Debby Hynx

PKBα/Akt1 Acts Downstream of DNA-PK in the DNA Double-Strand Break Response and Promotes Survival

Protein kinase B (PKB/Akt) is a well-established regulator of several essential cellular processes. Here, we report a route by which activated PKB promotes survival in response to DNA insults in vivo. PKB activation following DNA damage requires 3-phosphoinositide-dependent kinase 1 (PDK1) and DNA-dependent protein kinase (DNA-PK). Active PKB localizes in the nucleus of gamma-irradiated cells adjacent to DNA double-strand breaks, where it colocalizes and interacts with DNA-PK. Levels of active PKB inversely correlate with DNA damage-induced apoptosis. A significant portion of p53- and DNA damage-regulated genes are misregulated in cells lacking PKBalpha. PKBalpha knockout mice show impaired DNA damage-dependent induction of p21 and increased tissue apoptosis after single-dose whole-body irradiation. Our findings place PKB downstream of DNA-PK in the DNA damage response signaling cascade, where it provides a prosurvival signal, in particular by affecting transcriptional p21 regulation. Furthermore, this function is apparently restricted to the PKBalpha isoform.

Evidence of Placental Translation Inhibition and Endoplasmic Reticulum Stress in the Etiology of Human Intrauterine Growth Restriction

Unexplained intrauterine growth restriction of the fetus (IUGR) results from impaired placental development, frequently associated with maternal malperfusion. Some cases are complicated further by preeclampsia (PE+IUGR). Here, we provide the first evidence that placental protein synthesis inhibition and endoplasmic reticulum (ER) stress play key roles in IUGR pathophysiology. Increased phosphorylation of eukaryotic initiation factor 2alpha suggests suppression of translation initiation in IUGR placentas, with a further increase in PE+IUGR cases. Consequently, AKT levels were reduced at the protein, but not mRNA, level. Additionally, levels of other proteins in the AKT-mammalian target of rapamycin pathway were decreased, and there was associated dephosphorylation of 4E-binding protein 1 and activation of glycogen synthase kinase 3beta. Cyclin D1 and the eukaryotic initiation factor 2B epsilon subunit were also down-regulated, providing additional evidence for this placental phenotype. The central role of AKT signaling in placental growth regulation was confirmed in Akt1 null mice, which display IUGR. In addition, we demonstrated ultrastructural and molecular evidence of ER stress in human IUGR and PE+IUGR placentas, providing a potential mechanism for eukaryotic initiation factor 2alpha phosphorylation. In confirmation, induction of low-grade ER stress in trophoblast-like cell lines reduced cellular proliferation. PE+IUGR placentas showed elevated ER stress with the additional expression of the pro-apoptotic protein C/EBP-homologous protein/growth arrest and DNA damage 153. These findings may account for the increased microparticulate placental debris in the maternal circulation of these cases, leading to endothelial cell activation and impairing placental development.

Life with a single isoform of akt : Mice lacking Akt2 and Akt3 are viable but display impaired glucose homeostasis and growth deficiencies

ABSTRACT To address the issues of isoform redundancy and isoform specificity of the Akt family of protein kinases in vivo, we generated mice deficient in both Akt2 and Akt3. In these mice, only the Akt1 isoform remains to perform essential Akt functions, such as glucose homeostasis, proliferation, differentiation, and early development. Surprisingly, we found that Akt2−/−Akt3−/− and even Akt1+/−Akt2−/−Akt3−/− mice developed normally and survived with minimal dysfunctions, despite a dramatic reduction of total Akt levels in all tissues. A single functional allele of Akt1 appears to be sufficient for successful embryonic development and postnatal survival. This is in sharp contrast to the previously described lethal phenotypes of Akt1−/−Akt2−/− mice and Akt1−/−Akt3−/− mice. However, Akt2−/−Akt3−/− mice were glucose and insulin intolerant and exhibited an ∼25% reduction in body weight compared to wild-type mice. In addition, we found substantial reductions in relative size and weight of the brain and testis in Akt2−/−Akt3−/− mice, demonstrating an in vivo role for both Akt2 and Akt3 in the determination of whole animal size and individual organ sizes.

Akt/PKB-Mediated Phosphorylation of Twist1 Promotes Tumor Metastasis via Mediating Cross-Talk between PI3K/Akt and TGF-β Signaling Axes

UNLABELLED Metastatic breast tumor cells display an epithelial-mesenchymal transition (EMT) that increases cell motility, invasion, and dissemination. Although the transcription factor Twist1 has been shown to contribute to EMT and cancer metastasis, the signaling pathways regulating Twist1 activity are poorly understood. Here, we show that Twist1 is ubiquitously phosphorylated in 90% of 1,532 invasive human breast tumors. Akt/protein kinase B (PKB)-mediated Twist1 phosphorylation promotes EMT and breast cancer metastasis by modulating its transcriptional target TGF-β2, leading to enhanced TGF-β receptor signaling, which in turn maintains hyperactive phosphoinositide 3-kinase (PI3K)/Akt signaling. Preventing phosphorylation of Twist1, as well as depletion of TGF-β2, significantly impaired the metastatic potential of cancer cells in vivo, indicating a key role of phosphorylated Twist1 (phospho-Twist1) in mediating cross-talk between the PI3K/Akt and TGF-β/Smad signaling axes that supports metastatic tumor development. Our results describe a novel signaling event linking PI3K/Akt hyperactivation in tumor cells to direct regulation of Twist1 activation and tumor metastasis. SIGNIFICANCE We identified the first phospho-Twist1 transcriptional target TGF-β2, which mediates cross-talk between PI3K/Akt and TGF-β signaling and promotes tumor metastasis. Our results thus illustrate a direct role of PI3K/Akt signaling in metastatic cancer development and suggest that Twist1 phosphorylation could be a potential therapeutic target in clinical cancer treatment.

In Vivo Analysis of Protein Kinase B (PKB)/Akt Regulation in DNA-PKcs-null Mice Reveals a Role for PKB/Akt in DNA Damage Response and Tumorigenesis

Full activation of protein kinase B (PKB/Akt) requires phosphorylation on Thr-308 and Ser-473. It is well established that Thr-308 is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1). Ser-473 phosphorylation is mediated by both mammalian target of rapamycin-rictor complex (mTORC2) and DNA-dependent protein kinase (DNA-PK) depending on type of stimulus. However, the physiological role of DNA-PK in the regulation of PKB phosphorylation remains to be established. To address this, we analyzed basal, insulin-induced, and DNA damage-induced PKB Ser-473 phosphorylation in DNA-PK catalytic subunit-null DNA-PKcs-/- mice. Our results revealed that DNA-PK is required for DNA damage-induced phosphorylation but dispensable for insulin- and growth factor-induced PKB Ser-473 phosphorylation. Moreover, DNA-PKcs-/- mice showed a tissue-specific increase in basal PKB phosphorylation. In particular, persistent PKB hyperactivity in the thymus apparently contributed to spontaneous lymphomagenesis in DNA-PKcs-/- mice. Significantly, these tumors could be prevented by deletion of PKBα. These findings reveal stimulus-specific regulation of PKB activation by specific upstream kinases and provide genetic evidence of PKB deregulation in DNA-PKcs-/- mice.

Ablation of the Kinase NDR1 Predisposes Mice to the Development of T Cell Lymphoma

The kinase NDR1 functions as a haploinsufficient tumor suppressor by ensuring proper apoptotic responses. Lymphoma from Compromised Apoptosis The four members of the nuclear Dbf2–related (NDR) family of serine and threonine protein kinases act in a number of cellular processes, including proliferation, cytokinesis, and apoptosis. Two family members, LATS1 and LATS2, act as tumor suppressor proteins in flies and mice. Of the other two, NDR1 is abundant in organs of the immune system, whereas NDR2 is predominantly found in the gastrointestinal tract. Cornils et al. found that mouse lymphocytes deficient in NDR1 compensated for this loss by increasing the abundance of NDR2 protein in a posttranscriptional manner. Blockade of this compensatory mechanism resulted in increased resistance of cells to various extrinsic and intrinsic proapoptotic stimuli. Aged NDR1+/− and NDR1−/− mice were more susceptible to the development of T cell lymphoma than were their wild-type counterparts, and this was associated with a loss in total NDR protein. Correlating with this finding, the abundance of NDR proteins was less in samples of human T cell lymphomas than in normal T cells, suggesting that NDR1 acts as a tumor suppressor protein. Defective apoptosis contributes to the development of various human malignancies. The kinases nuclear Dbf2–related 1 (NDR1) and NDR2 mediate apoptosis downstream of the tumor suppressor proteins RASSF1A (Ras association domain family member 1A) and MST1 (mammalian Ste20-like kinase 1). To further analyze the role of NDR1 in apoptosis, we generated NDR1-deficient mice. Although NDR1 is activated by both intrinsic and extrinsic proapoptotic stimuli, which indicates a role for NDR1 in regulating apoptosis, NDR1-deficient T cells underwent apoptosis in a manner similar to that of wild-type cells in response to different proapoptotic stimuli. Analysis of the abundances of NDR1 and NDR2 proteins revealed that loss of NDR1 was functionally compensated for by an increase in the abundance of NDR2 protein. Despite this compensation, NDR1−/− and NDR1+/− mice were more prone to the development of T cell lymphomas than were wild-type mice. Tumor development in mice and humans was accompanied by a decrease in the overall amounts of NDR proteins in T cell lymphoma samples. Thus, reduction in the abundance of NDR1 triggered a decrease in the total amount of both isoforms. Together, our data suggest that a reduction in the abundances of the NDR proteins results in defective responses to proapoptotic stimuli, thereby facilitating the development of tumors.

Differential Effects of Protein Kinase B/Akt Isoforms on Glucose Homeostasis and Islet Mass

ABSTRACT Protein kinase B (PKB)/Akt is considered to be a key target downstream of insulin receptor substrate 2 (IRS2) in the regulation of β-cell mass. However, while deficiency of IRS2 in mice results in diabetes with insulin resistance and severe failure of β-cell mass and function, only loss of the PKBβ isoform leads to a mild metabolic phenotype with insulin resistance. Other isoforms were reported not to be required for metabolic regulation. To clarify the roles of the three PKB isoforms in the regulation of islet mass and glucose homeostasis, we assessed the metabolic and pancreatic phenotypes of Pkbα, Pkbβ, and Pkbγ-deficient mice. Our study uncovered a novel role for PKBα in the regulation of glucose homeostasis, whereas it confirmed that Pkbβ−/− mice are insulin resistant with compensatory increase of islet mass. Pkbα−/− mice displayed an opposite phenotype with improved insulin sensitivity, lower blood glucose, and higher serum glucagon concentrations. Pkbγ−/− mice did not show metabolic abnormalities. Additionally, our signaling analyses revealed that PKBα, but not PKBβ or PKBγ, is specifically activated by overexpression of IRS2 in β-cells and is required for IRS2 action in the islets.

NDR Functions as a Physiological YAP1 Kinase in the Intestinal Epithelium

Summary Background Phosphorylation of the transcriptional coactivator YAP1 is a key event in defining Hippo signaling outputs. Previous studies demonstrated that phosphorylation of YAP1 at serine 127 (S127) sequesters YAP1 in the cytoplasm and consequently inhibits YAP1 transcriptional activity. Mammalian tissue-culture experiments suggest that downstream of MST1/2 signaling, LATS1/2 function as YAP1-S127 kinases. However, studies of Mst1/2 knockout mouse models revealed that the identity of the physiological YAP1-S127 kinase(s) in certain tissues, such as the intestine, remains unknown. Results We show that mammalian NDR1/2 kinases phosphorylate YAP1 on S127 and thereby negatively regulate YAP1 activity in tissue-cultured cells. By studying NDR1/2-deficient mice, we demonstrate the in vivo relevance of NDR1/2-mediated regulation of YAP1. Specifically, upon loss of NDR1/2 in the intestinal epithelium, endogenous S127 phosphorylation is decreased whereas total YAP1 levels are increased. Significantly, ablation of NDR1/2 from the intestinal epithelium renders mice exquisitely sensitive to chemically induced colon carcinogenesis. Analysis of human colon cancer samples further revealed that NDR2 and YAP1 protein expression are inversely correlated in the majority of samples with high YAP1 expression. Collectively, we report NDR1/2 as physiological YAP1-S127 kinases that might function as tumor suppressors upstream of YAP1 in human colorectal cancer. Conclusions We establish mammalian NDR1/2 as bona fide kinases that target YAP1 on S127 in vitro and in vivo. Our findings therefore have important implications for a broad range of research efforts aimed at decoding and eventually manipulating YAP1 biology in cancer settings, regenerative medicine, and possibly also noncancer human diseases.

MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas

High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m7GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry-based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically.

Acyl coenzyme A thioesterase Them5/Acot15 is involved in cardiolipin remodeling and fatty liver development.

ABSTRACT Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/β-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5−/− mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and β-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions.