhibits binding to A. N. A. J.F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). It has been that the cyclin kinase 2 complex phosphorylates the Ser147 residue of pc3 with suggesting that is to regulation of cell cycle (8Guardavaccaro D. Corrente G. Covone F. Micheli L. D'Agnano I. Starace G. Caruso M. Tirone F. Mol. Cell. Biol. 2000; 20: 1797-1815Crossref PubMed Scopus (206) Google Scholar). Furthermore, be that BTG2 with and pc3 proteins the in the that of tis21 was for interaction and inhibition of Cdc2 activity and a to in vivo and in kinase activity of the cyclin B1-Cdc2 complex was significantly suggesting a mechanism of by tis21. on the of in vivo Cdc2 binding to cyclin B1 in the tis21 M.S. Lee M.S. E. Lim I.K. Cell Res. 2004; PubMed Scopus Google Scholar). Furthermore, tis21 through the cyclin E biosynthesis in the pRB cells (9Lim I.K. Lee M.S. Ryu M.S. Park T.J. Fujiki H. Eguchi H. Paik W.K. Mol. Carcinog. 1998; 23: 25-35Crossref PubMed Scopus (66) Google Scholar). tis21 be as cycle in both and the cells are pRB and p53 Pin-1 is an protein and to through the nuclear has nuclear protein in nuclear phosphorylation in its WW domain is shown to its in cells P.J. Zhou X.Z. Liou Y.C. J.P. Lu K.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In the that EGF-induced Ser147 phosphorylation of tis21 enhanced of Pin-1 and nuclear of U937 cells Pin-1 translocation in after EGF treatment was in with the that Pin-1 from was found after treatment in cells P.J. Zhou X.Z. Liou Y.C. J.P. Lu K.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Pin-1 into the in 4 after treatment was not in the U937 cells Pin-1 is a and in human such as and breast Pin-1 expression has been as an in prostate and inhibition of Pin-1 can and tumor cell growth L. A. G. Sowadski Lu K.P. Wang J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). can that tis21 function as a tumor through Pin-1 in cytoplasm, as a mitotic in the with p53 on the that the domain of that interacts with protein kinase interacts with tis21 and protein kinase W.J. Wang Huang Biochem. J. 2001; Google Scholar) and that protein can to in cells Yeh C.H. Huang W.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), mitochondrial depolarization in the cells. shown in EGF treatment significantly increased mitochondrial depolarization in U937 cells with tis21 suggesting that EGF-induced cell death with tis21 phosphorylation and Pin-1 which induces of mitochondrial potential as are data are the only the role of tis21 in strongly that EGF-induced Pin-1 binding to tis21 and mitochondrial depolarization a mechanism of EGF-induced growth inhibition of the human cancer such as J. Oncogene. 1996; Google Scholar), breast cancer J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. R. Biochem. Res. PubMed Scopus Google Scholar), and U937 cells H. H. 2001; PubMed Scopus Google Scholar), which the of EGF EGF-induced has been known to of in the or cells with EGF T.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and EGF induces of was L. Yang Q. J. Cell Biochem. 2004; PubMed Scopus Google Scholar). In of the to a expression and such as not It to be how Pin-1 interaction with phosphorylated tis21 the function of tis21 or of tis21 on the induction of cell death and of Pin-1 was by using tis21 shRNAi in U937 cells the of Pin-1 binding to tis21 has to be by on the regulation of mitochondrial depolarization. Paik for reading of the