Forkhead Family Transcription Factor FKHRL1 Is Expressed in Human Megakaryocytes

Abstract

FKHRL1, a member of the Forkhead transcription factor family, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt. This molecule is a mammalian homolog of DAF-16, which plays an important role in the longevity ofCaenorhabditis elegans. In this study we found that Akt and FKHRL1 proteins were detectable in highly purified normal human megakaryocytes and that these molecules were actually phosphorylated by thrombopoietin (TPO). To clarify the functional role of FKHRL1 in TPO signaling, we established a tetracycline-inducible system in the human TPO-dependent leukemia cell line UT-7/TPO. Induced expression of active FKHRL1 led to cell cycle arrest at G0/G1 phase in this cell line. These results suggest that FKHRL1 plays an important role in the cell cycle of megakaryocytic cells as one of the downstream target molecules of phosphatidylinositol 3-kinase-Akt, presumably mediated through the activation or inactivation of cell cycle-associated gene(s). FKHRL1, a member of the Forkhead transcription factor family, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt. This molecule is a mammalian homolog of DAF-16, which plays an important role in the longevity ofCaenorhabditis elegans. In this study we found that Akt and FKHRL1 proteins were detectable in highly purified normal human megakaryocytes and that these molecules were actually phosphorylated by thrombopoietin (TPO). To clarify the functional role of FKHRL1 in TPO signaling, we established a tetracycline-inducible system in the human TPO-dependent leukemia cell line UT-7/TPO. Induced expression of active FKHRL1 led to cell cycle arrest at G0/G1 phase in this cell line. These results suggest that FKHRL1 plays an important role in the cell cycle of megakaryocytic cells as one of the downstream target molecules of phosphatidylinositol 3-kinase-Akt, presumably mediated through the activation or inactivation of cell cycle-associated gene(s). phosphatidylinositol 3-kinase glycogen synthase kinase-3 glutathione S-transferase fetal calf serum bovine serum albumin stem cell factor thrombopoietin Iscove's modified Dulbecco's medium polyacrylamide gel electrophoresis fluorescein isothiocyanate fluorescence-activated cell sorter tetracycline erythropoietin FKHRL1, a mammalian homolog of DAF-16, belongs to the Forkhead transcription factor family (1Anderson M.J. Viars C.S. Czekay S. Cavenee W.K. Arden K.C. Genomics. 1998; 47: 187-199Crossref PubMed Scopus (290) Google Scholar). This family, characterized by the presence of a highly conserved forkhead domain having a winged-helix motif and DNA binding activity, is involved in embryogenesis, differentiation, and tumorigenesis (2Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (577) Google Scholar). DAF-16 plays an important role in the longevity of Caenorhabditis elegans (3Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-998Crossref PubMed Scopus (1551) Google Scholar). In C. elegans, the dauer lava ensures its survival in adverse conditions by lowering its metabolism and changing its shape and can live up to ten times longer than a normal adult (4Kops G.J Burgering B.M.T. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (253) Google Scholar). DAF-16 induces dauer formation and its activity is negatively regulated by DAF-2. Indeed, inC. elegans, a loss-of-function mutation in theDAF-2 gene causes developmental arrest at the dauer stage via the enhancement of DAF-16 transcription activity, resulting in an extension of the life span (5Kenyon C. Chang J. Gensch E. Ruder A. Tabtiang R.A.C. Nature. 1993; 366: 461-464Crossref PubMed Scopus (2523) Google Scholar). These observations strongly suggested that FKHRL1 plays an important role in mammalian biology. This notion is supported by the evidence that there is a highly conserved signaling pathway between C. elegans and human. DAF-2 is an insulin receptor-like protein, AGE-1 is a catalytic subunit of phosphatidylinositol 3-kinase (PI3K)1-like protein, DAF-23 is a PI3K-like protein, AKT1/AKT2 is a serine/threonine kinase protein kinase B (PKB) (also known as Akt)-like protein, and DAF-16 has high homology to the mammalian Forkhead subfamily FKHRL1, AFX, and FKHR (4Kops G.J Burgering B.M.T. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (253) Google Scholar). Akt has been identified as a downstream target of PI3K necessary for survival (6Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1528) Google Scholar). Akt activated by growth factors phosphorylates apoptosis-associated molecules including Bad, caspase-9, IKKα, and GSK-3 ensuring cell survival (7Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4957) Google Scholar, 8Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2735) Google Scholar, 9Romashkova J.A. Makarow S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1670) Google Scholar, 10Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1904) Google Scholar). In addition, the constitutively active form of Akt blocks apoptosis induced by growth factor deprivation (11Bao H. Jacobs-Helber S.M. Lawson A.E. Penta K. Wickrema A. Sawyer S.T. Blood. 1999; 93: 3757-3773Crossref PubMed Google Scholar, 12Kashii Y. Uchida M. Kirito K. Tanaka M. Nishijima K. Toshima M. Ando T. Koizumi K. Endoh T. Sawada Ki. Momoi M. Miura Y. Ozawa K. Komatsu N. Blood. 2000; 96: 941-949Crossref PubMed Google Scholar). Thus, the PI3K-Akt activation pathway appears to be a prerequisite for cell survival. Recently, FKHRL1, AFX, and FKHR have been identified as substrates of Akt (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 14Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 15Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (946) Google Scholar, 16Rena G. Guo S. Cichy S.C. Unterman T.G. Cohen P. J. Biol. Chem. 1999; 274: 17179-17183Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 17Takaishi H. Konishi H. Matsuzaki H. Ono Y. Shirai Y. Saito N. Kitamura T Ogawa W. Kasuga M. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11836-11841Crossref PubMed Scopus (217) Google Scholar, 18Kops G.J. de Ruiter N.D. De Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M.T. Nature. 1999; 398: 630-634Crossref PubMed Scopus (953) Google Scholar, 19Nakae J. Park B-C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Phosphorylation of these proteins by Akt regulates their nuclear translocation and targets gene transcription. There are two or three potential Akt phosphorylation sites (RXRXX(S/T)) on these three members. For example, FKHRL1 has three putative phosphorylation sites; Thr32 (RPRSCT32), Ser253 (RRRAVS253), and Ser315 (RSRTNS315). When cells are stimulated with serum or growth factors, a phosphorylated form of FKHRL1 is retained in the cytoplasm and interacts with 14-3-3 proteins, resulting in inhibition of target gene transcription. By contrast, when cells are deprived of serum or growth factors, a nonphosphorylated form of FKHRL1 translocates into nucleus and activates the transcription of target genes. These findings indicate that FKHRL1, when not phosphorylated by Akt, is an activator of transcription. Thus, phosphorylation by Akt is essential for suppressing the transcription activity of FKHRL1. In other words, Akt negatively regulates the transcription activity of FKHRL1 by phosphorylation (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). This is also true for both AFX and FKHR (14Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 15Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (946) Google Scholar, 16Rena G. Guo S. Cichy S.C. Unterman T.G. Cohen P. J. Biol. Chem. 1999; 274: 17179-17183Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 17Takaishi H. Konishi H. Matsuzaki H. Ono Y. Shirai Y. Saito N. Kitamura T Ogawa W. Kasuga M. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11836-11841Crossref PubMed Scopus (217) Google Scholar, 18Kops G.J. de Ruiter N.D. De Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M.T. Nature. 1999; 398: 630-634Crossref PubMed Scopus (953) Google Scholar). Very recently we identified FKHRL1 as one of the downstream molecules of the phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin (EPO) signal transduction (12Kashii Y. Uchida M. Kirito K. Tanaka M. Nishijima K. Toshima M. Ando T. Koizumi K. Endoh T. Sawada Ki. Momoi M. Miura Y. Ozawa K. Komatsu N. Blood. 2000; 96: 941-949Crossref PubMed Google Scholar), although the functional role of this molecule in erythropoiesis is still open to question. Based on several lines of evidence that there is a close relationship between erythropoiesis and megakaryopoiesis (20Longmore G.D. Pharr P. Neumann D. Lodish H.F. Blood. 1999; 82: 2386-2395Crossref Google Scholar, 21Komatsu N. Kirito K. Shimizu R. Kunitama M. Yamada M. Uchida M. Takatoku M. Eguchi M. Miura Y. Blood. 1997; 89: 4021-4033Crossref PubMed Google Scholar, 22Romeo P.H. Prandini M.H. Joulin V. Mignotte V. Prenant M. Vainchenker W. Marguerie G. Uzan G. Nature. 1990; 344: 447-449Crossref PubMed Scopus (323) Google Scholar), we examined whether or not TPO, a major regulator of megakaryopoiesis, induces phosphorylation of Akt and FKHRL1 in a human TPO-dependent leukemia cell line UT-7/TPO (23Komatsu N. Kunitama M. Yamada M. Hagiwara T. Kato T. Miyazaki H. Eguchi M. Yamamoto M. Miura Y. Blood. 1996; 87: 4552-4560Crossref PubMed Google Scholar) and highly purified normal human megakaryocytes. Moreover, to elucidate the function of FKHRL1 in TPO signaling, we established a tetracycline (Tet)-inducible expression system in UT-7/TPO cells. Our results suggest that FKHRL1 is phosphorylated by TPO stimulation via Akt activation and that unphosphorylated FKHRL1 negatively regulates the cell cycle, presumably via the activation or inactivation of cell cycle-associated gene(s). Recombinant TPO was kindly donated by Kirin Brewery Co. Ltd. (Tokyo, Japan). 2-mercaptoethanol, sodium orthovanadate, bovine serum albumin (BSA), DNase, Iscove's modified Dulbecco's medium (IMDM), saturated transferrin, oleic acid, l-α-phosphatidylcholine, cholesterol, and propidium iodide were purchased from Sigma. Vitamin B12 and folic acid were purchased from Sankyo Co. (Tokyo, and Co. with was from calf serum and were from activity was purchased from and were purchased from FKHRL1, protein, and for and FKHRL1 were kindly by (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). and were purchased from was purchased from Japan). UT-7/TPO was in with and TPO (23Komatsu N. Kunitama M. Yamada M. Hagiwara T. Kato T. Miyazaki H. Eguchi M. Yamamoto M. Miura Y. Blood. 1996; 87: 4552-4560Crossref PubMed Google Scholar). growth was examined by a to with T. J. PubMed Scopus Google Scholar). cells were at a of in in in the presence of TPO of at of was to a at of was to to the was at a of a UT-7/TPO cells were deprived of growth factor for stimulation with TPO at for a cells were and in of and sodium of on were by at for were with to protein for at in an were by a and times with of proteins were for in a the were by and were a were with in for and with the of including at a with the were with a of for at a the were with an to the In the were for in sodium orthovanadate, and a the were by and a were with in for at and with the of or FKHRL1 at a with the were with a of or for at a the were with an and to to were with and at for and were and were three times with and with the Akt kinase In kinase were with a GSK-3 or protein (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar) as of were by and by To we a system in which transcription of a target is by In UT-7/TPO cells were with expression for the by with of one was with a expression for with at a of the of the protein were examined and by and were to the target proteins were induced by in these were and in and for at a with the cells were with in and at at a with the cells were for with the cells were with and in were a system megakaryocytic cells were as with K. Sawada K. M. A. T. H. Y. M. E. T. N. S. T. 1998; Google Scholar). In human factor and was to by the of and the for the of as N. Sawada K. Y. S. T. S. Google Scholar). cells were K. S.T. S.T. J. Cell. 1990; PubMed Scopus Google Scholar, M. Sawada K. N. Koizumi K. S. T. 1997; PubMed Scopus Google Scholar). cells were and in a cells were and in and and were at for at cells were with and in cells were in as with K. Sawada K. M. A. T. H. Y. M. E. T. N. S. T. 1998; Google Scholar). In cells from to were in a human of transferrin, of oleic acid, l-α-phosphatidylcholine, and K. S.T. Sawyer S.T. J. PubMed Scopus Google Scholar), B12 at and folic acid at with TPO at in the presence of 2-mercaptoethanol, at at and in a for at in a the cells were and with of cells was by as K. Sawada K. M. A. T. H. Y. M. E. T. N. S. T. 1998; Google Scholar). were fluorescein isothiocyanate and Japan). cycle was by DNA with propidium iodide in for with the system In the we the UT-7/TPO cell line (23Komatsu N. Kunitama M. Yamada M. Hagiwara T. Kato T. Miyazaki H. Eguchi M. Yamamoto M. Miura Y. Blood. 1996; 87: 4552-4560Crossref PubMed Google Scholar). This cell line on TPO for growth and and has megakaryocytic as a in the high expression of the and and high DNA (23Komatsu N. Kunitama M. Yamada M. Hagiwara T. Kato T. Miyazaki H. Eguchi M. Yamamoto M. Miura Y. Blood. 1996; 87: 4552-4560Crossref PubMed Google Scholar). UT-7/TPO is a of megakaryocytic cells for the role of the activation pathway in TPO Based on the that Akt is activated by several including factor and (12Kashii Y. Uchida M. Kirito K. Tanaka M. Nishijima K. Toshima M. Ando T. Koizumi K. Endoh T. Sawada Ki. Momoi M. Miura Y. Ozawa K. Komatsu N. Blood. 2000; 96: 941-949Crossref PubMed Google Scholar, D. Cantley L.C. Kaplan D.R. Franke T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus (323) Google Scholar, Blood. 2000; PubMed Google Scholar), we examined whether or not TPO activates Akt in UT-7/TPO cells. UT-7/TPO cells were to TPO for of up to and for with was which a phosphorylated one of two sites on Akt phosphorylated in its active in phosphorylated Akt and its a at to and by Moreover, growth UT-7/TPO cells were to of TPO in phosphorylated Akt was at of TPO, and the a at of These findings indicate that TPO induced phosphorylation of Akt in a and growth UT-7/TPO cells were with of the for and stimulated with TPO the cells were for cell was in the phosphorylation of Akt was at and to the at This suggested that phosphorylation of Akt is mediated via PI3K To the activation of Akt by TPO, we in kinase GSK-3 as to TPO for of up to the cells were with and in was to the of the of phosphorylated GSK-3 was a to TPO, and its enhancement This that TPO induced Akt kinase activation in UT-7/TPO cells. was that FKHRL1 is a of Akt kinase in (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). also found that Akt kinase activated by phosphorylated the FKHRL1 protein (12Kashii Y. Uchida M. Kirito K. Tanaka M. Nishijima K. Toshima M. Ando T. Koizumi K. Endoh T. Sawada Ki. Momoi M. Miura Y. Ozawa K. Komatsu N. Blood. 2000; 96: 941-949Crossref PubMed Google Scholar). To that FKHRL1 downstream of Akt kinase in the TPO signaling we examined whether or not FKHRL1 is phosphorylated by TPO UT-7/TPO cells were to TPO for of up to or to of TPO for and the cells were for of cell was and that a phosphorylated and a phosphorylated FKHRL1 was phosphorylated at Thr32 and Ser253 at the a at and FKHRL1 was detectable at of TPO, and the a at of TPO These findings indicate that TPO induced phosphorylation of FKHRL1 in a and growth UT-7/TPO cells were with of the for and were stimulated with TPO the cells were for cell with and that the phosphorylation of FKHRL1 at Thr32 and Ser253 was at and at of This suggested that phosphorylation of FKHRL1 is mediated via PI3K To that FKHRL1 is by kinase activated by TPO in we an in kinase protein as a UT-7/TPO cells were to TPO for or and for with were with protein to the of the with in a phosphorylated FKHRL1 was with which phosphorylated Thr32 and phosphorylated that Akt activated by TPO phosphorylated FKHRL1 at Thr32 and examined whether or not FKHRL1 is in normal megakaryocytes. To a of megakaryocytes for human cells were in the presence of and TPO for human megakaryocytic cells the megakaryocytic at at and at not the cells these highly purified we examined whether or not Akt and its downstream molecule FKHRL1 are phosphorylated in megakaryocytic cells. a deprivation of growth factors, the cells were stimulated with TPO for and for in Akt and FKHRL1 proteins were in megakaryocytes. Akt at and FKHRL1 at Thr32 and Ser253 were phosphorylated by TPO that FKHRL1 downstream of the PI3K-Akt activation these results the that FKHRL1 is involved in the cell survival as a target molecule of the PI3K-Akt To this we the of FKHRL1 in UT-7/TPO by a in which expression of the target protein is induced by in which and Ser315 are to is to the nucleus and activates the gene in that is an form for activation of target gene(s). with and several and were for the led to the of protein in these of the cell that the with in of proteins in cells we the nuclear of in these study that induced was to the nucleus Induced expression of by led to the of into the cells on into a cell line that was with an expression not These results suggest that is actually functional in To elucidate the role of FKHRL1, we examined the of on cell of UT-7/TPO cells. a to in the of TPO, the cells were in for cell cycle cells was with TPO for and for cell cycle of cells at the G0/G1 phase with the cells These results strongly suggested that FKHRL1 activates or the transcription of the cell cycle-associated gene(s). was not by with the cells Indeed, the of cells was not in the cells with not results were in not active FKHRL1 induced cell cycle arrest at G0/G1 phase not apoptosis in UT-7/TPO FKHRL1 blocks at G0/G1 phase in UT-7/TPO cells. of on of UT-7/TPO and cells were at a of in with and with or at of TPO was of the of the at of TPO in are as the from of on cell a to in the of TPO, cells were stimulated with TPO for the and for cell cycle In this study we that Akt and FKHRL1 were and phosphorylated by TPO in a human leukemia cell line UT-7/TPO. found that PI3K the phosphorylation of Akt and FKHRL1 in UT-7/TPO that the activation of Akt and FKHRL1 is regulated by PI3K In addition, in kinase that Akt kinase activated by TPO phosphorylated FKHRL1 protein at and These findings indicate that FKHRL1 as a downstream molecule of the PI3K-Akt activation as is the for (12Kashii Y. Uchida M. Kirito K. Tanaka M. Nishijima K. Toshima M. Ando T. Koizumi K. Endoh T. Sawada Ki. Momoi M. Miura Y. Ozawa K. Komatsu N. Blood. 2000; 96: 941-949Crossref PubMed Google Scholar). we that unphosphorylated active FKHRL1 induced cell cycle arrest at the G0/G1 phase in UT-7/TPO cells. Akt and FKHRL1 were and phosphorylated in highly purified normal human megakaryocytes. This strongly suggested that these molecules are involved in normal megakaryopoiesis and presumably in is that expression of active FKHRL1 induced cell cycle arrest at G0/G1 phase in UT-7/TPO. Very recently was that AFX, a member of the Forkhead cell cycle in phase via activation of the gene at the G.J. Bos J.L. Burgering Nature. 2000; PubMed Scopus Google Scholar). This to whether or not activation of and is involved in cell cycle arrest at the G0/G1 phase in UT-7/TPO cells. that not or gene in the system not In addition, were at the protein not These findings indicate that cell cycle-associated other than and the cell cycle arrest at G0/G1 phase in UT-7/TPO cells. There was a in the of between the inhibition of phosphorylation of and of apoptosis in UT-7/TPO cells. a high of PI3K and of induced apoptosis in than of UT-7/TPO cells not phosphorylation of Akt and FKHRL1 by TPO was at at which apoptosis not in UT-7/TPO cells not is that phosphorylation of Akt and FKHRL1 is involved in the of This notion is in supported by the evidence that of of not apoptosis in UT-7/TPO cells B and not results suggest that FKHRL1 in a is not to apoptosis in UT-7/TPO cells. a factor be for apoptosis in this cell line. FKHRL1 was identified as a of in leukemia with translocation J. M. P. R. Blood. 1997; PubMed Google Scholar). has been that and are detectable in with and A. R. T. J. J. J. T. 1997; PubMed Scopus Google Scholar, N. S. III, G. Barr F.G. 1993; PubMed Scopus Google Scholar). expression or loss-of-function of these three appears to be involved in results that expression of of UT-7/TPO cells FKHRL1 function as a in normal although this is There is a close relationship between cell and cell cycle To whether or not FKHRL1 megakaryocytic of UT-7/TPO we cells in the presence of TPO and for expression of by not the expression of and which are for the megakaryocytic not This that cell cycle arrest at the G0/G1 phase is not for megakaryocytic There was a between Akt and FKHRL1 in the of TPO for their Phosphorylation of Akt by TPO was induced at of TPO, FKHRL1 was phosphorylated by TPO at In addition, the in kinase that the phosphorylation of FKHRL1 by Akt was than that in we the that the in phosphorylation and in is of the be of the of in this or that not Akt other protein are involved in the phosphorylation of FKHRL1 FKHRL1 has three putative Akt phosphorylation and Thr32 and Ser253 were phosphorylated by TPO stimulation in UT-7/TPO cells. Indeed, an in that both sites were phosphorylated by Akt activated by we not the phosphorylation of Ser315 induced by TPO was not To this we of the that phosphorylation of FKHRL1 at Ser315 not Thr32 or Ser253 a on the of FKHRL1 on an gel (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). in and TPO induced a up in the of FKHRL1. This strongly suggested that FKHRL1 at Ser315 was phosphorylated by TPO although is still whether or not Akt phosphorylated the three that FKHRL1 (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar), was that one of the target molecules of FKHRL1 in UT-7/TPO cells. Indeed, the nonphosphorylated active form of FKHRL1 can in and apoptosis in and T cells (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). Based on these we examined whether or not FKHRL1 can the gene a system in UT-7/TPO cells. that was the of not that gene is not a target molecule for FKHRL1 at in UT-7/TPO cells. To this is the that that Akt and FKHRL1 are in normal megakaryocytes and that these molecules are actually phosphorylated by evidence that FKHRL1 is in strongly that FKHRL1 is a molecule (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). In results that FKHRL1 induced cell cycle arrest at on and FKHRL1 an important role in the cells in the via the activation or inactivation of cell cycle-associated gene(s). A. and M. E. of for and FKHRL1 and the