PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Evidence of a Role for Phosphatidylinositol 3-Kinase Activation in the Blocking of Apoptosis by Polyomavirus Middle T Antigen

A polyomavirus mutant (315YF) blocked in binding phosphatidylinositol 3-kinase (PI 3-kinase) has previously been shown to be partially deficient in transformation and to induce fewer tumors and with a significant delay compared to wild-type virus. The role of polyomavirus middle T antigen-activated PI 3-kinase in apoptosis was investigated as a possible cause of this behavior. When grown in medium containing 1d-3-deoxy-3-fluoro-myo-inositol to block formation of 3′-phosphorylated phosphatidylinositols, F111 rat fibroblasts transformed by wild-type polyomavirus (PyF), but not normal F111 cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI 3-kinase, stimulated apoptosis in PyF cells but not in normal cells. Activation of Akt, a serine/threonine kinase whose activity has been correlated with regulation of apoptosis, was roughly twofold higher in F111 cells transformed by either wild-type virus or mutant 250YS blocked in binding Shc compared to cells transformed by mutant 315YF. In the same cells, levels of apoptosis were inversely correlated with Akt activity. Apoptosis induced by serum withdrawal in Rat-1 cells expressing a temperature-sensitive p53 was shown to be at least partially p53 independent. Expression of either wild-type or 250YS middle T antigen inhibited apoptosis in serum-starved Rat-1 cells at both permissive and restrictive temperatures for p53. Mutant 315YF middle T antigen was partially defective for inhibition of apoptosis in these cells. The results indicate that unlike other DNA tumor viruses which block apoptosis by inactivation of p53, polyomavirus achieves protection from apoptotic death through a middle T antigen–PI 3-kinase–Akt pathway that is at least partially p53 independent.Programmed cell death occurs during normal development and under certain pathological conditions. In mammalian cells, apoptosis can be induced by a variety of stimuli, including DNA damage (45), virus infection (54, 57), oncogene activation (25), and serum withdrawal (34, 37). Apoptosis can also be blocked by a number of factors, including adenovirus E1B 55- or 19-kDa proteins (9, 16), baculovirus p35 and iap genes (10), Bcl-2 (36, 61), and survival factors (12, 21). DNA tumor viruses have evolved mechanisms that both trigger and inhibit apoptosis. These frequently involve binding and inactivation of tumor suppressor proteins. E7 in some papillomaviruses (22), E1A in adenovirus (31, 43, 64), and large T antigen in simian virus 40 (SV40) (17) bind Rb and/or p300 and lead to upregulation of p53, which is thought to trigger apoptosis in virus-infected cells. The same viruses also inhibit apoptosis by inactivating p53 by various mechanisms (44, 63, 67). In contrast, the mechanism by which polyomavirus interacts with apoptotic pathways in the cell is not known; no direct interaction with p53 by any of the proteins encoded by this virus has been demonstrated (19, 62).The principal oncoprotein of polyomavirus is the middle T antigen. Neoplastic transformation by polyomavirus middle T antigen has as a central feature its association with and activation of members of the Src family of tyrosine kinases p60^c-src (13) and p62^c-yes (42). The major known consequence of these interactions is phosphorylation of middle T antigen on specific tyrosine residues creating binding sites for other signaling proteins. Phosphorylation at tyrosines 250, 315, and 322 promotes binding to Shc (18), the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (59), and phospholipase Cγ-1 (58), respectively. Recognition of multiple signaling pathways emanating from middle T antigen has led to a keen interest in identifying their downstream biochemical effects, which collectively lead to the emergence of neoplastic transformation and presumably underlie the dramatic ability of the virus to induce many kinds of tumors in the mouse.Previous work has shown that the binding of PI 3-kinase to middle T antigen is essential for full transformation of rat fibroblasts in culture (8) and for rapid development of a broad spectrum of tumors in mice (30), for translocation of the GLUT1 transporter (68), and activation of p70 S6 kinase (14). While the mutant 315YF (blocked in PI 3-kinase activation) was able to induce some tumors, it did so at reduced frequencies and with an average latency three times longer than that of either the wild-type virus or a mutant, 250YS, blocked in binding Shc (4, 30). Recent studies have indicated a role of PI 3-kinase in blocking apoptosis in nonviral systems. Growth factor receptors acting through protein tyrosine kinases may prevent apoptosis by activating PI 3-kinase in PC12 cells, T lymphocytes, hematopoietic progenitors, and rat fibroblasts (7, 48, 56, 65, 66). The failure of mutant 315YF to induce full transformation of cells in culture and to induce the rapid development of tumors in mice could therefore be related, at least in part, to a failure to block apoptosis. In this study, we focus on the question of whether middle T antigen–PI 3-kinase interaction is involved in blocking apoptosis in cells transformed by polyomavirus.     

Repair of Nitric Oxide-damaged DNA in ��-Cells Requires JNK-dependent GADD45�� Expression

Proinflammatory cytokines induce nitric oxide-dependent DNA damage and ultimately β-cell death. Not only does nitric oxide cause β-cell damage, it also activates a functional repair process. In this study, the mechanisms activated by nitric oxide that facilitate the repair of damaged β-cell DNA are examined. JNK plays a central regulatory role because inhibition of this kinase attenuates the repair of nitric oxide-induced DNA damage. p53 is a logical target of JNK-dependent DNA repair; however, nitric oxide does not stimulate p53 activation or accumulation in β-cells. Further, knockdown of basal p53 levels does not affect DNA repair. In contrast, expression of growth arrest and DNA damage (GADD) 45α, a DNA repair gene that can be regulated by p53-dependent and p53-independent pathways, is stimulated by nitric oxide in a JNK-dependent manner, and knockdown of GADD45α expression attenuates the repair of nitric oxide-induced β-cell DNA damage. These findings show that β-cells have the ability to repair nitric oxide-damaged DNA and that JNK and GADD45α mediate the p53-independent repair of this DNA damage.Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of insulin-secreting pancreatic β-cells found in the islets of Langerhans (1). Cytokines, released from invading leukocytes during insulitis, are believed to participate in the initial destruction of β-cells, precipitating the autoimmune response (2, 3). Treatment of rat islets with the macrophage-derived cytokine interleukin-1 (IL-1)² results in the inhibition of glucose-stimulated insulin secretion and oxidative metabolism and in the induction of DNA damage that ultimately results in β-cell death (4–6). Nitric oxide, produced in micromolar levels following enhanced expression of the inducible nitric-oxide synthase in β-cells, mediates the damaging actions of cytokines on β-cell function (7–9). Nitric oxide inhibits insulin secretion by attenuating the oxidation of glucose to CO₂, reducing cellular levels of ATP and, thereby, attenuating ATP-inhibited K⁺ channel activity (10, 11). The net effect is the inhibition of β-cell depolarization, calcium entry, and calcium-dependent exocytosis. In addition to the inhibition of β-cell function, nitric oxide induces DNA damage in β-cells (4, 12, 13). Nitric oxide or the oxidation products N₂O₃ and ONOO⁻ induce DNA damage through direct strand breaks and base modification (14–16) and by inhibition of DNA repair enzymes, thereby enhancing the damaging actions of nitric oxide (17, 18).Recent studies have shown that β-cells maintain a limited ability to recover from cytokine-mediated damage (19, 20). The addition of a nitric-oxide synthase inhibitor to islets treated for 24 h with cytokine and continued culture with the nitric-oxide synthase inhibitor and cytokine results in a time-dependent restoration of insulin secretion, mitochondrial aconitase activity, and the repair of nitric oxide-damaged DNA (20, 21). Nitric oxide plays a dual role in modifying β-cell responses to cytokines. Nitric oxide induces β-cell damage and also activates a JNK-dependent recovery response that requires new gene expression (22). The ability of β-cells to recover from cytokine-mediated damage is temporally limited because cytokine-induced β-cell damage becomes irreversible following a 36-h incubation, and islets at this point are committed to degeneration (19).The purpose of this study was to determine the mechanisms by which β-cells repair nitric oxide-damaged DNA. Previous reports have shown that DNA damage induced by oxidizing agents, such as nitric oxide, is repaired through the base excision repair pathway (23), but how this pathway is activated in response to nitric oxide is unknown. Similar to the recovery of metabolic function, we now show that the activation of JNK by nitric oxide is required for repair of cytokine-induced DNA damage in β-cells. p53 is a logical candidate to mediate this repair because it plays a central role in DNA repair, is a target of JNK, and is activated by nitric oxide (24–27). However, we show that cytokines do not stimulate p53 phosphorylation, and nitric oxide fails to stimulate p53 accumulation and phosphorylation. Growth arrest and DNA damage (GADD) 45α is a DNA damage-inducible gene that can be regulated by both p53-dependent and p53-independent mechanisms (28–31). In contrast to p53, we show that cytokines stimulate GADD45α expression in a nitric oxide- and JNK-dependent manner and that siRNA-mediated knockdown of GADD45α results in an attenuation in the repair of nitric oxide-mediated DNA damage. These findings support a role for JNK in the regulation of GADD45α-dependent and p53-independent repair of nitric oxide-damaged β-cell DNA.     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]