Phosphatidylinositol 3-kinase-related Kinase Research Articles

Abstract 5780: Development of potent inhibitors of the DNA-dependent protein kinase (DNA-PK)

Abstract The cellular response to DNA double-strand break (DSB) formation is an essential component of normal cell survival, following exposure to DNA-damaging chemicals and ionising radiation. The serine/threonine kinase DNA-dependent protein kinase (DNA-PK) is a member of the phosphatidylinositol (PI) 3-kinase related kinase (PIKK) family of enzymes, and plays an important role in DNA DSB repair via the non-homologous end-joining (NHEJ) pathway. DNA-PK inhibitors may, therefore, be useful as agents to improve the activity of radio- and chemo-therapy in the treatment of cancer. Identification of the lead benzo[h]chromen-4-one DNA-PK inhibitor NU7026 (IC50 = 0.23 uM) guided the development of the potent and selective ATP-competitive chromenone NU7441 (DNA-PK IC50 = 30 nM). Structure-activity relationship studies for DNA-PK inhibition by chromenone-derivatives were conducted in conjunction with homology modelling. Library synthesis was undertaken employing a solution multiple-parallel approach, by O-alkylation or N-acylation of the appropriately substituted NU7441 derivatives, respectively, followed by reaction with a range of amines to afford the target compounds. These studies resulted in the identification of the highly potent inhibitor KU-0060648 (IC50 = 8.6 nM). The further development of KU-0060648 and analogues will be described. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5780.

Abstract LB-243: Discovery of single amino acid changes that confer constitutive activation of mTOR in human cancer

Abstract Mammalian target of rapamycin (mTOR), a serine/threonine kinase that belongs to the PI3K related kinase family, plays critical roles in a variety of cellular processes including protein synthesis, growth, cell cycle and autophagy in response to growth factor, nutrient and energy conditions. Overactivation of the mTOR signaling is observed in a number of human cancer. Inhibitors of mTOR such as rapamycin and its derivatives are being evaluated in clinical trials. Despite these developments that point to the significance of mTOR in human cancer, no mutations in mTOR have so far been identified in human cancer. Here we report novel mTOR mutations identified in human cancer by mining human cancer genome database. Of 889 cancer samples mined, we identified 14 amino acid changes in mTOR. Characterization of the mTOR signaling after expressing some of these mutants led to the identification of two single amino acid changes (S2215Y and R2505P) that confer constitutive activation of mTOR as detected by the phosphorylation of S6K, 4E-BP1 and S6 under nutrient starvation conditions. The mutation directly affects mTOR complex 1 (mTORC1), as the complex isolated from cells expressing the mutant mTOR after nutrient starvation still retained the activity to phosphorylate 4E-BP1 in vitro. On the other hand, we did not observe significant effects on the activity of mTORC2. The cells expressing the mTOR mutant exhibited resistance to cell size decrease by amino acid starvation and showed increased percentage of S-phase cells. The cells expressing the activated mutant were resistance to 1-butanol treatment and partially resistant to hydrogen peroxide treatment. However, they were sensitive to rapamycin. The mutations are located within the kinase domain at positions that correspond to regions where oncogenic mutations of PI3 kinase were identified. It is interesting to point out that the mTOR S2215Y mutation was identified in large intestine adenocarcinoma together with K-rasG13D mutation, while the mTOR R2505P mutation was identified in renal cell carcinoma together with CDC25A F218I mutation, raising the possibility that the mTOR mutations may cooperate with mutations in other signaling proteins. Since “we are moving into an era in which it will be possible to obtain the complete DNA sequence of large numbers of cancer genomes” (Stratton et al., Nature 458, 719), our study points to the idea that mTOR activating mutations can be identified in a wide range of human cancer yielding valuable information on the significance of mTOR in human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-243.

Inhibition profiles of phosphatidylinositol 3-kinase inhibitors against PI3K superfamily and human cancer cell line panel JFCR39

As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in various diseases particularly cancer, considerable competition occurs in development of PI3K inhibitors. Consequently, novel PI3K inhibitors such as ZSTK474, GDC-0941 and NVP-BEZ235 have been developed. Even though all these inhibitors were reported to inhibit class I PI3K but not dozens of protein kinases, whether they have different molecular targets remained unknown. To investigate such molecular target specificity, we have determined the inhibitory effects of these novel inhibitors together with classical PI3K inhibitor LY294002 on PI3K superfamily (including classes I, II, and III PI3Ks, PI4K and PI3K-related kinases) by using several novel non-radioactive biochemical assays. As a result, ZSTK474 and GDC-0941 indicated highly similar inhibition profiles for PI3K superfamily, with class I PI3K specificity much higher than NVP-BEZ235 and LY294002. We further investigated their growth inhibition effects on JFCR39, a human cancer cell line panel which we established for molecular target identification, and analysed their cell growth inhibition profiles (fingerprints) by using COMPARE analysis programme. Interestingly, we found ZSTK474 exhibited a highly similar fingerprint with GDC-0941 ( r = 0.863), more similar than with that of either NVP-BEZ235 or LY294002, suggesting that ZSTK474 shares more in molecular targets with GDC-0941 than with either of the other two PI3K inhibitors, consistent with the biochemical assay result. The biological implication of the difference in molecular target specificity of these PI3K inhibitors is under investigation.

Immunohistochemical expression of mTOR protein in breast carcinoma tissues

Immunohistochemical expression of mTOR protein in breast carcinoma tissues

The Expression and Effect of mTOR During Mouse Oocyte Maturation

The mammalian target of rapamycin(mTOR) is a serine-threonine kinase downstream in the PI3K-Akt pathway, which belongs to the phosphatidylinositol kinase-related kinase(PIKK) family.The mTOR palys an important role in cell cycle regulation and protein synthesis.mTOR signaling pathway is evolutionarily conserved, which can integrate and converge a wide range of signals, including intracellular and extracellular nutrients, growth factors, energy and stress conditions, and has a crucial role in vertebrate growth control.The upstream regulation and downstream effects mediated by mTOR form a network of critical growth signaling pathways.Hence, dysregulation of components within mTOR signaling network result in impaired internal environment conditions, and first meiotic division can also be influenced if occurring during starfish and sea urchin oocyte maturation.However, its functions in mammalian meiosis are unclear.The localization and function of mTOR during mouse oocyte meiotic maturation were investigated with the oocytes of Kunming mouse.Location of mTOR was examined in Germinal vesicle(GV) stage, Germinal vesicle breakdown(GVBD) stage and second metaphase(M Ⅱ) stage during mouse oocyte maturation by immunofluorescence technology.The rate of GVBD and polar body extrusion(PB1) during mouse oocytes maturation was also detected by treatment with different concentration rapamycin(0.5 g/L, 2.5 g/L, 3.3 g/L, 5.0 g/L, 10 g/L ).Immunofluorescent staining showed that mTOR mainly is located on nuclear membrane during GV stage, distributed with the chromosome after GVBD, and distributed with the spindle apparatus during MⅡ stage.The oocyte maturation was inhibited by treatment with rapamycin, and the inhibitory action was concentration dependent, when the drug concentration reached to 3.3 g/L, the disparity was significant(P 0.05).Meanwhile, the location and morphous of mTOR were changed:under treatment with 3.3 g/L rapamycin, mTOR protein expression became obviously weaker and distributed more in intranuclear in the stage of GV;and didn't distribute with the chromosome after GVBD, the morphous of chromosome was changed.The mTOR can not be detected in MⅡ stage, and the chromosome arrangment was irregular.The results suggest that the expression and distribution of mTOR shows stage specificity during mouse oocyte maturation, rapamycin had an effect on mouse first meiotic division, including chromosome arrangment and spindle formation, demonstrating that a rapamycin-sensitive pathways is involved in this mechinism.mTOR plays an important role during procedure of GVBD and first polar body extrusion, which could regulate maturation process of mouse oocyte through the changes of its expression and localization.

Abstract A138: Development of potent inhibitors of the DNA-dependent protein kinase (DNA-PK)

Abstract The cellular response to DNA double-strand break (DSB) formation is an essential component of normal cell survival, following exposure to DNA-damaging chemicals and ionising radiation. The serine/threonine kinase DNA-dependent protein kinase (DNA-PK) is a member of the phosphatidylinositol (PI) 3-kinase related kinase (PIKK) family of enzymes, and plays an important role in DNA DSB repair via the non-homologous end-joining (NHEJ) pathway. DNA-PK inhibitors may, therefore, be useful as agents to improve the activity of radio- and chemo-therapy in the treatment of cancer. Identification of the lead benzo[h]chromen-4-one DNA-PK inhibitor NU7026 (IC50 = 0.23 uM) guided the development of the potent and selective ATP-competitive chromenone NU7441 (DNA-PK IC50 = 30 nM). Structure-activity relationship studies for DNA-PK inhibition by chromenone-derivatives were conducted in conjunction with homology modelling. Library synthesis was undertaken employing a solution multiple-parallel approach, by O-alkylation or N-acylation of the appropriately substituted NU7441 derivatives, respectively, followed by reaction with a range of amines to afford the target compounds. These studies resulted in the identification of KU-0060648, which combines high potency as a DNA-PK inhibitor (IC50 = 8.6 nM) with good aqueous solubility as an acid salt. The further development of KU-0060648 and analogues will be described. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A138.

Abstract C67: JFCR39 COMPARE system can predict molecular targets of phophatidylinositol 3-kinase inhibitors with high resolution

Abstract JFCR39 COMPARE system is an anticancer drug-activity database that we established by exploiting a panel of 39 human cancer cell lines. Molecular target of an agent can be predicted by comparing its cell growth inhibition profiles (fingerprint) with those of anticancer drugs using the COMPARE program. As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in various diseases, particularly in cancer, researchers in both industry and academics are in a race to develop PI3K inhibitors. Consequently, novel PI3K inhibitors such as ZSTK474, GDC-0941, NVP-BEZ235 and PI-103 have been developed in recent years. Even though all these inhibitors were reported to inhibit class I PI3K but not dozens of protein kinases, whether they have different molecular targets remained unknown. To predict such molecular target specificity, we have examined the fingerprints of these novel PI3K inhibitors and classical PI3K inhibitor LY294002 across JFCR39 panel, and analyzed the results using the COMARE system. Interestingly, we found highly similar fingerprints for ZSTK474 and GDC-0941 and cluster analysis distinguished them from the other three PI3K inhibitors, suggesting that ZSTK474 shared more in molecular targets with GDC-0941 than with any other PI3K inhibitors. To test this prediction, we determined their effects on PI3K superfamily (including classes I, II, and III PI3Ks, PI4K and PI3K-related kinases) by using several novel non-radioactive biochemical assays. As a result, ZSTK474 and GDC-0941 indicated highly similar inhibition profiles for PI3K superfamily, with class I PI3K inhibitory specificity much higher than the other three inhibitors, which supported the hypothesis derived from JFCR39 COMPARE result. In conclusion, JFCR39 system is able to provide valuable information regarding molecular target specificity, suggesting its utility in target identification with high resolution. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C67.

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin

The ubiquitin–proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.

Discovery of Potent and Selective Inhibitors of the Mammalian Target of Rapamycin (mTOR) Kinase

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compound collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compound 5a, employing an mTOR homology model based on an X-ray crystal structure of closely related PI3Kgamma led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC(50) = 9 nM; PI3Kalpha IC(50) = 1962 nM). Compound 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy.

Emerging common themes in regulation of PIKKs and PI3Ks

Phosphatidylinositol-3 kinase-related kinases (PIKKs) comprise a family of protein kinases that respond to various stresses, including DNA damage, blocks in DNA replication, availability of nutrients and errors in mRNA splicing. PIKKs are characterized by the presence of a conserved kinase domain (KD), whose activity is regulated by two C-terminal regions, referred to as PIKK-regulatory domain (PRD) and FRAP-ATM-TRRAP-C-terminal (FATC), respectively. Here, we review functional and structural data that implicate the PRD and FATC domains in regulation of PIKK activity, drawing parallels to phosphatidylinositol-3 kinases (PI3K), lipid kinases that have sequence similarity to PIKKs. The PI3K C-terminus, which we propose to be equivalent to the PRD and FATC domains of PIKKs, is in close proximity to the activation loop of the KD, suggesting that in PIKKs, the PRD and FATC domains may regulate kinase activity by targeting the activation loop.

PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin

The role of Mre11 phosphorylation in the cellular response to DNA double-strand breaks (DSBs) is not well understood. Here, we show that phosphorylation of Mre11 at SQ/TQ motifs by PIKKs (PI3 Kinase-related Kinases) induces MRN (Mre11–Rad50–Nbs1) complex dissociation from chromatin by reducing Mre11 affinity for DNA. Whereas phosphorylation of Mre11 at these residues is not required for DSB-induced ATM (Ataxia-Telangiectasia mutated) activation, abrogation of Mre11 dephosphorylation impairs ATM signaling. Our study provides a functional characterization of the DNA damage-induced Mre11 phosphorylation, and suggests that MRN inactivation participates in the down-regulation of damage signaling during checkpoint recovery following DSB repair.

The discovery and optimisation of pyrido[2,3- d]pyrimidine-2,4-diamines as potent and selective inhibitors of mTOR kinase

We describe a novel series of potent inhibitors of the kinase activity of mTOR. The compounds display good selectivity relative to other PI3K-related kinase family members and, in cellular assays, inhibit both mTORC1 and mTORC2 complexes and exhibit good antiproliferative activity.

TOR Complex 2 Controls Gene Silencing, Telomere Length Maintenance, and Survival under DNA-Damaging Conditions

The Target Of Rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR proteins are found at the core of two distinct evolutionarily conserved complexes, TORC1 and TORC2. Disruption of TORC1 or TORC2 results in characteristically dissimilar phenotypes. TORC1 is a major cell growth regulator, while the cellular roles of TORC2 are not well understood. In the fission yeast Schizosaccharomyces pombe, Tor1 is a component of the TORC2 complex, which is particularly required during starvation and various stress conditions. Our genome-wide gene expression analysis of Deltator1 mutants indicates an extensive similarity with chromatin structure mutants. Consistently, TORC2 regulates several chromatin-mediated functions, including gene silencing, telomere length maintenance, and tolerance to DNA damage. These novel cellular roles of TORC2 are rapamycin insensitive. Cells lacking Tor1 are highly sensitive to the DNA-damaging drugs hydroxyurea (HU) and methyl methanesulfonate, similar to mutants of the checkpoint kinase Rad3 (ATR). Unlike Rad3, Tor1 is not required for the cell cycle arrest in the presence of damaged DNA. Instead, Tor1 becomes essential for dephosphorylation and reactivation of the cyclin-dependent kinase Cdc2, thus allowing reentry into mitosis following recovery from DNA replication arrest. Taken together, our data highlight critical roles for TORC2 in chromatin metabolism and in promoting mitotic entry, most notably after recovery from DNA-damaging conditions. These data place TOR proteins in line with other PIKK members, such as ATM and ATR, as guardians of genome stability.

Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1

We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III-transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1-v allele. In W303 cells that carry the SSD1-d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm-to-nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm-to-nucleolus translocation. Together, these observations provide new insights into the chromatin-dependent mechanism by which TORC1 controls transcription by Pol III.

Kinome Sirna Screen Identifies SMG-1 as a Negative Regulator of Hypoxia-inducible Factor-1α in Hypoxia

Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1α. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1α-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1α activity in hypoxia, in part through blocking MAPK activation.

A tale of two tails: Activation of DNA damage checkpoint kinase Mec1/ATR by the 9-1-1 clamp and by Dpb11/TopBP1

The DNA damage and replication checkpoint kinase Mec1/ATR is a member of the PI3-kinase related kinases that function in response to various genotoxic stresses. The checkpoint clamp 9-1-1 (Rad9-Rad1-Hus1 in S. pombe and mammals; Ddc1-Rad17-Mec3 in S. cerevisiae) executes two distinct checkpoint functions. In S. cerevisiae, DNA-bound 9-1-1 directly activates Mec1 kinase activity, a function that has not been demonstrated in other organisms. A second, conserved activity of 9-1-1 is that of TopBP1/Cut5/Dpb11 recruitment to stalled replication sites; subsequent activation of Mec1/ATR is carried out by TopBP1/Cut5/Dpb11. Biochemical studies indicate that the mode of Mec1/ATR activation by S. cerevisiae 9-1-1 is analogous to activation by S. cerevisiae Dpb11 or by vertebrate TopBP1: activation is mediated by the intrinsically disordered C-terminal tail of each activator. The relative contributions made by multiple activators of Mec1/ATR are discussed.

The Shelterin Protein TRF2 Inhibits Chk2 Activity at Telomeres in the Absence of DNA Damage

The shelterin complex [1] shapes and protects telomeric DNA from being processed as double strand breaks (DSBs) [2, 3]. Here we show that in human undamaged cells, a fraction of the kinase Chk2, a downstream target of ATM and mediator of checkpoint responses and senescence [4, 5], physically interacts with the shelterin subunit TRF2 and colocalizes with this complex at chromosome ends. This interaction, enhanced by TRF2 binding to telomeric DNA, inhibits the activation and senescence-induced function of Chk2 by a mechanism in which TRF2 binding to the N terminus of Chk2 surrounding Thr68 hinders the phosphorylation of this priming site. In response to radiation-induced DSBs, but not chromatin-remodelling agents, the telomeric Chk2-TRF2 binding dissociates in a Chk2 activity-dependent manner. Moreover, active Chk2 phosphorylates TRF2 and decreases its binding to telomeric DNA repeats, corroborating the evidences on the specific TRF2 relocalization in presence of DSBs [6]. Altogether, the capacity of TRF2 to locally repress Chk2 provides an additional level of control by which shelterin restrains the DNA damage response from an unwanted activation [6, 7] and may explain why TRF2 overexpression acts as a telomerase-independent oncogenic stimulus [8].

The Mec1p and Tel1p checkpoint kinases allow humanized yeast to tolerate chronic telomere dysfunctions by suppressing telomere fusions

In this work we report that budding yeasts carrying human-type telomeric repeats at their chromosome termini show a chronic activation of the Rad53-dependent DNA damage checkpoint pathway and a G2/M cell cycle delay. Furthermore, in the absence of either TEL1/ATM or MEC1/ATR genes, which encodes phosphatidylinositol 3-kinase-related kinases (PIKKs), we detected telomere fusions, whose appearance correlates with a reduced cell viability and a high rate of genome instability. Based on sequence analysis, telomere fusions occurred primarily between ultrashort telomeres. Microcolony formation assays argue against the possibility that fusion-containing cells are eliminated by PIKK-dependent signalling.These findings reveal that humanized telomeres in yeast cells are sensed as a chronically damaged DNA but do not greatly impair cell viability as long as the cells have a functional DNA damage checkpoint.

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 PKBalpha. These findings reveal stimulus-specific regulation of PKB activation by specific upstream kinases and provide genetic evidence of PKB deregulation in DNA-PKcs(-/-) mice.

A Novel Tel1/ATM N-Terminal Motif, TAN, Is Essential for Telomere Length Maintenance and a DNA Damage Response

Tel1/ATM, a conserved phosphatidylinositol 3-kinase-related kinase (PIKK), acts in the response to DNA damage and regulates telomere maintenance. PIKK family members share an extended N-terminal region of low sequence homology. Sequence alignment of the N terminus of Tel1/ATM orthologs revealed a conserved, novel motif we term TAN (for Tel1/ATM N-terminal motif). Point mutations in conserved residues of the TAN motif resulted in telomere shortening, and its deletion caused the same short telomere phenotype as complete deletion of Tel1 did. Overexpressing Tel1 TAN mutants did not rescue telomere shortening. The TAN motif was also essential for the function of Tel1 in the response to DNA damage, as TAN-deleted Tel1 was indistinguishable from the complete lack of Tel1 in causing reduced viability and signaling through Rad53 upon DNA damage. Strikingly, TAN deletion reduced recruitment of Tel1 to a double-strand DNA break. Together, these results define a conserved sequence motif within an otherwise poorly defined region of the Tel1/ATM kinase family proteins that is essential for normal Tel1 function in Saccharomyces cerevisiae.