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1.
Res Pract Thromb Haemost ; 5(5): e12530, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34263099

ABSTRACT

BACKGROUND: Traumatic injury and the associated acute bleeding are leading causes of death in people aged 1 to 44 years. Acute bleeding in pathological and surgical settings also represents a significant burden to the society. Yet there are no approved hemostatic drugs currently available. While clinically proven as an effective pro-coagulant, activated factor VII (FVIIa) use in acute bleeding has been hampered by unwanted thromboembolic events. Enhancing the ability of FVIIa to quickly stop a bleed and clear rapidly from circulation may yield an ideal molecule suitable for use in patients with acute bleeding. OBJECTIVES: To address this need and the current liability of FVIIa, we produced a novel FVIIa molecule (CT-001) with enhanced potency and shortened plasma residence time by cell line engineering and FVIIa protein engineering for superior efficacy for acute bleeding and safety. METHODS: To address safety, CT-001, a FVIIa protein with 4 desialylated N-glycans was generated to promote active recognition and clearance via the asialoglycoprotein receptor. To enhance potency, the gamma-carboxylated domain was modified with P10Q and K32E, which enhanced membrane binding. RESULTS: Together, these changes significantly enhanced potency and clearance while retaining the ability to interact with the key hemostatic checkpoint proteins antithrombin and tissue factor pathway inhibitor. CONCLUSIONS: These results demonstrate that a FVIIa molecule engineered to combine supra-physiological activity and shorter duration of action has the potential to overcome the current limitations of recombinant FVIIa to be a safe and effective approach to the treatment of acute bleeding.

8.
Chem Biol Drug Des ; 74(1): 43-50, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19519743

ABSTRACT

Heat-shock protein-90 is an attractive target for anticancer drugs, as heat-shock protein-90 blockers such as the ansamycin 17-(allylamino)-17-demethoxygeldanamycin greatly reduce the expression of many signaling molecules that are disregulated in cancer cells and are key drivers of tumor growth and metastasis. While 17-(allylamino)-17-demethoxygeldanamycin has shown promise in clinical trials, this compound class has significant template-related drawbacks. In this paper, we describe a new, potent non-ansamycin small-molecule inhibitor of heat-shock protein-90, BX-2819, containing resorcinol and triazolothione rings. Structural studies demonstrate binding of BX-2819 to the ADP/ATP-binding pocket of heat-shock protein-90. The compound blocked expression of heat-shock protein-90 client proteins in cancer cell lines and inhibited cell growth with a potency similar to 17-(allylamino)-17-demethoxygeldanamycin. In a panel of four cancer cell lines, BX-2819 blocked growth with an average IC(50) value of 32 nM (range of 7-72 nM). Efficacy studies demonstrated that treatment with BX-2819 significantly inhibited the growth of NCI-N87 and HT-29 tumors in nude mice, consistent with pharmacodynamic studies showing inhibition of heat-shock protein-90 client protein expression in tumors for greater than 16 h after dosing. These data support further studies to assess the potential of BX-2819 and related analogs for the treatment of cancer.


Subject(s)
HSP90 Heat-Shock Proteins/antagonists & inhibitors , Triazoles/pharmacology , Animals , Benzoquinones/chemistry , Benzoquinones/pharmacology , Cell Line, Tumor , Computer Simulation , Crystallography, X-Ray , Drug Screening Assays, Antitumor , HSP90 Heat-Shock Proteins/metabolism , HT29 Cells , Humans , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/pharmacology , Mice , Mice, Nude , Transplantation, Heterologous , Triazoles/chemistry , Xenograft Model Antitumor Assays
9.
Bioorg Med Chem Lett ; 17(14): 3819-25, 2007 Jul 15.
Article in English | MEDLINE | ID: mdl-17544272

ABSTRACT

Based on the lead compound BX-517, a series of C-4' substituted indolinones have been synthesized and evaluated for PDK1 inhibition. Modification at C-4' of the pyrrole afforded potent compounds (7b and 7d) with improved solubility and ADME properties. In this letter, we describe the synthesis, selectivity profile, and pharmacokinetic data of selected compounds.


Subject(s)
Indoles/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Urea/analogs & derivatives , 3-Phosphoinositide-Dependent Protein Kinases , Cell Line, Tumor , Humans , Indoles/pharmacology , Protein Kinase Inhibitors/chemistry , Urea/chemistry , Urea/pharmacology
11.
J Biol Chem ; 280(20): 19867-74, 2005 May 20.
Article in English | MEDLINE | ID: mdl-15772071

ABSTRACT

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.


Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , 3-Phosphoinositide-Dependent Protein Kinases , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Catalytic Domain , Cell Division/drug effects , Cell Line, Tumor , Drug Evaluation, Preclinical , Female , HeLa Cells , Humans , In Vitro Techniques , Kinetics , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Melanoma, Experimental/drug therapy , Melanoma, Experimental/pathology , Melanoma, Experimental/secondary , Mice , Mice, Nude , Models, Molecular , Molecular Structure , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Pyrimidines/chemistry , Pyrimidines/pharmacology , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
12.
J Biol Chem ; 279(50): 52175-82, 2004 Dec 10.
Article in English | MEDLINE | ID: mdl-15469940

ABSTRACT

The tumor suppressor p53 is important in the decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 posttranslational modifications and association with other proteins have been implicated in the regulation of its stability and transactivation activity. Here we show that p53 is phosphorylated by the mitotic kinase Aurora-A at serine 215. Unlike most identified phosphorylation sites of p53 that positively associate with p53 function (Brooks, C. L., and Gu, W. (2003) Curr. Opin. Cell Biol. 15, 164-171), the phosphorylation of p53 by Aurora-A at Ser-215 abrogates p53 DNA binding and transactivation activity. Downstream target genes of p53, such as p21Cip/WAF1 and PTEN, were inhibited by Aurora-A in a Ser-215 phosphorylation-dependent manner (i.e. phosphomimic p53-S215D lost and non-phosphorylatable p53-S215A retained normal p53 function). As a result, Aurora-A overrides the apoptosis and cell cycle arrest induced by cisplatin and gamma-irradiation, respectively. However, the effect of Aurora-A on p53 DNA binding and transactivation activity was not affected by phosphorylation of Ser-315, a recently identified Aurora-A phosphorylation site of p53 (Katayama, H., Sasai, K., Kawai, H., Yuan, Z. M., Bondaruk, J., Suzuki, F., Fujii, S., Arlinghaus, R. B., Czerniak, B. A., and Sen, S. (2004) Nat. Genet. 36, 55-62). Our data indicate that phosphorylation of p53 at Ser-215 by Aurora-A is a major mechanism to inactivate p53 and can provide a molecular insight for Aurora-A function.


Subject(s)
DNA/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Aurora Kinases , Cell Line , DNA/genetics , Genes, p53 , Humans , In Vitro Techniques , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine/chemistry , Transcriptional Activation , Tumor Suppressor Protein p53/genetics
13.
Cancer Res ; 64(13): 4394-9, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15231645

ABSTRACT

Accumulated studies have shown that activation of the Akt pathway plays a pivotal role in malignant transformation and chemoresistance by inducing cell survival, growth, migration, and angiogenesis. Therefore, Akt is believed to be a critical target for cancer intervention. Here, we report the discovery of a small molecule Akt pathway inhibitor, Akt/protein kinase B signaling inhibitor-2 (API-2), by screening the National Cancer Institute Diversity Set. API-2 suppressed the kinase activity and phosphorylation level of Akt. The inhibition of Akt kinase resulted in suppression of cell growth and induction of apoptosis in human cancer cells that harbor constitutively activated Akt due to overexpression of Akt or other genetic alterations such as PTEN mutation. API-2 is highly selective for Akt and does not inhibit the activation of phosphatidylinositol 3'-kinase, phosphoinositide-dependent kinase-1, protein kinase C, serum- and glucocorticoid-inducible kinase, protein kinase A, signal transducer and activators of transcription 3, extracellular signal-regulated kinase-1/2, or c-Jun NH(2)-terminal kinase. Furthermore, API-2 potently inhibited tumor growth in nude mice of human cancer cells in which Akt is aberrantly expressed/activated but not of those cancer cells in which it is not. These findings provide strong evidence for pharmacologically targeting Akt for anticancer drug discovery.


Subject(s)
Enzyme Inhibitors/pharmacology , Nuclear Proteins , Nucleosides/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pyridazines/pharmacology , Animals , Apoptosis/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Female , Humans , Immediate-Early Proteins , JNK Mitogen-Activated Protein Kinases , MAP Kinase Signaling System/drug effects , Mice , Mice, Nude , Mitogen-Activated Protein Kinases/antagonists & inhibitors , NIH 3T3 Cells , Protein Kinase C/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-akt , Signal Transduction/drug effects , Signal Transduction/physiology , Substrate Specificity , Xenograft Model Antitumor Assays , p38 Mitogen-Activated Protein Kinases
14.
Cancer Res ; 64(2): 463-7, 2004 Jan 15.
Article in English | MEDLINE | ID: mdl-14744757

ABSTRACT

Aurora-A kinase is frequently overexpressed/activated in human ovarian and breast cancers. A rat mammary tumor model study indicates that alterations of Aurora-A are early events during mammary tumor development (T. M. Goepfert et al., Cancer Res., 62: 4115-4122, 2002), suggesting that Aurora-A plays a pivotal role in transformation. However, the molecular mechanism by which Aurora-A induces ovarian and breast cell transformation remains elusive. Here we show that ectopic expression of Aurora-A induces telomerase activity in human ovarian and breast epithelial cell lines HIOSE118 and MCF-10A. The mRNA and promoter activities of human telomerase reverse transcriptase (hTERT) are stimulated by Aurora-A. Furthermore, we have demonstrated that the c-Myc binding sites of hTERT promoter are required for Aurora-A-induced hTERT promoter activity. Ectopic expression of Aurora-A up-regulates c-Myc. Knockdown of c-Myc by RNA interference attenuates Aurora-A-stimulated hTERT expression and telomerase activity. To our knowledge, these findings demonstrate, for the first time, that Aurora-A induces telomerase activity and hTERT by up-regulation of c-Myc and provides an additional mechanism for the role of Aurora-A in malignant transformation in addition to its cell cycle control.


Subject(s)
Ovary/physiology , Protein Kinases/metabolism , Proto-Oncogene Proteins c-myc/genetics , Telomerase/genetics , Telomerase/metabolism , Aurora Kinase A , Aurora Kinases , Breast/cytology , Breast/enzymology , Breast/physiology , Cell Cycle Proteins , Cell Line , DNA Primers , DNA-Binding Proteins , Epithelial Cells/enzymology , Epithelial Cells/physiology , Female , Gene Expression Regulation , Humans , Kinetics , Ovary/enzymology , Promoter Regions, Genetic , Protein Serine-Threonine Kinases , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Xenopus Proteins
15.
J Biol Chem ; 279(7): 5405-12, 2004 Feb 13.
Article in English | MEDLINE | ID: mdl-14645242

ABSTRACT

Akt negatively regulates apoptotic pathways at a premitochondrial level through phosphorylation and modulation of proteins such as Bad, Forkhead proteins, and GSK-3beta. Akt has also been shown to protect cell death at a post-mitochondrial level, although its downstream targets have not been well documented. Here, we demonstrate that Akt, including AKT1 and AKT2, interacts with and phosphorylates X-linked inhibitor of apoptosis protein (XIAP) at residue serine-87 in vitro and in vivo. Phosphorylation of XIAP by Akt protects XIAP from ubiquitination and degradation in response to cisplatin. Moreover, autoubiquitination of XIAP is also inhibited by Akt. Consistent with this, an XIAP mutant introduced into cells which mimics the Akt-phosphorylated form (i.e. XIAP-S87D) displays reduced ubiquitination and degradation as compared with wild type XIAP. The greater stability of XIAP-S87D in cells translated to increased cell survival after cisplatin treatment. Conversely, a mutant that could not be phosphorylated by Akt (XIAP-S87A) was more rapidly degraded and showed increased cisplatin-induced apoptosis. Furthermore, suppression of XIAP by either siRNA or adenovirus of antisense of XIAP induced programmed cell death and inhibited Akt-stimulated cell survival in ovarian cancer cells. These data identify XIAP as a new downstream target of Akt and a potentially important mediator of the effect of Akt on cell survival.


Subject(s)
Protein Serine-Threonine Kinases , Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Animals , Apoptosis , Blotting, Western , COS Cells , Caspase 3 , Caspases/metabolism , Cell Line , Cell Line, Tumor , Cell Survival , Cisplatin/pharmacology , Cysteine Endopeptidases/metabolism , Dose-Response Relationship, Drug , Glutathione Transferase/metabolism , Humans , Immunoblotting , In Situ Nick-End Labeling , Mice , Multienzyme Complexes/metabolism , Mutation , Oligonucleotides, Antisense/pharmacology , Phosphorylation , Plasmids/metabolism , Precipitin Tests , Proteasome Endopeptidase Complex , Protein Biosynthesis , Proteins/metabolism , Proto-Oncogene Proteins c-akt , RNA, Small Interfering/metabolism , Rats , Recombinant Fusion Proteins/metabolism , Serine/chemistry , Transcription, Genetic , Ubiquitin/metabolism , X-Linked Inhibitor of Apoptosis Protein
16.
J Biol Chem ; 278(44): 42992-3000, 2003 Oct 31.
Article in English | MEDLINE | ID: mdl-12933816

ABSTRACT

Recent studies have demonstrated that the cell growth and antiapoptotic actions of androgen could be dissociated from the transcriptional activity of the receptor and were, instead, mediated by activation of a mitogen-activated protein kinase pathway. This finding suggests an important cellular function of androgen receptor (AR) outside the nucleus. In this report, we demonstrate that androgen activates phosphatidylinositol 3-kinase (PI3K) and Akt, including AKT1 and AKT2, in AR-positive cells. Androgen-induced cell growth and survival were inhibited by PI3K inhibitor and dominant-negative Akt. AR interacts with the p85alpha regulatory subunit of PI3K, and its binding affinity is increased after androgen stimulation. The sites of interaction on the two proteins were mapped to the C-terminal Src-homology 2 domain of p85alpha and N terminus of AR. Activation of PI3K/Akt by androgen was inhibited by dominant-negative Src. Neither N-terminal truncated nor proline-rich region-deleted AR mutants, which are unable to bind to p85alpha and Src, respectively, was able to mediate androgen-induced PI3K/Akt activation. AR with deletion of C-terminal region including ligand binding domain, however, retains the ability to activate PI3K/Akt upon androgen stimulation, which supports the notion that nongenomic function of androgen is mediated by its interaction with membrane receptors (1, 3, 4). These findings indicate that a triple complex between AR, p85alpha, and Src is required for androgen-stimulated PI3K/Akt activation, and that the PI3K/Akt pathway, in addition to mitogen-activated protein kinase, mediates androgen-induced cell growth and cell survival.


Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Receptors, Androgen/metabolism , src-Family Kinases/metabolism , Apoptosis , Blotting, Western , Cell Division , Cell Line , Cell Line, Tumor , Cell Survival , Enzyme Activation , Glutathione Transferase/metabolism , Humans , Ligands , MAP Kinase Signaling System , Models, Biological , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary
17.
J Biol Chem ; 278(26): 23432-40, 2003 Jun 27.
Article in English | MEDLINE | ID: mdl-12697749

ABSTRACT

Cisplatin and its analogues have been widely used for treatment of human cancer. However, most patients eventually develop resistance to treatment through a mechanism that remains obscure. Previously, we found that AKT2 is frequently overexpressed and/or activated in human ovarian and breast cancers. Here we demonstrate that constitutively active AKT2 renders cisplatin-sensitive A2780S ovarian cancer cells resistant to cisplatin, whereas phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 sensitizes A2780S and cisplatin-resistant A2780CP cells to cisplatin-induced apoptosis through regulation of the ASK1/JNK/p38 pathway. AKT2 interacts with and phosphorylates ASK1 at Ser-83 resulting in inhibition of its kinase activity. Accordingly, activated AKT2 blocked signaling down-stream of ASK1, including activation of JNK and p38 and the conversion of Bax to its active conformation. Expression of nonphosphorylatable ASK1-S83A overrode the AKT2-inhibited JNK/p38 activity and Bax conformational changes, whereas phosphomimic ASK1-S83D inhibited the effects of cisplatin on JNK/p38 and Bax. Cisplatin-induced Bax conformation change was inhibited by inhibitors or dominant negative forms of JNK and p38. In conclusion, our data indicate that AKT2 inhibits cisplatin-induced JNK/p38 and Bax activation through phosphorylation of ASK1 and thus, plays an important role in chemoresistance. Further, regulation of the ASK1/JNK/p38/Bax pathway by AKT2 provides a new mechanism contributing to its antiapoptotic effects.


Subject(s)
Cisplatin/pharmacology , Drug Resistance, Neoplasm , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/physiology , Apoptosis/drug effects , Humans , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase Kinase 5 , MAP Kinase Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation/drug effects , Protein Conformation , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/drug effects , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Signal Transduction/drug effects , Tumor Cells, Cultured , bcl-2-Associated X Protein , p38 Mitogen-Activated Protein Kinases
18.
J Biol Chem ; 277(38): 35364-70, 2002 Sep 20.
Article in English | MEDLINE | ID: mdl-12167664

ABSTRACT

Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Here we show that insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. We further demonstrate that Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27(kip1), thereby promoting CDK2 activity and cellular proliferation. Our results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function.


Subject(s)
Genes, Tumor Suppressor , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/genetics , Proto-Oncogene Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Amino Acid Sequence , Animals , Blood , Cell Line , Humans , Hydrolysis , Insulin/metabolism , Insulin-Like Growth Factor I/metabolism , Phosphorylation , Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Substrate Specificity , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins
19.
J Biol Chem ; 277(33): 29973-82, 2002 Aug 16.
Article in English | MEDLINE | ID: mdl-12048203

ABSTRACT

Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor alpha (TNFalpha) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNF alpha-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNF alpha-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates I kappa B kinase alpha. The phosphorylation of I kappa B kinase alpha and activation of NF kappa B mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNF alpha-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFalpha -induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NF kappa B pathway.


Subject(s)
Mitogen-Activated Protein Kinases/antagonists & inhibitors , NF-kappa B/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Necrosis Factor-alpha/physiology , Apoptosis , Cell Line , Epithelial Cells/metabolism , Heat-Shock Response , Humans , I-kappa B Kinase , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , Osmolar Concentration , Phosphorylation , Precipitin Tests , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins c-akt , Ultraviolet Rays , p38 Mitogen-Activated Protein Kinases
20.
J Biol Chem ; 277(26): 23230-5, 2002 Jun 28.
Article in English | MEDLINE | ID: mdl-11948187

ABSTRACT

Akt2 is a member of the Akt/PKB family, which is involved in a variety of cellular events including cell survival, proliferation, and differentiation. During skeletal muscle differentiation, the Akt2 but not Akt1 expression was significantly increased. Microinjection of anti-Akt2 but not anti-Akt1 antibody efficiently abrogated myogenesis, indicating that Akt2 plays a specific role in muscle differentiation. It has been well documented that ectopic expression of MyoD is sufficient to induce muscle differentiation in myoblasts. However, the mechanism of induction of Akt2 during muscle differentiation and the significance of Akt2 protein in MyoD-induced myogenesis are largely unknown. In this study, we provide direct evidence that Akt2 is transcriptionally regulated by MyoD and activates MyoD-myocyte enhancer binding factor-2 (MEF2) transactivation activity. The Akt2 promoter was isolated and found to contain nine putative E-boxes (CANNTG), which are putative MyoD binding sites. Electrophoretic mobility shift analyses revealed that MyoD bound to eight of the sites. The expression of MyoD significantly enhanced Akt2 promoter activity and up-regulated Akt2 mRNA and protein levels. Moreover, Akt2 but not Akt1 was activated during differentiation. The expression of Akt2 activated MyoD-MEF2 transcriptional activity and induced myogenin expression. These data indicate that there is a positive feedback regulation loop between Akt2 and MyoD-MEF2 during muscle differentiation, which is essential for MyoD-induced myogenesis.


Subject(s)
Muscles/embryology , MyoD Protein/physiology , Promoter Regions, Genetic , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/genetics , Base Sequence , Binding Sites , Cell Differentiation , Cells, Cultured , Cloning, Molecular , DNA-Binding Proteins/physiology , Feedback , Humans , MEF2 Transcription Factors , Molecular Sequence Data , Myogenic Regulatory Factors , Myogenin/genetics , Proto-Oncogene Proteins c-akt , Transcription Factors/physiology , Transcriptional Activation
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