Mdm2 phosphorylation by Akt regulates the p53 response to oxidative stress to promote cell proliferation and tumorigenesis
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We have shown previously that phosphorylation of Mdm2 by ATM and c-Abl regulates Mdm2-p53 signaling and alters the effects of DNA damage in mice, including bone marrow failure and tumorigenesis induced by ionizing radiation. Here, we examine the physiological effects of Mdm2 phosphorylation by Akt, another DNA damage effector kinase. Surprisingly, Akt phosphorylation of Mdm2 does not alter the p53-mediated effects of ionizing radiation in cells or mice but regulates the p53 response to oxidative stress. Akt phosphorylation of Mdm2 serine residue 183 increases nuclear Mdm2 stability, decreases p53 levels, and prevents senescence in primary cells exposed to reactive oxidative species (ROS). Using multiple mouse models of ROS-induced cancer, we show that Mdm2 phosphorylation by Akt reduces senescence to promote KrasG12D-driven lung cancers and carcinogen-induced papilloma and hepatocellular carcinomas. Collectively, we document a unique physiologic role for Akt-Mdm2-p53 signaling in regulating cell growth and tumorigenesis in response to oxidative stress.Akt is commonly overexpressed and activated in cancer cells and plays a pivotal role in cell survival, protection, and chemoresistance. Therefore, Akt is one of the target molecules in understanding characters of cancer cells and developing anticancer drugs. Here we examined whether a newly developed photo-activatable Akt (PA-Akt) probe, based on a light-inducible protein interaction module of plant cryptochrome2 (CRY2) and cryptochrome-interacting basic helixloophelix (CIB1), can regulate Akt-associated cell functions. By illuminating blue light to the cells stably transfected with PA-Akt probe, CRY2-Akt (a fusion protein of CRY2 and Akt) underwent a structural change and interacted with Myr-CIBN (myristoylated N-terminal portion of CIB1), anchoring it at the cell membrane. Western blot analysis revealed that S473 and T308 of the Akt of probe-Akt were sequentially phosphorylated by intermittent and continuous light illumination. Endogenous Akt and GSK-3, one of the main downstream signals of Akt, were also phosphorylated, depending on light intensity. These facts indicate that photo-activation of probe-Akt can activate endogenous Akt and its downstream signals. The photo-activated Akt conferred protection against nutritional deprivation and H 2 O 2 stresses to the cells significantly. Using the newly developed PA-Akt probe, endogenous Akt was activated easily, transiently, and repeatedly. This probe will be a unique tool in studying Akt-associated specific cellular functions in cancer cells and developing anticancer drugs.
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O-GlcNAcylation of proteins is a recently discovered post-translational modification of nuclear and cytoplasmic proteins. This modification is similar to protein phosphorylation rather than to classical protein glycosylation of membrane and secreted proteins. Both O-GlcNAcylation and phosphorylation modify the hydroxyl group of serine or threonine residues of tau, the effect of O-GlcNAcylation on phosphorylation of tau was studied. The level of O-GlcNAcylation in differenciated PC12 cells was modulated by changing the concentration of the donor of O-GlcNAcylation and activities of the key enzymes, then, the consequent changes of tau phosphorylation at various phosphorylation sites were examined by using Western blot developed with phosphorylation-dependent and site-specific tau antibodies. It was found that O-GlcNAcylation modulated phosphorylation of tau at many phosphorylation sites and in a site-specific manner. Increased protein O-GlcNAcylation induced a decrease in tau phosphorylation at most of phosphorylation sites, and vise versa. These results suggest that O-GlcNAcylation negatively modulates tau phosphorylation at most phosphorylation sites. Therefore, these studies provide novel insight into the regulation of tau phosphorylation and the molecular mechenism of abnormal hyperphosphorylation of tau in Alzheimer disease brain.
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AbstractRecent advances in the field of pharmacological activation of the p53 tumor suppressor are beginning to be translated into the clinic. In addition, small molecules that activate p53 through established mechanisms of action are proving invaluable tools for basic research. Here we analyze and compare the effects of nutlin-3, tenovin-6 and low doses of actinomycin-D on p53 and its main negative regulator, mdm2. We reveal striking differences in the speed at which these compounds increase p53 protein levels, with nutlin-3 having a substantial impact within minutes. We also show that nutlin-3 is very effective at increasing the synthesis of mdm2 mRNA, mdm2 being not only a modulator of p53 but also a transcriptional target. In addition, we show that nutlin-3 stabilizes mdm2’s conformation and protects mdm2 from degradation. These strong effects of nutlin-3 on mdm2 correlate with a remarkable rate of recovery of p53 levels upon removal of the compound. We discuss the potential application of our results as molecular signatures to assess the on-target effects of small-molecule mdm2 inhibitors. To conclude, we discuss the implications of our observations for using small-molecule p53 activators to reduce the growth of tumors retaining wild-type p53 or to protect normal tissues against the undesired side effects of conventional chemotherapy.
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The p53 inhibitor murine double-minute gene 2 (Mdm2) is a target for potential cancer therapies, however increased p53 function can be lethal. To directly address whether reduced Mdm2 function can inhibit tumorigenesis without causing detrimental side effects, we exploited a hypomorphic murine allele of mdm2 to compare the effects of decreased levels of Mdm2 and hence increased p53 activity on tumorigenesis and life span in mice. Here we report that mice with decreased levels of Mdm2 are resistant to tumor formation yet do not age prematurely, supporting the notion that Mdm2 is a promising target for cancer therapeutics.
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肿瘤 suppressor p53 在阻止肿瘤形成起一个中央作用。p53 的层次和活动在紧密的 regulationto 下面保证它的合适的功能。鼠科的双分钟 2 (MDM2 ) ， p53 目标基因，是 E3 ubiquitin ligase。MDM2 是 p53 蛋白质的一个 keynegative 管理者，并且与 p53 形成一个自动调整的反馈环。MDM2 是有两 p53-dependentand 的 oncogene p53 独立的 oncogenic 活动，并且经常在许多人的癌症增加了表示层次。ishighly 调整的 MDM2；MDM2 的层次和功能在 transcriptional 被调整，翻译并且 post-translationallevels。这评论提供 MDM2 的规定的概述。MDM2 的 Dysregulation 在 p53functions 之上显著地影响，并且接着 tumorigenesis。就 MDM2 在人的癌症起的关键作用而言，更好的 understandingof MDM2 的规定将帮助我们得新奇、更有效的癌症在房间指向 MDM2 andactivate p53 的治疗学的策略。
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Inactivation of the Arf-Mdm2-p53 tumor suppressor pathway is a necessary event for tumorigenesis. Arf controls Mdm2, which in turn regulates p53, but Arf and Mdm2 also have p53-independent functions that affect tumor development. Moreover, inhibition of oncogene-induced tumorigenesis relies on Arf and p53, but the requirements of Arf and p53 in tumor development initiated in the absence of overt oncogene overexpression and the role of Mdm2 in this process remain unclear. In a series of genetic experiments in mice with defined deficiencies in Arf, Mdm2 and/or p53, we show Mdm2 haploinsufficiency significantly delayed tumorigenesis in mice deficient in Arf and p53. Mdm2 heterozygosity significantly inhibited tumor development in the absence of Arf, and in contrast to Myc oncogene-driven cancer, this delay in tumorigenesis could not be rescued with the presence of one allele of Arf. Notably, Mdm2 haploinsufficieny blocked the accelerated tumor development in Arf deficient mice caused by p53 heterozygosity. However, tumorigenesis was not inhibited in Mdm2 heterozygous mice lacking both alleles of p53 regardless of Arf status. Surprisingly, loss of Arf accelerated tumor development in p53-null mice. Tumor spectrum was largely dictated by Arf and p53 status with Mdm2 haploinsufficiency only modestly altering the tumor type in some of the genotypes and not the number of primary tumors that arose. Therefore, the significant effects of Mdm2 haploinsufficiency on tumor latency were independent of Arf and required at least one allele of p53, and an Mdm2 deficiency had minor effects on the types of tumors that developed. These data also demonstrate that decreased levels of Mdm2 are protective in the presence of multiple genetic events in Arf and p53 genes that normally accelerate tumorigenesis.
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Akt plays a central role in the regulation of cellular anti-apoptosis underlying various human neoplastic diseases. We have demonstrated previously that TCL1 (a proto-oncogene underlying human T cell prolymphocytic leukemia) interacts with Akt and functions as an Akt kinase co-activator. With the aim to develop an Akt kinase inhibitor, we hypothesized that a peptide, which spans the Akt-binding site, binds to Akt and modulates Akt kinase activity and its downstream biological responses. Indeed, we demonstrated that a peptide, named "Akt-in" (Akt inhibitor, NH2-AVTDHPDRLWAWEKF-COOH, encompassing the βA strand of human TCL1), interacted with Akt and specifically inhibited its kinase activity. Nuclear magnetic resonance studies suggested that interaction of Akt-in with the pleckstrin homology domain (PH) of Akt caused conformational changes on the variable loop 1 of Akt, the locus mediating phosphoinositide binding. Consistently, interaction of Akt-in with the Akt PH domain prevented phosphoinositide binding and hence inhibited membrane translocation and activation of Akt. Moreover, Akt-in inhibited not only cellular proliferation and anti-apoptosis in vitro but also in vivo tumor growth without any adverse effect. The roles of Akt, which possesses a PH domain, in intracellular signaling were well established. Hence, Akt inhibitors create an attractive target for anticancer therapy. However, no effective inhibitors specific for Akt have been developed. Akt-in, which inhibits association of phosphatidylinositol with Akt, is the first molecule to demonstrate specific Akt kinase inhibition potency. This observation will facilitate the design of specific inhibitors for Akt, a core intracellular survival factor underlying various human neoplastic diseases. Akt plays a central role in the regulation of cellular anti-apoptosis underlying various human neoplastic diseases. We have demonstrated previously that TCL1 (a proto-oncogene underlying human T cell prolymphocytic leukemia) interacts with Akt and functions as an Akt kinase co-activator. With the aim to develop an Akt kinase inhibitor, we hypothesized that a peptide, which spans the Akt-binding site, binds to Akt and modulates Akt kinase activity and its downstream biological responses. Indeed, we demonstrated that a peptide, named "Akt-in" (Akt inhibitor, NH2-AVTDHPDRLWAWEKF-COOH, encompassing the βA strand of human TCL1), interacted with Akt and specifically inhibited its kinase activity. Nuclear magnetic resonance studies suggested that interaction of Akt-in with the pleckstrin homology domain (PH) of Akt caused conformational changes on the variable loop 1 of Akt, the locus mediating phosphoinositide binding. Consistently, interaction of Akt-in with the Akt PH domain prevented phosphoinositide binding and hence inhibited membrane translocation and activation of Akt. Moreover, Akt-in inhibited not only cellular proliferation and anti-apoptosis in vitro but also in vivo tumor growth without any adverse effect. The roles of Akt, which possesses a PH domain, in intracellular signaling were well established. Hence, Akt inhibitors create an attractive target for anticancer therapy. However, no effective inhibitors specific for Akt have been developed. Akt-in, which inhibits association of phosphatidylinositol with Akt, is the first molecule to demonstrate specific Akt kinase inhibition potency. This observation will facilitate the design of specific inhibitors for Akt, a core intracellular survival factor underlying various human neoplastic diseases. Akt (also known as protein kinase B), a central component of the phosphoinositide 3-kinase (PI3K) 1The abbreviations used are: PI3K, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol; PH, pleckstrin homology; GSK, glycogen synthesis kinase; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; PDGF, platelet-derived growth factor; GST, glutathione S-transferase; GFP, green fluorescent protein; TUNEL, terminal dUTP nick-end labeling; H & E, hematoxylin and eosin; FKHR, fork head transcription factor; HA, hemagglutinin; MAP, mitogen-activated protein; MAPK, MAP kinase; PDK, phosphoinositide-dependent protein kinase. signaling pathways, has emerged as a pivotal regulator of many cellular processes (1Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (882) Google Scholar, 2Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4770) Google Scholar, 3Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar, 4Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (973) Google Scholar). Three highly homologous Akt isoforms (Akt1, Akt2, and Akt3) exist in mammals. Akt is composed of three functionally distinct regions: an N-terminal pleckstrin homology (PH) domain, a central catalytic domain, and a C-terminal hydrophobic region. The PH domain is a small 100–120-residue module found in many proteins involved in cell signaling or cytoskeletal rearrangement. The PH domain of Akt is similar to other proteins, and it consists of seven β strands forming two orthogonal antiparallel β-sheets that are closed with the C-terminal α-helix (5Auguin D. Barthe P. Auge-Senegas M.T. Stern M.H. Noguchi M. Roumestand C. J. Biomol. NMR. 2004; 28: 137-155Crossref PubMed Scopus (46) Google Scholar, 6Yang J. Cron P. Thompson V. Good V.M. Hess D. Hemmings B.A. Barford D. Mol. Cell. 2002; 9: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 7Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (443) Google Scholar, 8Milburn C.C. Deak M. Kelly S.M. Price N.C. Alessi D.R. Van Aalten D.M. Biochem. J. 2003; 375: 531-538Crossref PubMed Scopus (234) Google Scholar, 9Thomas C.C. Deak M. Alessi D.R. van Aalten D.M. Curr. Biol. 2002; 12: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In response to growth factors and other extracellular stimuli, Akt is activated by the lipid products (PtdIns (3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2) of PI3K, which phosphorylates the 3-OH position of the inositol core of inositol phospholipids (PtdIns) (10Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar, 11Frech M. Andjelkovic M. Ingley E. Reddy K.K. Falck J.R. Hemmings B.A. J. Biol. Chem. 1997; 272: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 12James S.R. Downes C.P. Gigg R. Grove S.J. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (275) Google Scholar). Recent structural studies have located the binding pocket of PtdIns(1,3–5) P4 (the polar head group of PtdIns(3,4,5)P3) in variable loop 1 (VL1, the loop between the β1 and β2strands) of the Akt PH domain (5Auguin D. Barthe P. Auge-Senegas M.T. Stern M.H. Noguchi M. Roumestand C. J. Biomol. NMR. 2004; 28: 137-155Crossref PubMed Scopus (46) Google Scholar, 7Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (443) Google Scholar, 8Milburn C.C. Deak M. Kelly S.M. Price N.C. Alessi D.R. Van Aalten D.M. Biochem. J. 2003; 375: 531-538Crossref PubMed Scopus (234) Google Scholar, 13Ferguson K.M. Kavran J.M. Sankaran V.G. Fournier E. Isakoff S.J. Skolnik E.Y. Lemmon M.A. Mol. Cell. 2000; 6: 373-384Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). An association with PtdIns(3,4,5)P3 induces a conformational change of Akt, which allows phosphoinositide-dependent protein kinase 1 (PDK1) to access and phosphorylate threonine 308 (Thr-308) (14Lawlor M.A. Mora A. Ashby P.R. Williams M.R. Murray-Tait V. Malone L. Prescott A.R. Lucocq J.M. Alessi D.R. EMBO J. 2002; 21: 3728-3738Crossref PubMed Scopus (276) Google Scholar, 15Collins B.J. Deak M. Arthur J.S. Armit L.J. Alessi D.R. EMBO J. 2003; 22: 4202-4211Crossref PubMed Scopus (161) Google Scholar) in the so-called activation loop. It is also regulated by phosphorylation events within the conserved C-terminal hydrophobic motif (10Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar, 15Collins B.J. Deak M. Arthur J.S. Armit L.J. Alessi D.R. EMBO J. 2003; 22: 4202-4211Crossref PubMed Scopus (161) Google Scholar). Both serine 473 (Ser-473) phosphorylation and membrane anchoring are required for Thr-308 phosphorylation (16Scheid M.P. Woodgett J.R. FEBS Lett. 2003; 546: 108-112Crossref PubMed Scopus (348) Google Scholar) and complete activation of Akt (10Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar, 15Collins B.J. Deak M. Arthur J.S. Armit L.J. Alessi D.R. EMBO J. 2003; 22: 4202-4211Crossref PubMed Scopus (161) Google Scholar). Over 20 molecules have been identified as potential physiological substrates of Akt, including GSK3α (glycogen synthesis kinase3α), GSK3β, fork head transcription factor (FKHR), BAD, and endothelial nitric-oxide synthase (1Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (882) Google Scholar, 17Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3754) Google Scholar). Activation of Akt promotes cell survival (18Chen W.S. Xu P.Z. Gottlob K. Chen M.L. Sokol K. Shiyanova T. Roninson I. Weng W. Suzuki R. Tobe K. Kadowaki T. Hay N. Genes Dev. 2001; 15: 2203-2208Crossref PubMed Scopus (792) Google Scholar); thus, it could be the underlying mechanism for numerous human neoplastic diseases including lung, ovarian, and prostate cancers (1Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (882) Google Scholar, 19Luo J. Manning B.D. Cantley L.C. Cancer Cells. 2003; 4: 257-262Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar). Activation of Akt is also induced in the mutation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene. PTEN antagonizes PI3K function by the reduction in the levels of both PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Mutations of PTEN are implicated in several tumor types, including glioblastoma, endometrial tumors, and Cowden's syndrome (20Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Crossref PubMed Scopus (1768) Google Scholar, 21Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2146) Google Scholar). We have demonstrated that the proto-oncogene TCL1 is an Akt kinase co-activator (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 23Pekarsky Y. Koval A. Hallas C. Bichi R. Tresini M. Malstrom S. Russo G. Tsichlis P. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3028-3033Crossref PubMed Scopus (317) Google Scholar, 24Gold M.R. Trends Immunol. 2003; 24: 104-108Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 25Auguin D. Barthe P. Royer C. Stern M.H. Noguchi M. Arold S.T. Roumestand C. J. Biol. Chem. 2004; 279: 35890-35902Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). TCL1 contains two distinct functional motifs responsible for Akt association and homodimerization. Both Akt association and homodimerization of TCL1 are required for the complete function of TCL1 to enhance Akt kinase activity. TCL1 binds to Akt and activates Akt via a transphosphorylation reaction (26Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (84) Google Scholar, 27Laine J. Kunstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). TCL1 oncogene was first implicated in human T cell prolymphocytic leukemia, a chronic adulthood leukemia (28Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (83) Google Scholar). Under physiological conditions, TCL1 expression is limited to early developmental stages such as the immune system (24Gold M.R. Trends Immunol. 2003; 24: 104-108Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 28Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (83) Google Scholar, 29Narducci M.G. Fiorenza M.T. Kang S.M. Bevilacqua A. Di Giacomo M. Remotti D. Picchio M.C. Fidanza V. Cooper M.D. Croce C.M. Mangia F. Russo G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11712-11717Crossref PubMed Scopus (81) Google Scholar). Because the PI3K-Akt pathway is involved in various human neoplastic diseases, Akt represents an attractive target for drug development (19Luo J. Manning B.D. Cantley L.C. Cancer Cells. 2003; 4: 257-262Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 30Lock R.B. Int. J. Biochem. Cell Biol. 2003; 35: 1614-1618Crossref PubMed Scopus (11) Google Scholar). A small peptide was proven to effectively modulate activity of kinases effectively (31Barr R.K. Boehm I. Attwood P.V. Watt P.M. Bogoyevitch M.A. J. Biol. Chem. 2004; 279: 36327-36338Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 32Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 33Datta K. Sundberg C. Karumanchi S.A. Mukhopadhyay D. Cancer Res. 2001; 61: 1768-1775PubMed Google Scholar). One class of Akt inhibitors under development is based on the cross-reactivity between known kinase inhibitors (e.g. cyclic AMP-dependent protein kinase (PKA) or PI3K) (34Reuveni H. Livnah N. Geiger T. Klein S. Ohne O. Cohen I. Benhar M. Gellerman G. Levitzki A. Biochemistry. 2002; 41: 10304-10314Crossref PubMed Scopus (114) Google Scholar, 35Hu Y. Qiao L. Wang S. Rong S.B. Meuillet E.J. Berggren M. Gallegos A. Powis G. Kozikowski K. J. Med. Chem. 2000; 43: 3045-3051Crossref PubMed Scopus (199) Google Scholar, 36Niv M.Y. Rubin H. Cohen J. Tsirulnikov L. Licht T. Peretzman-Shemer A. Cna'an E. Tartakovsky A. Stein I. Albeck S. Weinstein I. Goldenberg-Furmanov M. Tobi D. Cohen E. Laster M. Ben-Sasson S.A. Reuveni H. J. Biol. Chem. 2003; 279: 1242-1255Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar); however, these drugs are not specific for Akt. With the goal to develop a putative Akt kinase inhibitor, we hypothesized that a peptide, which is spanning the Akt-binding site, binds to Akt and modulates Akt kinase activity along with its downstream biological responses. Based on the binding domain of TCL1 with Akt, we identified and characterized a peptide that encompassed the βA strand of TCL1, interacted with Akt, and inhibited Akt kinase activity. Akt-in prevented PtdIns binding to Akt, and consequently it inhibited membrane translocation of Akt and its downstream biological responses. Given the pivotal role of Akt kinase as a core intracellular survival factor implicated in the molecular mechanisms of human neoplastic diseases, the results could help to design Akt kinase-specific inhibitors for therapeutic approaches. Peptide Design—For the Akt-in peptides, the amino acid positions 10–24 of human TCL1, NH2-AVTDHPDRLWAWEKF-COOH are used. For TAT-FLAG Akt-in, the sequence is YGRKKRRQRRRDYKDDDDKAVTDHPDRLWAWEKF-COOH. Control Peptides—For βC peptides, the amino acid positions 29–40 of human TCL1 are used, NH2-EKQHAWLPLTIE-COOH. For TAT-βC, the sequence is H2-YGRKKRRQRRREKQHAWLPLTIE-COOH; and for TAT-FLAG, the sequence is NH2-YGRKKRRQRRR-DYKDDDDK-COOH. For functional assays, the Akt-in peptide was fused with TAT (YGRKKRRQRRR) (37Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (891) Google Scholar). The peptides were either purchased from Sigma Genosys and Applied Biosystems (SynthAssist®) or synthesized using FMOC (N-(9-fluorenyl)methoxycarbonyl)-protected amino acids and standard 1-benzotriazolyloxy-trisdemethylamino-phosphonium-hexafluorophosphate-N-hydroxybenzotriazole-coupling methods as reported previously (38Obata T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Co-immunoprecipitation Assay—Co-immunoprecipitation assays were performed as described previously (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Briefly, Akt1, Akt2, or Akt3 in pCMV6 was transfected into 293 cells (ATCC). The cells were then harvested, lysed, and pre-cleaned with protein G/A-agarose mixture (50% v/v, Pro-G/A, Amersham Biosciences). FLAG-Akt-in or control peptides (βC) at 400 μm were added to the cell lysates, incubated at 4 °C for 3 h, and incubated with Pro-G/A preconjugated with anti FLAG M2 antibody (Sigma). The resultant immune precipitants were washed and run on SDS-PAGE and immunoblotted with anti-HA antibody (3F10, Roche Applied Science). GST Pull-down Assay—293T cells (ATCC) were transfected with 10 μg of FLAG-tagged wild type Akt3, PH domain, or C-terminal Akt3 (27Laine J. Kunstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The cell lysates were immunoprecipitated with anti-FLAG antibody (FLAG M2, Sigma) bound to Pro-G/A (27Laine J. Kunstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Fifty ng of GST fusion proteins were incubated with 20 μl of immobilized Akt3, PH domain, or C-terminal Akt. The samples were run on SDS gels and immunoblotted with anti-GST antibody (Amersham Biosciences). The results were consistent in at least three independent experiments. GST fusion Akt-in was generated by subcloning with the corresponding nucleotide into pGEX4T-2 vectors (Amersham Biosciences). All nucleotide sequences were verified before the experiments. GST Competition Assay—Recombinant GST-Akt-in fusion protein was generated by pGEX 4T-2 Vector (Amersham Biosciences) using oligonucleotide pairs (5′-aattcgcagtcaccgaccacccggaccgcctgtgggcctgggagaagttctagg-3′). 0.1 μg of Akt (activated, Upstate Biotechnology, Inc.) was incubated with TAT-FLAG, TAT-Akt-in, or TAT-βC at the concentration of 0, 50, 100, or 250 μm in HEPES Binding Buffer (20 mm HEPES (pH 7.0), 150 mm NaCl, 0.5 μg/μl bovine serum albumin). 0.1 μg of GST-Akt-in was then added and incubated for an additional 20 min at 4 °C. Twenty μl of immobilized Akt beads (Cell Signaling) were added to the sample, washed five times with HEPES Binding Buffer in the presence of 0.1% Nonidet P-40, resolved onto an SDS gel, and immunoblotted by anti-Akt (Cell Signaling) or anti-GST (Amersham Biosciences) antibodies using ECL (Amersham Biosciences). Kinetics of Akt-in—The kinetics of Akt-in with the human Akt2-PH domain (amino acid 1–125 of human Akt2) was performed using the Applied Biosystems 8500 Affinity Chip Analyzer. Briefly, His fusion protein of human Akt2-PH domain was generated using pQE30 (Qiagen) by PCR. 1.25 pg of GST fusion proteins (Akt-in or wild type TCL1) were spotted onto the protein-A/G Affinity Chips preconjugated with anti-GST antibody (Sigma). Fifty μm of His-Akt2-PH domain was applied, and the dissociation constant was calculated by using data analysis software (Applied Biosystems). The values (mean ± S.D.) were calculated from the 80 measurements. In Vitro Akt Kinase Assay—In vitro Akt kinase assays were performed using the Akt kinase assay kit (Cell Signaling) (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Briefly, the immobilized Akt was incubated with 0, 200, or 400 μm of indicated peptides for 2 h, and then an in vitro kinase assay reaction was performed for 4 min at 30 °C. The samples were heat-denatured, separated on SDS-PAGE, and immunoblotted with anti-phospho-GSK or anti-Akt (Cell Signaling) using ECL (Amersham Biosciences). PKA Kinase Assay—In vitro PKA kinase assays were performed using Peptag (Promega) as described previously (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). The indicated concentrations of peptides (Akt-in or TAT-FLAG control peptide) were incubated with 25 ng of PKA with 100 ng of bovine serum albumin for 1 h in the presence or absence of 2 μm PKA inhibitor (Calbiochem catalog number 116805), followed by the kinase reaction for 20 min at 26 °C, and then separated on 0.8% TBE-agarose gel. In Vitro PDK1 Kinase Assay—Purified baculovirus-derived recombinant His-PDK-1-WT (1.0 μg/reaction (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar)) was incubated with or without the indicated concentrations of peptides (Akt-in or TAT-FLAG) in 20 μl of reaction mixture containing 20 mm HEPES-NaOH (pH 7.4), 100 mm NaCl, 10 mm MgCl2, 0.5 mm EGTA, 1 mm dithiothreitol, 100 μm cold ATP, and 3 μCi of [γ-32P]ATP for 10 min at 30 °C. The reactions were analyzed by SDS-PAGE with Coomassie staining followed by autoradiography (shown in Fig. 3D). Phosphorylation of Akt, BAD, FKHR, or p44/42 MAP Kinase in 293 Cells—293 cells (ATCC) were cultured in a 60-mm dish and transfected (or nontransfected in Fig. 3B) with 5 μg of m-BAD (Fig. 3C, pEBGmBad, Cell Signaling) using calcium phosphate transfection as described previously (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). 24 h after transfection, the cells were serum-starved (0.2% fetal bovine serum) and treated with either control (TAT-FLAG) or Akt-in (TAT-Akt-in) at 50 μm for additional 12 h. The cells were stimulated with or without 20 ng/ml PDGF for 8 (in Fig. 3C) or 5 min (in Fig. 3B) and lysed with Brij lysis buffer in the presence of phosphatase inhibitors (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), and the resultant samples were resolved on 4–20% SDS gel (Kyoto Daiichi Kagaku Co., Ltd.). They were then immunoblotted with the indicated antibodies purchased from Cell Signaling (Akt, 9272, phospho-Ser-473 Akt 9271, phospho-Ser-308 Akt 9275, Ser-256 FKHR 9461, FKHR 9462, phospho-Ser-136 BAD 9295, BAD 9292, anti-p44/42 MAP kinase 9102, anti-phospho-p44/42 MAP kinase 9106, anti-38 MAP kinase 9212, and phospho-p38 MAP kinase antibody 9216), and detected by ECL (Amersham Biosciences). NMR Experiment—NMR spectra were recorded on 0.25–0.3-ml (Shigemi tubes pre-coated with a silicon solution (Sigma)) samples of 0.05 mm15N-labeled Akt2-PH dissolved in the conditioning buffer (10 mm Tris/H2O (pH 7.4), 300 mm NaCl, 0.1 mm benzamidine, 0.1 mm EDTA, with 5–10% 2H2O for the lock), in the presence or absence of 20 mmAkt-in. NMR experiments were carried out at 10 °C on a Bruker AVANCE 600 spectrometer equipped with 5-mm z-shielded gradient 1H-13C-15N triple resonance cryogenic probe. 1H chemical shifts were directly referenced to the resonance of 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt, and 15N chemical shifts were indirectly referenced with the absolute frequency ratios (15N/1H) = 0.101329118. In all experiments, the 1H carrier was centered on the water resonance, and a WATERGATE sequence was incorporated to suppress the solvent resonance. All NMR spectra were acquired in the phase-sensitive mode with Digital Quadrature Detection in the F2 dimension and hypercomplex States-TPPI method in F1 dimension and processed using Gifa (version 4.22) software. 1H,15N-HSQC spectra were recorded using a time domain data size of 64 t1 × 1K t2 complex points and 32 transients per complex t1 increment. PtdIns(3,4,5)P3 Lipid-Protein Pull-down Assay—A lipid-protein pull-down assay was performed using PIP Beads (PtdIns(3,4,5)P3, Echelon Bioscience Inc.). Indicated peptides (Akt-in or βC control) were incubated with 50 ng of Akt kinase (unactivated, Upstate Biotechnology Inc., catalog number 14-279) with 400 ng/ml bovine serum albumin for 2 h with gentle agitation at 4 °C. TAT-FLAG control was added to adjust the final peptide concentration to be equal throughout the samples. 25 μl of PIP Beads were then added to each sample and incubated for an additional 16 h. The reactions were then washed four times with washing buffer (10 mm HEPES (pH 7.4), 0.25% Nonidet P-40, 140 mm NaCl), resolved on SDS gel, and immunoblotted by ECL (Amersham Biosciences). Membrane Translocation Experiment—293 cells (ATCC) were grown on a poly-l-lysine-coated cover glass and were transfected with 1 μg of HA-Akt1 or Akt-PH-GFP or Btk (Bruton tyrosine kinase)-PH-GFP in a mammalian expression vector (39Varnai P. Rother K.I. Balla T. J. Biol. Chem. 1999; 274: 10983-10989Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) using FuGENE 6 (Roche Applied Science). Six hours after the transfection, 50 μmAkt-in or TAT-FLAG control was added and incubated for 16 h. The cells were then serum-starved (0.5%) and then incubated for an additional 24 h. The cells were treated with or without 50 nm wortmannin for 20 min, stimulated with 50 ng/ml PDGF-AB (Sigma, 3226) for 10 min, fixed with 4% paraformaldehyde, stained with 10 ng/ml fluorescein isothiocyanate-conjugated anti-HA antibody (12CA5, MBL) or phospho-Ser-473 antibody (587F11, Cell Signaling), and examined using a confocal microscope (Nikon). Proliferation Assay—Cell growth was assessed by a colorimetric method using WST-8 regent (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) (347-07621, Dojin) according to the manufacturer's instructions. Briefly, 2000 T4 cells were seeded into each well of 96-well plate. The peptides (TAT-βC or TAT-Akt-in) were added at the indicated concentrations. Three days later, WST-8 regent was added and incubated for an additional 4 h at 37 °C, and the absorbance was determined using a microplate reader (Bio-Rad). Cell Death Assay and Mitochondrial Permeability Transition Assay—T4 cells (human T cell leukemia cells) were treated with the indicated concentrations of peptide (TAT-FLAG control or TAT-Akt-in) for 24 h. The cells were transfected with either myr-Akt (Upstate Biotechnology, Inc., catalog number 17-253) or a control. Cell death was assessed by staining with 2 μg/ml propidium iodide. Mitochondrial permeability transition was verified by staining with rhodamine 123 (Molecular Probe) at 5 μm for 15 min at 37 °C (26Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (84) Google Scholar) and analyzed using fluorescence-activated cell sorter (Cell Quest). In Vivo Tumor Growth—Fibrosarcoma cells (QRsP-11 cells, 2 × 105 cells per mouse) were subcutaneously transplanted into syngeneic C57BL/6 mice (eight mice in each group) (40Okada F. Hosokawa M. Hamada J.I. Hasegawa J. Kato M. Mizutani M. Ren J. Takeichi N. Kobayashi H. Br. J. Cancer. 1992; 66: 635-639Crossref PubMed Scopus (62) Google Scholar). Two micromoles of the indicated peptides (TAT-Akt-in, TAT-FLAG, or phosphate-buffered saline) per mouse were injected directly into the tumor on days 5, 7, 10, 12, 14, 17, and 19. In vivo cell growth was calculated based on the diameter of the tumor. On day 9 after transplantation, the tumors were resected, fixed in formalin, embedded in paraffin, stained with hematoxylin and eosin (H & E), TUNEL (terminal dUTP nick-end labeling, MK500, Takara Shuzo), or phospho-Akt (Ser-473) monoclonal antibody (587F11, Cell Signaling). Peptide Design of Akt-in and the Structure of TCL1—TCL1 forms a closed symmetrical β-barrel structure, consisting of eight antiparallel β strands (41Hoh F. Yang Y.S. Guignard L. Padilla A. Stern M.H. Lhoste J.M. van Tilbeurgh H. Structure (Lond.). 1998; 6: 147-155Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) (Fig. 1A). In our previous studies, we showed that the surface composed of βA and βE strands of TCL1 mediated the interaction with Akt (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 26Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (84) Google Scholar, 42French S.W. Shen R.R. Koh P.J. Malone C.S. Mallick P. Teitell M.A. Biochemistry. 2002; 41: 6376-6382Crossref PubMed Scopus (34) Google Scholar) (Fig. 1A, top surface). Both dimerization and Akt interaction are essential for the full function of TCL1 to activate Akt (26Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (84) Google Scholar). We hypothesized that a peptide, which spans the Akt-binding sequences, can modulate Akt kinase activity and its downstream signals. We designed a peptide (named Akt-in, Akt inhibitor, positions 10–24 of human TCL1, NH2-AVTDHPDRLWAWEKF-COOH), which encompasses the βA strand of TCL1 for further study (Fig. 1B). For functional assays, the Akt-in peptide (amino acid positions 10–24 of TCL1, Fig. 1B) was fused with TAT (YGRKKRRQRRR) and/or FLAG epitope (DYKDDDDK).
Pleckstrin homology domain
LY294002
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