The Death Effector Domain-containing DEDD Supports S6K1 Activity via Preventing Cdk1-dependent Inhibitory Phosphorylation

Cell cycle regulation and biochemical responses upon nutrients and growth factors are the major regulatory mechanisms for cell sizing in mammals. Recently, we identified that the death effector domain-containing DEDD impedes mitotic progression by inhibiting Cdk1 (cyclin-dependent kinase 1) and thus maintains an increase of cell size during the mitotic phase. Here we found that DEDD also associates with S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase, and supports its activity by preventing inhibitory phosphorylation of S6K1 brought about by Cdk1 during the mitotic phase. DEDD-/- cells showed reduced S6K1 activity, consistently demonstrating decreased levels in activating phosphorylation at the Thr-389 site. In addition, levels of Cdk1-dependent inhibitory phosphorylation at the C terminus of S6K1 were enhanced in DEDD-/- cells and tissues. Consequently, as in S6K1-/- mice, the insulin mass within pancreatic islets was reduced in DEDD-/- mice, resulting in glucose intolerance. These findings suggest a novel cell sizing mechanism achieved by DEDD through the maintenance of S6K1 activity prior to cell division. Our results also suggest that DEDD may harbor important roles in glucose homeostasis and that its deficiency might be involved in the pathogenesis of type 2 diabetes mellitus. Cell cycle regulation and biochemical responses upon nutrients and growth factors are the major regulatory mechanisms for cell sizing in mammals. Recently, we identified that the death effector domain-containing DEDD impedes mitotic progression by inhibiting Cdk1 (cyclin-dependent kinase 1) and thus maintains an increase of cell size during the mitotic phase. Here we found that DEDD also associates with S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase, and supports its activity by preventing inhibitory phosphorylation of S6K1 brought about by Cdk1 during the mitotic phase. DEDD-/- cells showed reduced S6K1 activity, consistently demonstrating decreased levels in activating phosphorylation at the Thr-389 site. In addition, levels of Cdk1-dependent inhibitory phosphorylation at the C terminus of S6K1 were enhanced in DEDD-/- cells and tissues. Consequently, as in S6K1-/- mice, the insulin mass within pancreatic islets was reduced in DEDD-/- mice, resulting in glucose intolerance. These findings suggest a novel cell sizing mechanism achieved by DEDD through the maintenance of S6K1 activity prior to cell division. Our results also suggest that DEDD may harbor important roles in glucose homeostasis and that its deficiency might be involved in the pathogenesis of type 2 diabetes mellitus. Cell size is closely related to specialized cell function and to the specific patterning of tissues in the body. Cell sizing is regulated mainly by two mechanisms: cell cycle control and the biochemical response to nutrients and/or growth factors (1Conlon I. Raff M. Cell. 1999; 96: 235-244Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, 2Montagne J. Mol. Cell. Biol. Res. Commun. 2000; 4: 195-202Crossref PubMed Scopus (26) Google Scholar, 3Cooper S. BMC Cell Biol. 2004; 5: 35Crossref PubMed Scopus (34) Google Scholar, 4Jorgensen P. Tyers M. Curr. Biol. 2004; 14: 1014-1027Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 5De Virgilio C. Loewith R. Oncogene. 2006; 25: 6392-6415Crossref PubMed Scopus (182) Google Scholar). During cell cycle progression, both the G1 (which is believed to be dominant) and the G2 periods are important for cells to increase their volume (6Jorgensen P. Nishikawa J.L. Breitkreutz B.J. Tyers M. 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The DEDD molecule was initially described as a member of the death effector domain (DED) 2The abbreviations used are: DED, death effector domain; rRNA, ribosomal RNA; S6K1, S6 kinase 1; PI3K, phosphatidylinositol 3-kinase; TOR, target of rapamycin; mTOR, mammalian TOR; MEF, mouse embryonic fibroblast; siRNA, small interfering RNA; GST, glutathione S-transferase; rpS6, S6 ribosomal protein.2The abbreviations used are: DED, death effector domain; rRNA, ribosomal RNA; S6K1, S6 kinase 1; PI3K, phosphatidylinositol 3-kinase; TOR, target of rapamycin; mTOR, mammalian TOR; MEF, mouse embryonic fibroblast; siRNA, small interfering RNA; GST, glutathione S-transferase; rpS6, S6 ribosomal protein.-containing protein family (12Stegh A.H. Schickling O. Ehret A. Scaffidi C. Peterhänsel C. Hofmann T.G. Grummt I. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 5974-5986Crossref PubMed Scopus (114) Google Scholar). Although the absence of DEDD did not apparently influence progression of apoptosis (10Arai S. Miyake K. Voit R. Nemoto S. Wakeland E.K. Grummt I. Miyazaki T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2289-2294Crossref PubMed Scopus (25) Google Scholar), we found that during mitosis, DEDD is associated with Cdk1-cyclin B1 and that it decreases the kinase activity of Cdk1. This response impedes the Cdk1-dependent mitotic program to shut off synthesis of ribosomal RNA (rRNA) and protein and is consequently useful in gaining sufficient cell growth prior to cell division. Depletion of DEDD consistently results in a shortened mitotic duration and an overall reduction in the amount of cellular rRNA and protein and, furthermore, in cell and body size (10Arai S. Miyake K. Voit R. Nemoto S. Wakeland E.K. Grummt I. Miyazaki T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2289-2294Crossref PubMed Scopus (25) Google Scholar, 11Miyazaki T. Arai S. Cell Cycle. 2007; 6: 1419-1425Crossref PubMed Scopus (3) Google Scholar). Of the biochemical responses responsible for cell sizing, the signaling cascade involving phosphatydilinositol 3-kinase (PI3K) and its downstream target of rapamycin (TOR) is most crucial (13Oldham S. Hafen E. Trends Cell Biol. 2003; 13: 79-85Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 14Valentinis B. Baserga R. Mol. Pathol. 2001; 54: 133-137Crossref PubMed Scopus (297) Google Scholar, 15Vogt P.K. Trends Mol. Med. 2001; 7: 482-484Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In mammals, upon stimulation by growth factors, including insulin, the mammalian TOR (mTOR) cooperates with PI3K-dependent effectors to activate S6K1, thereby phosphorylating the 40 S ribosomal protein S6, and subsequently enhances translation of the 5′-terminal oligopyrimidine sequences that encode components of the translational machinery. This reaction increases the number of ribosomes and the efficacy of protein synthesis, thus critically promoting cell growth (16Huang S. Houghton P.J. Curr. Opin. Pharmacol. 2003; 3: 371-377Crossref PubMed Scopus (404) Google Scholar, 17Shamji A.F. Nghiem P. Schreiber S.L. Mol. Cell. 2003; 12: 271-280Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 18Fingar D.C. Salama S. Tsou C. Harlow E. Blenis J. Genes Dev. 2002; 16: 1472-1487Crossref PubMed Scopus (860) Google Scholar). Therefore, mice deficient for S6K1 (S6K1-/-) had reduced cell and body size (19Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar, 20Montagne J. Stewart M.J. Stocker H. Hafen E. Kozma S.C. Thomas G. Science. 1999; 285: 2126-2129Crossref PubMed Scopus (624) Google Scholar, 21Pende M. Kozma S.C. Jaquet M. Oorschot V. Burcelin R. Le Marchand-Brustel Y. Klumperman J. Thorens B. Thomas G. Nature. 2000; 408: 994-997Crossref PubMed Scopus (377) Google Scholar, 22Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1056) Google Scholar, 23Ohanna M. Sobering A.K. Lapointe T. Lorenzo L. Praud C. Petroulakis E. Sonenberg N. Kelly P.A. Sotiropoulos A. Pende M. Nat. Cell Biol. 2005; 7: 286-294Crossref PubMed Scopus (318) Google Scholar). This effect also involves S6K1 in maintenance of glucose tolerance. S6K1 significantly supports the size of insulin-producing β cells within pancreatic Langerhans islets (24Um S.H. D'Alessio D. Thomas G. Cell Metab. 2006; 3: 393-402Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 25Dann S.G. Selvaraj A. Thomas G. Trends Mol. Med. 2007; 13: 252-259Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Thus, in S6K1-/- mice, the insulin mass was diminished, which resulted in ineffective secretion of insulin upon glucose administration (21Pende M. Kozma S.C. Jaquet M. Oorschot V. Burcelin R. Le Marchand-Brustel Y. Klumperman J. Thorens B. Thomas G. Nature. 2000; 408: 994-997Crossref PubMed Scopus (377) Google Scholar, 23Ohanna M. Sobering A.K. Lapointe T. Lorenzo L. Praud C. Petroulakis E. Sonenberg N. Kelly P.A. Sotiropoulos A. Pende M. Nat. Cell Biol. 2005; 7: 286-294Crossref PubMed Scopus (318) Google Scholar). The activation of S6K1 proceeds through chronological phosphorylation at various residues, toward the crucial phosphorylation of Thr-389, present within the linker domain between the catalytic domain and the carboxyl tail, to obtain maximal enzymatic activity (26Pullen N. Thomas G. FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (484) Google Scholar). Interestingly, phosphorylation at several Ser/Thr residues within the C-terminal autoinhibitory tail appears to either activate or inhibit S6K1, depending on the cell cycle phase. Shah et al. (27Papst P.J. Sugiyama H. Nagasawa M. Lucas J.J. Maller J.L. Terada N. J. Biol. Chem. 1998; 273: 15077-15084Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) demonstrated that phosphorylation of those residues (featured by the Thr-421/Ser-424 site) during mitosis pursued by Cdk1 inactivates S6K1 to terminate protein synthesis prior to cell division (28Shah O.J. Ghosh S. Hunter T. J. Biol. Chem. 2003; 278: 16433-16442Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). A recent report by Schmidt et al. (29Schmidt T. Wahl P. Wüthrich R.P. Vogetseder A. Picard N. Kaissling B. Le Hir M. Histochem. Cell Biol. 2007; 127: 123-129Crossref PubMed Scopus (13) Google Scholar) demonstrating that phosphorylation of Thr-421/Ser-424 is specifically increased during the G2/M phase may also support the finding, whereas during the G1 phase, there is consensus that the phosphorylation at the autoinhibitory domain is requisite for S6K1 activation (26Pullen N. Thomas G. FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (484) Google Scholar), as also recently demonstrated by Hou et al. (30Hou Z. He L. Qi R.Z. J. Biol. Chem. 2007; 282: 6922-6928Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), where the Cdk5 phosphorylates the Ser-411 site, leading to activation of S6K1. In contrast to such inhibitory regulation of S6K1 during mitosis, however, a recent report by Boyer et al. (31Boyer D. Quintanilla R. Lee-Fruman K.K. Mol. Cell. Biochem. 2007; 307: 59-64Crossref PubMed Scopus (23) Google Scholar) sharply demonstrated that the activity of S6K1 peaks at mitosis, suggesting that S6K1 may also have some roles during the mitotic phase. If so, how its activity is supported against the inhibitory regulation caused by Cdk1 remains an open question. Hence, the two observations above that both DEDD-/- and S6K1-/- situations decrease the efficacy of ribosome and protein synthesis, resulting in smaller cell and body size, and that mitotic Cdk1 has a functional interaction with both S6K1 and DEDD led us here to assess a possible role of DEDD in the context of the functional regulation of S6K1. Mice—DEDD-/- mice (10Arai S. Miyake K. Voit R. Nemoto S. Wakeland E.K. Grummt I. Miyazaki T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2289-2294Crossref PubMed Scopus (25) Google Scholar) had been backcrossed to C57BL/6 (B6) for 17 generations before they were used in experiments. Mice were maintained under a specific pathogen-free condition. Antibodies—Antibodies used for experiments are as follows: anti-S6K1 phosphorylated at Thr-421/Ser-424, anti-S6K1 phosphorylated at Thr-389, anti-total rpS6 (clone 54D2), anti-rpS6 phosphorylated at Ser-240/244, anti-total Akt (clone 11E7), anti-Akt phosphorylated at Thr-308 (clone 244F9) (all are from Cell Signaling Technology, Beverly, MA); anti-S6K1 phosphorylated at Ser-411 (clone SC-7983R), anti-α-tubulin and anti-insulin (clone H-86) (from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-cyclin B1 (clone GNS-11) and anti-total S6K1 (clone 16) (from BD Biosciences); anti-Hsp90 (clone SPA-830) and anti-Cdk1 (clone A17) (from Stressgen (Victoria, Canada) and Zymed Laboratories Inc. (South San Francisco, CA)). S6K1 Kinase Assay—DEDD+/+ or DEDD-/- mouse embryonic fibroblast (MEF) cells were lysed in lysis buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm Na3VO4, 50 mm NaF, 5 mm EDTA, 0.1% Nonidet P-40, supplemented with a mixture of protease inhibitors). The cell lysates were clarified by centrifugation for 30 min at 14,000 rpm and immunoprecipitated with anti-S6K1 antibody preabsorbed to protein G-agarose beads at 4 °C for 4 h. The immune complexes were washed twice with lysis buffer and once with kinase assay buffer (20 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 0.1 mg/ml bovine serum albumin, and 0.4 mm dithiothreitol). The kinase reaction was performed at 30 °C for 15 min in the presence of 100 μm ATP, 200 μCi/ml [γ-32P]ATP, and 125 μm S6 peptide substrate (RRRLSSLRA; Upstate Biotechnology Inc., Lake Placid, NY) and was terminated by the addition of 20 μl of 250 mm EDTA and boiling for 5 min. Stopped reactions were loaded onto P-81 phosphocellulose membrane (Whatman, Maidstone, UK) and were washed with 75 mm phosphoric acid. The labeled probe was measured by liquid scintillation counting. siRNA Transfection—Double-stranded siRNA targeting DEDD or Cdk1 were purchased from Applied Biosystems or Sigma, respectively. Wild-type MEF cells at a 50% confluent state were transfected with 10 μm siRNA using Lipofectamine 2000 (Invitrogen). the cells were and by or of the were as follows: DEDD siRNA DEDD siRNA Cdk1 siRNA Cdk1 siRNA Cdk1 siRNA a an targeting the was In mouse or S6K1 were in or S6K1. These were to described in the or was in the cell (Invitrogen). In 200 of or 100 μl of cell lysates were or cell were with the anti-S6K1 or anti-cyclin B1 in lysis buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm MgCl2, 1 mm Na3VO4, mm NaF, 1 mm 0.1% Nonidet P-40, supplemented with a mixture of protease at 4 °C for 4 and with protein beads The were washed with lysis The and cell lysates were by using that the antibody was by us and that antibody is for of not of DEDD by and of islets from mice and of insulin within cells were as described N. K. Y. K. T. R. Y. K. R. H. S. H. S. M. S. R. S. Y. T. 2000; PubMed Scopus Google Scholar). the at a to its the Applied was the pancreatic The was and at °C for min. The was by and cells were washed were through a These islets were used for the experiments was measured as described N. K. Y. K. T. R. Y. K. R. H. S. H. S. M. S. R. S. Y. T. 2000; PubMed Scopus Google Scholar). islets were in at and with the glucose for 20 of 10 was performed at °C for 1 h. levels were with an insulin assay for used are as follows: and and and and and and and of of the was performed using phosphorylation levels to those in control as are at were was used to DEDD of S6K1 and DEDD is involved in regulation of S6K1 activity, we the of DEDD influences S6K1 levels of phosphorylation at Thr-389 of S6K1, a of S6K1, was in DEDD-/- with MEF cells that had been in the mitotic phase by a Interestingly, reduction in Thr-389 phosphorylation was also in DEDD-/- MEF cells Thus, such a DEDD activating effect on S6K1 at the mitotic phase appears to influence the overall S6K1 activity in a kinase activity assay on the of demonstrated that S6K1 from DEDD-/- MEF cells had 50% activity on a specific substrate of S6K1, rpS6, with that from cells by the amount of S6K1 a of This was also supported by a reduction in phosphorylation levels of rpS6 in DEDD-/- with MEF by The reduction of the rpS6 phosphorylation in the absence of DEDD was by the from the kinase activity This might be to a possible functional caused by in the phosphorylation of rpS6 J.R. T. L. O. S. Pende M. Blenis J. Thomas G. S. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). DEDD mitotic Cdk1 (10Arai S. Miyake K. Voit R. Nemoto S. Wakeland E.K. Grummt I. Miyazaki T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2289-2294Crossref PubMed Scopus (25) Google Scholar), increased Cdk1 activity in the absence of DEDD might the inhibitory regulation of S6K1, leading to S6K1 activity in DEDD-/- of Cdk1 siRNA increased Thr-389 phosphorylation in DEDD-/- cells the phosphorylation of the inhibitory of S6K1, Thr-421/Ser-424, which is by mitotic Cdk1. in phosphorylation at Thr-421/Ser-424 was enhanced in mitotic DEDD-/- MEF cells with MEF In in the absence of the activity of S6K1 was to at the inhibitory Ser/Thr The phosphorylation at Ser-411 within the autoinhibitory was also increased in DEDD-/- cells Although is with the by Shah et al. (28Shah O.J. Ghosh S. Hunter T. J. Biol. Chem. 2003; 278: 16433-16442Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) suggesting the presence of Cdk1-dependent inhibitory phosphorylation the mitotic phosphorylation at Ser-411 decreases S6K1 activity remains DEDD with S6K1 through DEDD associates with S6K1. DEDD in cells and S6K1 is with S6K1 was to DEDD S6K1 with DEDD was deficient in phosphorylation at both Thr-421/Ser-424 and whereas the Thr-389 was suggesting that the of DEDD with S6K1 inhibitory phosphorylation of S6K1 that is caused by mitotic Cdk1 in assay using supported the that DEDD associates with S6K1 through Cdk1-cyclin DEDD was with S6K1 in the presence of both Cdk1 and B1 in to the involved in the of DEDD with S6K1, we a number of DEDD and their with S6K1. in a DEDD the did not to S6K1, a of the of DEDD for the with S6K1. that in the of the DEDD was with that of in cells is possible that the DEDD may be in β Cell and to S6K1 in DEDD-/- is involved in control of glucose by the size of insulin-producing β cells in the (21Pende M. Kozma S.C. Jaquet M. Oorschot V. Burcelin R. Le Marchand-Brustel Y. Klumperman J. Thorens B. Thomas G. Nature. 2000; 408: 994-997Crossref PubMed Scopus (377) Google Scholar). in the of DEDD with S6K1 was in β cells using a mouse pancreatic β cell J. K. E. H. T. Y. Y. K. 127: PubMed Scopus Google Scholar). In with finding, activating phosphorylation of S6K1 at Thr-389 was significantly decreased in cells DEDD was by siRNA in of S6K1 phosphorylation at the Thr-389 was in the DEDD-/- with the the DEDD-/- also an increase in phosphorylation levels at Thr-421/Ser-424 and Ser-411 residues, as in MEF cells pancreatic islets were smaller in DEDD-/- mice in mice, as in S6K1-/- to the size of β cell was reduced in DEDD-/- mice two as also by a of β cells within the the insulin by the of β cells to mass of pancreatic for insulin, was reduced by 50% in DEDD-/- mice two and In with insulin within β cells from DEDD-/- mice was significantly decreased by in using pancreatic islets analysis demonstrated that the amount of insulin in the of DEDD-/- mice was decreased with that in the of the did not in DEDD-/- and suggesting influence of the of DEDD on apoptosis of cells not upon in DEDD-/- we insulin secretion and glucose in DEDD-/- mice in we the insulin levels in response to glucose administration mice were for 2 h. from the reduction in β cell size and insulin in insulin levels were at 2 or 20 min the glucose in DEDD-/- mice with mice insulin secretion in DEDD-/- mice resulted in glucose intolerance. in at the glucose glucose levels were in DEDD-/- in mice, they were in both of mice before glucose administration the of the glucose decreased to the in mice, it was twice as as that under the in DEDD-/- mice Thus, in S6K1-/- mice, DEDD-/- mice glucose to insulin secretion upon glucose which is for by a reduction in insulin mass in pancreatic we demonstrated that DEDD is for of S6K1 activity during mitosis and that reaction increases overall S6K1 activity in This may suggest that the maintenance of mitotic S6K1 activity by the of appears to be important to cell and body size in mammals. This is with the by Boyer et al. (31Boyer D. Quintanilla R. Lee-Fruman K.K. Mol. Cell. Biochem. 2007; 307: 59-64Crossref PubMed Scopus (23) Google Scholar), in which S6K1 activity the maximal levels in the mitotic phase. in (10Arai S. Miyake K. Voit R. Nemoto S. Wakeland E.K. Grummt I. Miyazaki T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2289-2294Crossref PubMed Scopus (25) Google Scholar), DEDD appears to be involved in cell growth control prior to cell division mechanisms of mitosis progression and maintenance of S6K1 both are achieved through of mitotic are to the mechanism of how DEDD mitotic Cdk1 such may the mitotic phase during the cell cycle as a crucial period involved in mammalian cell Although the were initially to be involved in apoptosis evidence has that they have L. M.J. Nat. 2003; 4: PubMed Scopus Google Scholar, and J. Cell. Mol. in Scholar). et al. A.F. S. M. Z. Y. R. J. 2007; PubMed Scopus (26) Google Scholar) that and also family roles in S6K1 activity during by in protein also a kinase that levels through the thereby promoting the S. D. R. S. K. J.L. O. EMBO PubMed Scopus (13) Google Scholar). with findings of the of DEDD in the mitotic phase, observations may that the to be involved in the control of the cascade as as in the progression of cell cycle the is crucial in the regulation of cell might the role of in various DEDD-/- mice glucose it might be to assess of DEDD is either in the body or in specific in a of type 2 diabetes et al. K. I. D. J. M. A. D. A. T. M. B. P.A. T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: PubMed Scopus Google Scholar) that the where are is involved in β cell Thus, might also influence homeostasis through apoptosis DEDD-/- cells or mice showed in apoptosis (10Arai S. Miyake K. Voit R. Nemoto S. Wakeland E.K. Grummt I. Miyazaki T. Proc. Natl. Acad. Sci. U. S. 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