Phosphatidylinositol 3-kinase Domain Research Articles

Site-Specific Phosphorylation of RTK KIT Kinase Insert Domain: Interactome Landscape Perspectives

The kinase insert domain (KID) of RTK KIT is a key recruitment region for downstream signalling proteins (DSPs). KID, as a multisite phosphorylation region, provides alternative recognition sites for DSPs and activates them by binding a phosphotyrosine (pY) to their SH2 domains. Significant steric, biochemical, and biophysical requirements must be fulfilled by each pair of interacting proteins as the adaptation of their configurations is mandatory for the selective activation of DSPs. The accurate 3D atomistic models obtained by modelling and molecular dynamics (MD) simulations of phosphorylated KID (p-KID) have been delivered to describe KID INTERACTOME. By taking phosphorylated KIDpY721 and the N-terminal SH2 domain of phosphatidylinositol 3-kinase (PI3K), a physiological partner of KID, we showed the two proteins are intrinsically disordered. Using 3D models of both proteins, we probe alternative orientations of KIDpY721 relative to the SH2 binding pocket using automatic docking (HADDOCK) and intuitive user-guided docking. This modelling yields to two possible models of the functionally related non-covalent complex KIDpY721/SH2, where one can be regarded as the first precursor to probe PI3K activation via KIT KID. We suggest that such generation of a KID/SH2 complex is best suited for future studies of the post-transduction effects of RTK KIT.

Binding-induced folding under unfolding conditions: Switching between induced fit and conformational selection mechanisms

The chemistry of protein-ligand binding is the basis of virtually every biological process. Ligand binding can be essential for a protein to function in the cell by stabilizing or altering the conformation of a protein, particularly for partially or completely unstructured proteins. However, the mechanisms by which ligand binding impacts disordered proteins or influences the role of disorder in protein folding is not clear. To gain insight into this question, the mechanism of folding induced by the binding of a Pro-rich peptide ligand to the SH3 domain of phosphatidylinositol 3-kinase unfolded in the presence of urea has been studied using kinetic methods. Under strongly denaturing conditions, folding was found to follow a conformational selection (CS) mechanism. However, under mildly denaturing conditions, a ligand concentration-dependent switch in the mechanism was observed. The folding mechanism switched from being predominantly a CS mechanism at low ligand concentrations to being predominantly an induced fit (IF) mechanism at high ligand concentrations. The switch in the mechanism manifests itself as an increase in the reaction flux along the IF pathway at high ligand concentrations. The results indicate that, in the case of intrinsically disordered proteins too, the folding mechanism is determined by the concentration of the ligand that induces structure formation.

Structural characterization of ANGPTL8 (betatrophin) with its interacting partner lipoprotein lipase

Angiopoietin-like protein 8 (ANGPTL8) (also known as betatrophin) is a newly identified secretory protein with a potential role in autophagy, lipid metabolism and pancreatic beta-cell proliferation. Its structural characterization is required to enhance our current understanding of its mechanism of action which could help in identifying its receptor and/or other binding partners. Based on the physiological significance and necessity of exploring structural features of ANGPTL8, the present study is conducted with a specific aim to model the structure of ANGPTL8 and study its possible interactions with Lipoprotein Lipase (LPL). To the best of our knowledge, this is the first attempt to predict 3-dimensional (3D) structure of ANGPTL8. Three different approaches were used for modeling of ANGPTL8 including homology modeling, de-novo structure prediction and their amalgam which is then proceeded by structure verification using ERRATT, PROSA, Qmean and Ramachandran plot scores. The selected models of ANGPTL8 were further evaluated for protein–protein interaction (PPI) analysis with LPL using CPORT and HADDOCK server. Our results have shown that the crystal structure of iSH2 domain of Phosphatidylinositol 3-kinase (PI3K) p85β subunit (PDB entry: 3mtt) is a good candidate for homology modeling of ANGPTL8. Analysis of inter-molecular interactions between the structure of ANGPTL8 and LPL revealed existence of several non-covalent interactions. The residues of LPL involved in these interactions belong from its lid region, thrombospondin (TSP) region and heparin binding site which is suggestive of a possible role of ANGPTL8 in regulating the proteolysis, motility and localization of LPL. Besides, the conserved residues of SE1 region of ANGPTL8 formed interactions with the residues around the hinge region of LPL. Overall, our results support a model of inhibition of LPL by ANGPTL8 through the steric block of its catalytic site which will be further explored using wet lab studies in future.

Abstract A07: Decline in the growth of murine T-cell lymphoma via modulation of PI3K signaling pathway: Key role of quercetin and PI-103

Abstract Deregulation in cell proliferation, oxidative status, antiapoptosis and survival are involved in development of cancer. The cellular signaling cascades mediated by Phosphatidylinositol 3-kinase (PI3K) with their ability to stimulate cell proliferation and suppress apoptosis, has been considered to play key role in tumor initiation, promotion, and progression. Therefore, use of inhibitors or antioxidants like natural flavonoids could be promising strategy to target PI3K pathway for suppression of carcinogenic process. The present study was aimed to investigate the anti-cancer activity of quercetin, a natural flavonoid and PI-103, a synthetic inhibitor in transplantable murine T-cell lymphoma. Cytotoxicity, apoptotic potential, cell survival, cell proliferation as well as antioxidant defense are evaluated in ascite cells of murine T-cell lymphoma in vivo and in primary culture. Significant decline in level of AKT1, PKCα, COX 2 as well as regulatory domain of PI3K (p85α), AKT1 and Bad, suggested implication of quercetin in regression of lymphoma by down regulation of cell proliferation and survival. Improvement of active PKCδ, p53, active caspase 3, 9 and cleaved PARP supported its cell apoptotic mechanism in cancer prevention. Additionally, decreased ROS level and LDH A activity contributes to reduced survival of cancer cells. Inhibition of p110α by PI-103 resulted in suppression of phospho-AKT and p85α level in primary culture. Modulation of various molecular targets including PI3K/AKT signaling by quercetin and PI-103 in vitro and in vivo tumor models suggests the importance and contribution of antioxidant quercetin in the prevention of cancer. Citation Format: Akhilendra Kumar Maurya, Manjula Vinayak. Decline in the growth of murine T-cell lymphoma via modulation of PI3K signaling pathway: Key role of quercetin and PI-103. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A07.

Structure and Assembly of the PI3K-like Protein Kinases (PIKKs) Revealed by Electron Microscopy

The phosphatidylinositol 3-kinase-like kinases (PIKKs) are large serine-threonine protein kinases with a catalytic domain homologous to the phosphatidylinositol 3-kinase (PI3K). All PIKK family members share a general organization comprising a conserved C-terminus that contains the PI3K domain, which is preceded by a large N-terminal region made of helical HEAT repeats. In humans, the PIKK family includes six members, which play essential roles in various processes including DNA repair and DNA damage signalling (ATM, ATR, DNA-PKcs), control of cell growth (mTOR), nonsense-mediated mRNA decay (SMG1) and transcriptional regulation (TRRAP). High-resolution structural information is limited due to the large size (approx. 280-470 kDa) and structural complexity of these kinases. Adding further complexity, PIKKs work as part of larger assemblies with accessory subunits. These complexes are dynamic in composition and protein-protein and protein-DNA interactions regulate the kinase activity and functions of PIKKs. Moreover, recent findings have shown that the maturation and correct assembly of the PIKKs require a large chaperon machinery, containing RuvBL1 and RuvBL2 ATPases and the HSP90 chaperon. Single-particle electron microscopy (EM) is making key contributions to our understanding of the architecture of PIKKs and their complex regulation. This review summarizes the findings on the structure of these kinases, focusing mainly on medium-low resolution structures of several PIKKs obtained using EM, combined with X-ray crystallography of DNA-PKcs and mTOR. In addition, EM studies on higher-order complexes have revealed some of the mechanisms regulating the PIKKs, which will also be addressed. The model that emerges is that PIKKs, through their extensive interacting surfaces, integrate the information provided by multiple accessory subunits and nucleic acids to regulate their kinase activity in response to diverse stimuli.

Piperlongumine, an alkaloid causes inhibition of PI3K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human triple-negative breast cancer cells.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth and survival under physiological conditions. However, aberrant PI3K/Akt/mTOR signaling has been implicated in many human cancers, including human triple negative breast cancer. Therefore, dual inhibitors of PI3K/Akt and mTOR signaling could be valuable agents for treating breast cancer. The objective of this study was to investigate the effect of piperlongumine (PPLGM), a natural alkaloid on PI3K/Akt/mTOR signaling, Akt mediated regulation of NF-kB and apoptosis evasion in human breast cancer cells. Using molecular docking studies, we found that PPLGM physically interacts with the conserved domain of PI3K and mTOR kinases and the results were comparable with standard dual inhibitor PF04691502. Our results demonstrated that treatment of different human triple-negative breast cancer cells with PPLGM resulted in concentration- and time-dependent growth inhibition. The inhibition of cancer cell growth was associated with G1-phase cell cycle arrest and down-regulation of the NF-kB pathway leads to activation of the mitochondrial apoptotic pathway. It was also found that PPLGM significantly decreased the expression of p-Akt, p70S6K1, 4E-BP1, cyclin D1, Bcl-2, p53 and increased expression of Bax, cytochrome c in human triple-negative breast cancer cells. Although insulin treatment increased the phosphorylation of Akt (Ser473), p70S6K1, 4E-BP1, PPLGM abolished the insulin mediated phosphorylation, it clearly indicates that PPLGM acts through PI3k/Akt/mTOR axis. Our results suggest that PPLGM may be an effective therapeutic agent for the treatment of human triple negative breast cancer.

Abstract 2589: The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer.

Abstract Verifying potential target proteins of phytochemicals is important for explaining their health benefits. In the present study, we examined the chemopreventive effect and molecular target of acacetin, which is a flavonoid from Robinia Psuedoacacia also known as Black Locust. Treatment with acacetin significantly inhibits EGF-induced transformation of JB6 P+ cells. Western blot assay results revealed that acacetin suppresses EGF-induced phosphorylation of Akt/p70S6K, which is a well-known downstream signaling molecule of phosphatidylinositol 3-kinase (PI3-K). In vitro kinase and pull-down assay results showed that acacetin significantly inhibits PI3-K activity by direct binding. Additionally, acacetin also attenuated EGF-induced PI3-K activity and binds with PI3-K in JB6 P+ cells. Acacetin showed significant effects against anchorage-dependent and -independent cell growth in cells displaying higher PI3-K activity compared with those exhibiting lower PI3-K activity. Binding assay data and computational modeling showed that acacetin binds in an ATP-competitive manner with the p110 kinase domain of PI3-K and interacts with Val828, Glu826 Asp911 Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900. Acacetin reduced SK-MEL-28 xenograft tumor growth and phosphorylation of Akt in vivo. Overall, these results indicate that acacetin is an ATP competitive PI3-K inhibitor and might be a promising chemopreventive agent targeting PI3-K activity. Citation Format: Jon Eun Kim, Sung Keun Jung, Sanguine Byun, Sung-Young Lee, Myoung Ok Kim, Tae Gyu Lim, Ann M. Bode, Hyong Joo Lee, Ki Won Lee, Zigang Dong. The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2589. doi:10.1158/1538-7445.AM2013-2589

P5-01-05: Activating Mutations in PIK3CA or AKT1 in the I-SPY 1 Trial (CALGB 150007/150012; ACRIN 6657).

Abstract Background Mutations in the catalytic domain of phosphatidylinositol 3-kinase (PIK3CA) are among the most frequently observed activating mutations in breast cancer. We used the I SPY 1 TRIAL, a group of biologically and clinically high risk patients molecularly profiled and treated with neoadjuvant chemotherapy, to determine the frequency of mutations and their relationship to pathologic complete response (pCR) and outcomes, within the entire cohort and within subtypes defined by growth and hormone receptor (HR) expression. Methods: Patients enrolled in the I-SPY 1 TRIAL had a tumor size ≥3.0cm and were administered a doxorubicin-containing regimen, followed by a taxane, prior to surgery. Sequenom single nucleotide polymorphism (SNP) profiling was performed on breast tumor genomic DNA isolated from a subset of patients (n=152). A total of 149 SNPs covering 16 genes (including PIK3CA and AKT1/2/3) were analyzed. Mutations were tested for association with estrogen receptor (ER), progesterone receptor (PgR), and HER2 status, as well as pCR, using Fisher's exact test; associations between mutations and recurrence-free survival (RFS) were measured by log-rank tests. pCR was defined as no invasive tumor present in either the breast or axillary lymph nodes following neoadjuvant treatment. Results: Of 149 mutations profiled in the cohort, 13 of the SNPs were observed. PIK3CA mutations were the most frequently observed in the panel (15.1%), followed by AKT1(E17K; 2.7%), CTNNB1 (D32; 1.4%), NRAS(Q61; 0.7%), and FGFR2(N549; 0.7%). Mutations in PIK3CA or AKT1 was associated with ER-positivity (p=0.0047) and PgR-positivity (p=0.044). Within receptor subtypes, the frequencies of PIK3CA/AKT1 mutations were also significantly different (HR+HER2−: 27%:(18/68); HER2+ 20% (8/40) [HR+HER2+: 26%, HR-HER2+: 14%]; HR-HER2−: 0% (0/36), p<0.0008). Unlike previous reports (Loi et al, PNAS 2010), no significant association between PIK3CA/AKT mutation status and RFS was observed when we restricted our analysis to the adjuvant endocrine treated subset of the HR+HER2− patients (n=49; log rank p = 0.369). In contrast, and similar to cell line reports (Junttila et al, Cancer Cell 2009), PIK3CA mutations appears to associate with worse RFS within the small subset of trastuzumab treated HER2+ patients (n=22, 13 HR-HER2+, 9 HR+HER2−; log rank p=0.001), suggesting mutations may influence response. Similar analyses of a larger cohort are planned to confirm these observations. Conclusions: Within the I-SPY 1 TRIAL cohort, PIK3CA and AKT1 mutations are much more frequent in the HR+ and HER2 subsets but are not predictive of response to therapy or outcome except potentially within the HER2+ subset. The potential link observed between activating PIK3CA/AKT mutations and trastuzumab resistance merits further investigation, as it may provide a clinical rationale for testing PIK3CA mutation status in HER2+ patients and investigating combinational therapies targeting this pathway, particularly in the HER2+HR+ subset which have an elevated risk for recurrence despite pCR and trastuzumab therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-05.

The nonsense-mediated mRNA decay SMG-1 kinase is regulated by large-scale conformational changes controlled by SMG-8

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression of many physiological transcripts. SMG-1 is a large kinase essential to NMD that phosphorylates Upf1, which seems to be the definitive signal triggering mRNA decay. However, the regulation of the kinase activity of SMG-1 remains poorly understood. Here, we reveal the three-dimensional architecture of SMG-1 in complex with SMG-8 and SMG-9, and the structural mechanisms regulating SMG-1 kinase. A bent arm comprising a long region of HEAT (huntington, elongation factor 3, a subunit of PP2A and TOR1) repeats at the N terminus of SMG-1 functions as a scaffold for SMG-8 and SMG-9, and projects from the C-terminal core containing the phosphatidylinositol 3-kinase domain. SMG-9 seems to control the activity of SMG-1 indirectly through the recruitment of SMG-8 to the N-terminal HEAT repeat region of SMG-1. Notably, SMG-8 binding to the SMG-1:SMG-9 complex specifically down-regulates the kinase activity of SMG-1 on Upf1 without contacting the catalytic domain. Assembly of the SMG-1:SMG-8:SMG-9 complex induces a significant motion of the HEAT repeats that is signaled to the kinase domain. Thus, large-scale conformational changes induced by SMG-8 after SMG-9-mediated recruitment tune SMG-1 kinase activity to modulate NMD.

Are we missing the mTOR target in breast cancer?

Abnormalities in the PI3K/Akt/mTOR intracellular signalling pathway have been implicated in several forms of human cancer. The central component of the pathway is the phosphatidylinositol 3-kinase (PI3K) heterodimer which comprises the p85 regulatory and p110 catalytic subunit, and mutations in the catalytic domain of PI3K have been identified in 20–25% of breast cancers [1, 2]. A further 15–35% of breast cancer patients demonstrate reduced expression of PTEN (phosphatase and tensin homologue deleted on chromosome ten), an endogenous inhibitor of the PI3K/AKT pathway [3]. Akt is the ultimate effector of the pathway and directs a multitude of intracellular effects related to growth, glucose metabolism, protein synthesis and cell survival. Activation of the PI3K/Akt pathway provides a cell with unrestricted growth and survival signals, and one important downstream consequence is the alleviation of the suppression by the tuberous sclerosis protein complex (TSC1/2) of the mammalian target of rapamycin (mTOR) [4]. There are at least two groups of mTOR proteins, and the key protein involved in transmitting signals from PI3K/Akt is the mTORC1 complex. Within cells, mTORC1 plays a critical role in the transduction of proliferative signals through phosphorylation of the translational regulator 4E-BP1 (eukaryotic initiation factor 4E-binding protein) and the ribosomal protein p70 (the 70-kDa S6 kinase) that facilitate translation of mRNAs into proteins [5, 6]. In recent years our understanding of this pathway in breast cancer has been exploited by the development of a number of targeted anti-cancer therapeutics, with mTOR inhibitors being the most clinically developed to date. Rapamycin is the original inhibitor of mTOR that was found to be a potent fungicide with immunosuppressive properties, and therapeutics were developed for clinical use in transplantation to prevent graft rejection. Subsequently rapamycin was found to have anti-proliferative effects in a range of experimental tumours [7], and rapamycin analogues with a more favourable pharmacological profile were synthesised including CCI-779 (temsirolimus), RAD001 (everolimus) and AP23573 (ridaforolimus). In various breast cancer cell lines with constitutive activation of the PI3K/Akt pathway due to either HER2 amplification, ER activation or PTEN deletions temsirolimus showed specific sensitivity [8], whilst everolimus (an oral hydroxyethyl ether of rapamycin) demonstrated impressive anti-proliferative activity against a wide variety of tumour models in vivo [9]. In the clinic mTOR antagonists were found to be relatively well tolerated in early phase I/II trials [10, 11], and development programmes were established in various solid malignancies, in particular renal cell cancer and breast cancer. But has the development of mTOR antagonists for breast cancer moved ahead too fast before it was really appreciated how best to utilise these drugs as targeted therapeutics? As a single agent temsirolimus showed only modest activity in the advanced breast cancer setting [12], and subsequently both temsirolimus and everolimus were developed in combination with endocrine therapy following encouraging evidence for additive or synergistic effects from several pre-clinical models of endocrine resistant breast cancer [13, 14]. However, results from randomised clinical trials of mTOR antagonists in combination with This is an invited commentary to articles doi: 10.1007/s10549-010-0967-z, 10.1007/s10549-010-0986-9, 10.1007/s10549-010-1058-x.

Patterns of PIK3CA alterations in familial colorectal and endometrial carcinoma

While the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is known to be activated in multiple sporadic cancers, the role of this pathway in familial tumors is mostly unknown. We searched for alterations in the catalytic domain of PI3K (PIK3CA), PTEN and KRAS, all of which may contribute to PI3K/AKT pathway activation, in a total of 160-familial colorectal (CRC) and endometrial carcinomas (EC), stratified by the presence vs. absence of germline mutations in DNA mismatch repair (MMR) genes. PIK3CA alterations (consisting of point mutations or low-level amplification, which were mutually exclusive with 1 exception) occurred in 10/70 (14%) of CRCs and 19/90 (21%) of ECs. Within ECs, amplification was significantly associated with the subgroup lacking germline mutations in MMR genes (familial site-specific endometrial cancer) (p = 0.015). Decreased or lost PTEN expression was characteristic of endometrial tumourigenesis (51/81, 63%, in EC compared with 24/62, 39%, in CRC, p = 0.004) and KRAS mutations of colorectal tumourigenesis (19/70, 27% in CRC vs. 9/89, 10%, in EC, p = 0.006) regardless of the MMR gene mutation status. PIK3CA alterations frequently coexisted with PTEN or KRAS changes. Combined with published studies on sporadic tumors, our data broaden the understanding of the role for PI3K pathway genes in human tumorigenesis.

DYRK2 Is Targeted to the Nucleus and Controls p53 via Ser46 Phosphorylation in the Apoptotic Response to DNA Damage

Genotoxic stress exerts biological activity by activating downstream effectors, including the p53 tumor suppressor. p53 regulates cell-cycle checkpoint and induction of apoptosis in response to DNA damage; however, molecular mechanisms responsible for committing to these distinct functions remain to be elucidated. Recent studies demonstrated that phosphorylation of p53 at Ser46 is associated with induction of p53AIP1 expression, resulting in commitment to apoptotic cell death. In this regard, the role for Ser46 kinases in p53-dependent apoptosis has been established; however, the kinases responsible for Ser46 phosphorylation have yet to be identified. Here, we demonstrate that the dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) directly phosphorylates p53 at Ser46. Upon exposure to genotoxic stress, DYRK2 translocates into the nucleus for Ser46 phosphorylation. Consistent with these results, DYRK2 induces p53AIP1 expression and apoptosis in a Ser46 phosphorylation-dependent manner. These findings indicate that DYRK2 regulates p53 to induce apoptosis in response to DNA damage.

TbVps34, the Trypanosome Orthologue of Vps34, Is Required for Golgi Complex Segregation

Phosphoinositides are important regulators of numerous cellular functions. The yeast class III phosphatidylinositol 3-kinase Vps34p, and its human orthologue hVPS34, are implicated in control of several key pathways, including endosome to lysosome transport, retrograde endosome to Golgi traffic, multivesicular body formation, and autophagy. We have identified the Vps34p orthologue in the African trypanosome, TbVps34. Knockdown of TbVps34 expression by RNA interference induces a severe growth defect, with a post-mitotic block to cytokinesis accompanied by a variety of morphological abnormalities. GFP2xFYVE, a chimeric protein that specifically binds phosphatidylinositol 3-phosphate, localizes to the trypanosome endosomal system and is delocalized under TbVps34 RNA interference (RNAi), confirming that TbVps34 is an authentic phosphatidylinositol 3-kinase. Expression of GFP2xFYVE enhances the TbVps34 RNAi-associated growth defect, suggesting a synthetic interaction via competition for phosphatidylinositol 3-phosphate-binding sites with endogenous FYVE domain proteins. Endocytosis of a fluid phase marker is unaffected by TbVps34 RNAi, but receptor-mediated endocytosis of transferrin and transport of concanavalin A to the lysosome are both impaired, confirming a role in membranous endocytic trafficking for TbVps34. TbVps34 knockdown inhibits export of variant surface glycoprotein, indicating a function in exocytic transport. Ultrastructural analysis revealed a highly extended Golgi apparatus following TbVps34 RNAi, whereas expression of the Golgi marker red fluorescent protein-GRASP (Grp1 (general receptor for phosphoinositides-1)-associated scaffold protein) demonstrated that trypanosomes are able to duplicate the Golgi complex but failed to complete segregation during mitosis, despite faithful replication and segregation of basal bodies and the kinetoplast. These observations implicate TbVps34 as having a role in coordinating segregation of the Golgi complex at cell division.

A role for PKCζ in potentiation of the topoisomerase II activity and etoposide cytotoxicity by wortmannin

Enhanced cytotoxicity of etoposide by wortmannin, an inhibitor of enzymes holding a phosphatidylinositol 3-kinase domain, was investigated in eight cell lines proficient or deficient for DNA double-strand break repair. Wortmannin stimulated the decatenating activity of topoisomerase II, promoted etoposide-induced accumulation of DNA double-strand breaks, shifted the specificity for cell killing by etoposide from the S to G1 phase of the cell cycle, and potentiated the cytotoxicity of etoposide through two mechanisms. (a) Sensitization to high, micromolar amounts of etoposide required integrity of the nonhomologous end-joining repair pathway. (b) Wortmannin dramatically increased the susceptibility to low, submicromolar amounts of etoposide in a large fraction of the cell population irrespective of the status of ATM, Ku86, and DNA-PKCS. It is shown that this process correlates depression of phosphatidylinositol 3-kinase-dependent phosphorylation of the atypical, zeta isoform of protein kinase C (PKCzeta). Stable expression of a dominant-negative, kinase-dead mutant of PKCzeta in a tumor cell line reproduced the hypersensitivity pattern induced by wortmannin. The results are consistent with up-regulation of the topoisomerase II activity in relation to inactivation of PKCzeta and indicate that PKCzeta may be a useful target to improve the efficiency of topoisomerase II poisons at low concentration.

Alterations of DNA damage-response genes ATM and ATR in pyothorax-associated lymphoma

Pyothorax-associated lymphoma (PAL) is non-Hodgkin's lymphoma that develops from chronic inflammation. Free radicals and oxidative stress generated in the inflammatory lesions could cause DNA damage and thus provide a basis for lymphomagenesis. Ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) genes are responsive genes for DNA damage, therefore potential involvement of these genes in PAL lymphomagenesis was examined in eight PAL cell lines and clinical samples from five cases. ATM mutations were detected in five of eight PAL lines. All but one of these mutations affected the phosphatidylinositol 3-kinase domain, indicating the loss-of-function mutation of ATM gene. Heterozygous mutations of ATR were found in two of eight lines; one a missense and the other a truncation mutation. ATR mutations were also detected in two of five cases in clinical samples from PAL. PAL cells with ATR mutation showed a delay or abrogation in repair for ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) or ultraviolet (UV)-induced DNA single-strand breaks (SSBs), and exhibited a defect in p53 accumulation and failure in cell cycle checkpoint at G1–S phase. These findings showed that mutations of ATR gene result in failure for DNA DSB and SSB repair, suggesting the role of ATM and ATR gene mutations in PAL lymphomagenesis.

Full Activation of PKB/Akt in Response to Insulin or Ionizing Radiation Is Mediated through ATM

The gene mutated in ataxia telangiectasia, ATM, has been implicated in several cell functions such as cell cycle control and response to DNA damage and insulin. PKB/Akt has also been implicated in the cellular response to insulin, gamma-radiation, and cell cycle control. Interestingly, lack of PKB/Akt function in vivo is able to mimic some phenotypic abnormalities associated with ataxia telangiectasia (AT). Here we show that ATM is a major determinant of full PKB/Akt activation in response to insulin or gamma-radiation. This effect is mediated through the phosphatidylinositol 3-kinase domain of ATM that specifically affects Akt serine 473 phosphorylation. This conclusion was inferred from the results obtained in transient transfection assays using exogenous PKB/Akt and ATM in Cos cells. Moreover, the use of ATM inhibitors or small interfering RNA confirmed our observation. Further supporting these results, we also observed that biological responses tightly regulated by Akt, such as transcription factor of the forkhead family activity after insulin treatment or gamma-radiation response, were altered in cell lines derived from AT patients and knockout mice for ATM in which phosphorylation in serine 473 was almost abolished. This study proposes new clues in the search of the unknown PDK2 and new explanations for the radiosensitivity or insulin intolerance described more than 30 years ago in AT patients.

Identification of a Molecular Recognition Role for the Activation Loop Phosphotyrosine of the Src Tyrosine Kinase

A human cDNA phage display library screen, using a phosphopeptide designed to mimic the activation loop phosphotyrosine of the Src tyrosine kinase, has identified the N-terminal SH2 domain of the p85 regulatory subunit of phosphatidyl inositol-3 kinase (PI3K) as an interacting recognition domain. Activation loop phosphorylation is known to play a conformational role in kinase activation, but is largely not thought to play a role in protein/protein recognition. Affinity chromatography and biochemical evaluation in mouse fibroblast cells has confirmed the dependence of this interaction on both the Src activation loop phosphotyrosine and the N-terminal SH2 domain of PI3K.

Transient ischemia enhances tyrosine phosphorylation and binding of the NMDA receptor to the Src homology 2 domain of phosphatidylinositol 3-kinase in the rat hippocampus.

Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3-kinase (PI3-kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3-kinase p85 was co-precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3-kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5-fold relative to sham) was greater than in CA1 (1.7-fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3-kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.

ATM gene inactivation in mantle cell lymphoma mainly occurs by truncating mutations and missense mutations involving the phosphatidylinositol-3 kinase domain and is associated with increasing numbers of chromosomal imbalances

The ataxia-telangiectasia mutated (ATM) gene codifies for a protein critically involved in the cellular response to DNA damage. ATM alterations have been observed in some sporadic lymphoproliferative disorders. The recurrent 11q22-23 deletions found in mantle cell lymphoma (MCL) suggest that ATM could be inactivated in these lymphomas. In this study, ATM gene alterations and protein expression were examined in 20 and 17 MCL tumor specimens, respectively. Previously, these patients had been examined for p53 and p14(ARF) gene status and analyzed by comparative genomic hybridization. Nine patients had 11q22-23 losses. Eight ATM gene mutations were detected in 7 patients. These alterations were 3 missense mutations in the phosphatidylinositol-3 kinase (PI-3K) domain and 5 truncating mutations, including 3 frameshifts, a nonsense mutation, and a substitution of the initial methionine. All truncating mutations were associated with lack of protein expression. Somatic origin was demonstrated in 3 mutations, whereas one mutation was carried heterozygously in the patient germ line. Chromosomal imbalances were significantly higher in typical MCL with ATM inactivation (7.8 +/- 1.3) than in tumors with the wild-type gene (3 +/- 1.1) (P =.001). Moreover, tumors with bi-allelic ATM alteration were associated with 3q gains (P =.015) and frequent extranodal involvement (P =.049). ATM gene alterations were not related to the histologic variant of the tumors, p53/p14(ARF) gene status, survival, or other clinicopathologic features of the patients. These findings indicate that ATM gene mutations in MCL are mainly truncating or missense mutations involving the PI-3K domain, and that may play a role in the pathogenesis of a subset of these tumors with increased numbers of chromosomal imbalances.

Identification of four highly conserved regions in DNA-PKcs.

The gene for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is responsible for severe combined immune deficiency (SCID) and its products associate with Ku70/Ku86 autoantigens, c-Abl, and other factors to exert its roles. Investigations to date suggest that DNA-PKcs comprises several regions which specifically interact with these known and other as yet unidentified factors. Nevertheless, the relationship between the structure and function of the DNA-PKcs molecule is poorly understood. Here we report cloning of the entire DNA-PKcs cDNA from chicken and Xenopus laevis. Comparative study of the DNA-PKcs polypeptides from four different vertebrates revealed at least three novel conserved regions in addition to the carboxyl-terminal region containing the phosphatidylinositol-3 kinase domain. We also demonstrated that the four vertebrates share the common genomic organization between a licensing factor, MCM4, and DNA-PKcs, both of which locate in a head-to-head manner within a few kilobase intervals. These data provide useful clues for the further genetic study of this huge multifunctional enzyme.