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.     

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]     

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.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Complex Lipid Requirements for SNARE- and SNARE Chaperone-dependent Membrane Fusion

Membrane fusion without lysis has been reconstituted with purified yeast vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE chaperones Sec17p/Sec18p and the multifunctional HOPS complex, which includes a subunit of the SNARE-interactive Sec1-Munc18 family, and vacuolar lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), ergosterol (ERG), diacylglycerol (DAG), and phosphatidylinositol 3-phosphate (PI3P). We now report that many of these lipids are required for rapid and efficient fusion of the reconstituted SNARE proteoliposomes in the presence of SNARE chaperones. Omission of either PE, PA, or PI3P from the complete set of lipids strongly reduces fusion, and PC, PE, PA, and PI3P constitute a minimal set of lipids for fusion. PA could neither be replaced by other lipids with small headgroups such as DAG or ERG nor by the acidic lipids PS or PI. PA is needed for full association of HOPS and Sec18p with proteoliposomes having a minimal set of lipids. Strikingly, PA and PE are as essential for SNARE complex assembly as for fusion, suggesting that these lipids facilitate functional interactions among SNAREs and SNARE chaperones.Biological membrane fusion is the regulated rearrangement of the lipids in two apposed sealed membranes to form one bilayer while mixing lumenal contents without leakage or lysis. It is fundamental for intracellular vesicular traffic, cell growth and division, regulated secretion of hormones and other blood proteins, and neurotransmission and thus has attracted wide and sustained study (1, 2). Its fundamental mechanisms are conserved and employ a Rab-family GTPase, proteins which bind to the GTP-bound form of a Rab, termed its “effectors” (3), and SNARE³ (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins (4) with their attendant chaperones. SNAREs are integral or peripheral membrane proteins with characteristic heptad-repeat domains, which can associate in 4-helical coiled-coils (5), termed “cis-SNARE complexes,” if they are all anchored to the same membrane bilayer, or “trans-SNARE complexes” if they are anchored to apposed membranes.Stable membrane proximity (docking) does not suffice for fusion. Studies in model systems have shown that fusion can be promoted by any of several agents, which promote bilayer rearrangement, such as diacylglycerol (6), high levels of calcium (7), viral-encoded fusion proteins (8, 9), or SNAREs (10, 11). These studies frequently employed liposomes or proteoliposomes of simple lipid composition, suggesting that fusion may not have stringent requirements of lipid head group species. However, each of these model fusion reactions is accompanied by substantial lysis (12–15), whereas the preservation of subcellular compartments is a hallmark of physiological membrane fusion.We have studied membrane fusion with the vacuole (lysosome) of Saccharomyces cerevisiae (reviewed in Ref. 16). The fusion of isolated vacuoles requires the Rab Ypt7p, 4 SNAREs (Vam3p, Vti1p, Vam7p, and Nyv1p), the SNARE chaperones Sec17p (α-soluble N-ethylmaleimide-sensitive factor attachment protein)/Sec18p (N-ethylmaleimide-sensitive factor) and the hexameric HOPS complex (17), and key “regulatory” lipids including ERG, phosphoinositides, and DAG (18). HOPS interacts physically or functionally with each component of this fusion system. HOPS stably associates with Ypt7p in its GTP-bound state (19). One HOPS subunit, Vps33p, is a member of the Sec1-Munc18 family of SNARE-binding proteins, and HOPS exhibits direct affinity for SNAREs (17, 20–22) and proofreads correct vacuolar SNARE pairing (23). HOPS also has direct affinity for phosphoinositides (17). The SNAREs on isolated vacuoles are in cis-complexes, which are disassembled by Sec17p, Sec18p, and ATP (24). Docking requires Ypt7p (25) and HOPS (17). During docking, vacuoles are drawn against each other until each has a substantial membrane domain tightly apposed to the other. Each of the proteins (26) and lipids (18) required for fusion becomes enriched in a ring-shaped microdomain, the “vertex ring,” which surrounds the two tightly apposed membrane domains. Not only do the proteins depend on each other, in a cascade fashion, for vertex ring enrichment, and the lipids depend on each other for their vertex ring enrichment as well, but the lipids and proteins are mutually interdependent for their enrichment at this ring-shaped microdomain (18, 27). Fusion occurs around the ring, joining the two organelles. The fusion of vacuoles bearing physiological fusion constituents does not cause measurable organelle lysis, although fusion supported exclusively by higher levels of SNARE proteins is accompanied by massive lysis (28), in accord with model liposome studies (14). Thus fusion microdomain assembly and the coordinate action of SNAREs with other proteins and lipids to promote fusion without lysis are central topics in membrane fusion studies.Reconstitution of fusion with pure components allows chemical definition of essential elements of this biologically important reaction. Although SNAREs can drive a slow fusion of PC/PS proteoliposomes (29), this was not stimulated by HOPS and Sec17p/Sec18p (30). SNARE proteoliposomes bearing all the vacuolar lipids (18, 31–33), PC, PE, PI, PS, CL, PA, ERG, DAG, PI3P, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), showed rapid and efficient fusion that was fully dependent on Sec17p/Sec18p and HOPS (30). The omission of either DAG, ERG, or phosphoinositide from the liposomes caused a marked reduction in fusion (30). We now report that PE and PA are also necessary for rapid and efficient fusion, function in distinct manners, and are required for efficient assembly of newly formed SNARE complexes by the SNARE chaperones Sec17p/Sec18p and HOPS.