Nuclear Phospholipase C Gamma: Punctate Distribution and Association with the Promyelocytic Leukemia Protein Brian J. Ferguson,†,# Claire L. Dovey,†,‡,# Kathryn Lilley,§ Andrew H. Wyllie,† and Tina Rich*,†,| Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, Department of Biochemistry, Proteomics Centre, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom, and Institute of Comparative Medicine, Faculty of Veterinary Sciences, University of Glasgow, United Kingdom Received December 20, 2006
The marriage between transducers of cell stress stimuli and their nuclear targets is likely to be achieved in part by some spatial-temporal compartmentalization of the relevant effectors. A candidate compartment for these events is the promyelocytic leukemia nuclear domain (PML-ND), within which are found numerous effectors of damage recognition, repair, and cell death. We predicted that the identification of PML-ND cargo proteins would clarify those biochemical pathways that straddle the recognition of cellular damage and cell fate. We now use mass spectrometry of peptides eluted from PML coprecipitates to demonstrate that the gamma 1 (γ1) isoform of PLC associates with nuclear PML. Though thought to act primarily in the cytoplasm, we use biochemical fractionation combined with immunocytochemistry to verify the nuclear expression of PLC-γ1 and its interaction with PML. These are the first data to show an interaction between endogenous levels of a phosphoinositide metabolizing protein and the biophysically labile PML-ND by mass spectrometry and add weight to the view that PML-NDs may act as tumor suppressors by sequestering mitogenic effectors. Keywords: Promyelocytic leukemia nuclear domain • Nucleus • Phospholipase C gamma • Mitogen
Introduction It is now widely appreciated that the mammalian nucleus contains multiple, distinct, subnuclear bodies; some have welldefined functions, while others are more enigmatic.1,2 The most conspicuous subnuclear body is the nucleolus,3 the ribosome factory of the cell that assembles and disassembles with each cellular division.4 However, nucleoli appear to house many more nucleoprotein activities than those purely associated with ribosome biosynthesis, many of which have now been identified by mass spectrometry (MS).5 Improved MS coupled with enrichment by affinity techniques6,7 has permitted the characterization of a number of large protein complexes, including the BRCA1-associated genome surveillance (BASC) complex8 in the nucleus, death inducing signaling complex (DISC) at the cell membrane,9 and the apoptosome complex associated with the mitochondrial outer membrane,10 each of which appears * To whom correspondence should be addressed. E-mail: T.Rich@ vet.gla.ac.uk, Institute of Comparative Medicine, Faculty of Veterinary Sciences, University of Glasgow, UK. Tel: (44) 0161 330 8613. † Department of Pathology, University of Cambridge. ‡ Department of Molecular Biology, The Scripps Research Institute (present address). # These authors contributed equally and should be considered joint first authors. § Department of Biochemistry, Proteomics Centre, University of Cambridge. | Institute of Comparative Medicine, Faculty of Veterinary Sciences, University of Glasgow (present address). 10.1021/pr060684v CCC: $37.00
2007 American Chemical Society
to combine incoming “sensory” with downstream effector signaling functions. While the density of nucleoli readily allows their enrichment by centrifugation, no such option exists for another subnuclear body, the promyelocytic leukemia nuclear domain (PML-ND).11 Thirty or more of these structures may be expressed in individual nuclei, each being between 0.2 and 1 µm in diameter with the PML protein itself acting as the principal structural component. Rather like nucleoli, PML-NDs also contain multiple protein activities, collectively termed the PML-ND cargo.11 Unlike nucleoli, no singular role has been found for the PMLND. Instead, the physical organization of the PML-ND itself appears to provide the basis of its function as an environment for the storage and modification of cargo proteins.11,12 Acting as a giant scaffold, each PML-ND is the nexus of multiple stress pathways, whose signals are communicated to select constituent proteins.13,14 The exquisite sensitivity of PML-NDs to stress is demonstrable by their modification in terms of size, number, distribution, and, importantly, nucleoprotein composition, following cell injury.13-16 The dynamism of PML-NDs may well contribute to the capacity of the cell to withstand injury, a relationship that is underscored by the aberrant survival of irradiated PML null thymocytes.17 The frequent loss of PML protein in solid tumors presumably also confers a similar growth advantage.18 Revealing the identity of PML-ND resident and facultative proteins has long been the aim of several groups. However, Journal of Proteome Research 2007, 6, 2027-2032
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letters conventional cell fractionation approaches have largely failed to enrich PML-NDs and their protein cargo. There are several reasons for this. First is the fragility of the PML-ND. The biochemical modifications that render PML-NDs responsive to stress, for example, desumoylation,13,14,19,20 are equally welltriggered by any attempt to extract intact PML-NDs from the matrix and then nucleus. Desumoylation is catalyzed by isopeptidases21 whose activity is difficult to inhibit with specificity. Second, unlike nucleoli, PML-NDs are not amenable to isolation by gradient centrifugation being of comparable magnitude and density to many other subnuclear structures. Third, and most importantly, desumoylation of PML-NDs provokes the release of their cargo proteins, the very targets that we are attempting to identify.13,14 We now report the identification of the gamma 1 isoform of phospholipase C (PLC-γ1) as a novel PML-ND cargo protein. We adapted and combined two protocols, one to enrich nuclei for cell free replication22 and one to isolate intact Cajal bodies,23 to extract intact PML-NDs. PML protein was then immunoprecipitated. and the proteins captured from these and control immunoprecipitates were separated by one-dimensional SDSPAGE. Proteins bands were subjected to in-gel trypsinization, and the eluted peptides were identified by tandem mass spectrometry. The association of PML and PLC-γ1 was subsequently corroborated by a combination of microscopic and biochemical methodologies.
Experimental Section Cell Lines. Human diploid lung fibroblasts (WI38 and IMR90) were obtained from the ATCC, and HCT116 human colon carcinoma cells were from Dr. Bert Vogelstein (The Johns Hopkins University Medical Institution, Baltimore, MD). WI38 and HCT116 cells were grown in DMEM (with GLUTAMAX) standard medium. The promyelocytic leukemia cell line NB4 was cultured as a suspension in RPMI standard medium. All cell lines were cultured in a humidified incubator at 37 °C and 5% CO2. Preparation of Nuclear Lysates. Cells grown on 15 cm dishes until 70% confluent were transferred to a 4 °C cold room where all further steps were carried out. Medium was removed; cells were rinsed in LS buffer (20 mM K-HEPES, pH 7.8, 5 mM K-Ac, 0.5 mM MgCl2, and 0.5 mM DTT), then overlaid with 20 mL LS buffer, and allowed to swell for 10 min. LS buffer was removed, and cells were scraped into 1 mL dounce homogenizers (Wheaton Scientific Products), containing 1× protease inhibitor cocktail. Cells were disrupted by 25 strokes of the homogenizer, using a loose fitting pestle, and the cell extract was spun at 1000g for 2 min. The nuclear pellet was resuspended in 2 vol of sonication buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM MgCl2, 1× protease inhibitor cocktail, and 20 mM iodoacetamide) and pulsed for 3 × 6 s using a Sanyo MSE Soniprep 150 ultrasonic homogenizer. Sodium chloride and NP-40 were subsequently added to final concentrations of 150 mM and 0.1%, respectively. Immunoprecipitations and Mass Spectrometry. For largescale experiments, nuclear lysates were clarified (15 min microfuge spin at 13 000 rpm) and precleared by incubation for 30 min with 50 µL of protein-G Sepharose (Sigma). A total of 50 mg of nuclear lysate was used per immunoprecipitation, with 200 µg of immunoglobulin. The following precipitating antibodies were used: goat polyclonal IgG and mouse monoclonal IgG1K were controls (Sigma), monoclonal anti-PLC-γ1 (E12, Santa Cruz, CA), monoclonal anti-PML (PGM3, Santa Cruz Biotech), and anti-PML (N19, Santa Cruz Biotech). 2028
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For small-scale co-immunoprecipitation experiments, HCT116 cells were lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 0.2% Triton-X100 plus Complete Mini protease inhibitors (Roche, Mannheim, Germany)) and clarified, and total protein was quantified by BCA assay (Pierce, Rockford, IL). A total of 100 µg of lysate was precleared with 50 µL of protein-G Sepharose for 1 h before immunoprecipitation with 10 µg of anti-PML (PGM3) and 50 µL of protein-G Sepharose for 3 h. Immunoprecipitated proteins were separated by SDS-PAGE and detected by Western blotting. One-dimensional SDS-PAGE gels were fixed in Sypro Ruby (Molecular Probes) fixation solution (40% methanol and 10% glacial acetic acid) for 1 h then stained overnight. The gel was transferred to wash solution (10% methanol and 7% glacial acetic acid) and then scanned using a Typhoon 9400 imager (GE Healthcare), with excitation at 532 nm and emission at 610 nm. Proteins within the gel pieces were first reduced and carboxyamidomethylated using DTT and iodoacetamide, and then digested to peptides using trypsin on a MassPrepStation (Waters, Manchester, U.K.). The resulting peptides were applied to a LC-MS/MS. For LC-MS/MS, the reverse-phase liquid chromatographic separation of peptides was achieved with a PepMap C18 reverse-phase, 75 mm i.d., 15-cm column (LC Packings, Amsterdam) on a capillary LC system (Waters) attached to a QTof2 (Waters) mass spectrometer. The MS/MS fragmentation data achieved was used to search the National Center for Biotechnology Information database using the MASCOT search engine (http://www.matrixscience.com). Probability-based MASCOT scores were used to evaluate identifications. Only matches with P < 0.05 for random occurrence were considered significant (further explanation of MASCOT scores can be found at http://www.matrixscience.com). Size-Exclusion Chromatography. Size-exclusion chromatography was performed using a Sephacryl 500 column (Amersham Biosciences, Uppsala, Sweden) of 30 cm height and 10 mm diameter. Nuclear lysates were prepared in an identical fashion to those used in the large-scale immunoprecipitation experiments. Samples were eluted isocratically with a buffer of 10 mM Tris-HCl (pH 7.4) and 100 mM NaCl at a flow rate of 0.3 mL/min. One-milliliter fractions were collected for analysis by Western blotting. To estimate molecular masses, 100 µg each of thyroglobulin (667 kDa), ferritin (440 kDa), aldolase (168 kDa), and bovine serum albumin (67 kDa) (Amersham Biosciences) were applied to the same column, and identical elution was performed. SDS-PAGE Western Blotting. SDS-PAGE Western blotting was performed according to standard protocols using the following antibodies; anti-PML polyclonal Ab1370 (Chemicon), anti-PML (N19, Santa Cruz), anti PLC-γ1 (E12, Santa Cruz) diluted 1/500, anti-DAXX (M112, Santa Cruz) diluted 1/250, and anti-SUMO (clone 21C7- Invitrogen, Paisley, U.K.) diluted 1/200. HRP conjugates were purchased from Dako (Glostrup, Denmark). Immunofluorescence. Immunocytochemistry was as previously described.24 Anti PML (N19, Santa Cruz, CA) was used at 1/250 and anti anti-PLC-γ1 at 1/100. Alexa-fluor-conjugated secondary antibodies (Invitrogen) were used at a dilution of 1/500.
Results and Discussion Mass Spectrometry identification of PLC-γ1 as a PML Binding Protein. Nuclear extracts of HCT116 colon carcinoma cells were subject to immunoprecipitation (IP) using anti-PML
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Figure 1. Identification of PLC-γ1 as a PML interacting protein. Left, upper panel; nuclear extracts of HCT116 cells were immunoprecipitated using either PGM3 (+ lane) or an isotype control antibody (- lane) and resolved by one-dimensional SDS-PAGE. Filled arrow heads denote the position of putative PLC-γ1 and PML-1 bands; hc and lc indicate immunoglobulin heavy and light chain signals. Boxed regions A and C indicate areas from which bands were excised and subject to tandem mass spectrometry; though shown superposed (for clarity), these regions derive from multiple IPs run in parallel (not shown). Magnified views of A and C (upper right) indicate the positions of bands H1 and G3, which cross reference to the most significant Mowse scores. Left, middle panel; Western blot with N19 confirms PML enrichment by PGM3; lower panel, Western blot to detect SUMO-1 reveals substantive SUMO-1 enriched band at the same apparent molecular weight as PML-1 following IP with PGM3. Table 1. Protein Identities Were Acquired by Entering MS/MS Fragmentation Data into the MASCOT Search Engine sample
H1
D2 G3
possible identities based on Mowse scorea
Phospholipase C, gamma 1 (human) Phospholipase C, gamma 1 (mouse) Phospholipase C, gamma 1 (Bos taurus) PML-1 putative zinc finger protein (human) Phospholipase C, gamma 1 (human) Phospholipase C, gamma 1 (mouse) Phospholipase C, gamma 1 (B.taurus)
peptide Mowse size matches score kDa
30 26 22 6
1200 1025 830 248
150 137 150 97
26 22 18
988 803 656
150 137 150
Score of >51 indicates identity or extensive homology, p < 0.05. Keratin and trypsin contaminants are omitted. a
(PGM3) and an isotype control antibody (Figure 1). The success of the IP was confirmed by the identification of 6 peptides of PML1 in the anti-PML but not control immunoprecipitates. These PML1 peptides gave a Mowse score of 248 (Table 1) and molecular mass of 97 kDa (Mowse > 51 indicates identity or extensive homology, p < 0.05). While a majority of other bands had high background levels of contaminants (trypsin and keratin derivatives), or were masked by traces of immunoglobulin, two other protein identities were obtained, one of which is further characterized in this paper. Phospholipase C-γ1 peptides were uniquely identified in samples analyzed from the anti-PML immunoprecipitates, from gel slices H1, and G3, excised from similar gel positions in parallel IPs. These regions of the gel were chosen as they corresponded to a prominent band in the antiPML, but not control, precipitates. The Mowse scores for our PLC-γ1 ID were 988 over 26 peptides (G3 slice) and 1200 from 30 peptides (H1). A molecular weight of 150 kDa was recorded for both bands (Table 1). With the caveat that contaminating immunoglobulin may mask peptide signatures in our negative controls, we proceeded to confirm our mass spectrometry data
Figure 2. PML coprecipitates with PLC-γ1 in HCT116 but not NB4 cells. The upper panel shows that PLC-γ1 coprecipitates with PML following its capture with PGM3 but not an isotype control antibody in lysates of HCT116 nuclei. The middle panel shows no PML/PLC-γ1 coprecipitation in NB4 nuclear lysates. The LH track was loaded with cell lysate as a positive control for the blotting antibodies. Successful capture of PML from NB4 cells using PGM3 as the precipitating antibody is confirmed in the bottom panel.
by Western blot. Again, we found coprecipitation of PLC-γ1 with PML by small scale IPs and Western blot (Figure 2) in our anti-PML, but not isotype control, precipitates. However, immunoprecipitation with an anti-PLC-γ1 antibody, then Western blot with anti-PML failed to reveal any detectable association between the two proteins. The relative concentrations of either antigen or antibody affinity could contribute to the unidirectional success of the co-IP. A relatively low concentration of PLC-γ1 associating with PML, with the major pool elsewhere, could prevent detection of PML following an IP against PLC-γ1. An abundance of PLC-γ1 will also bias toward its detection following IP. PLC-γ1 activity is known to be high in HCT116 colon carcinoma cells. Likewise, PLC-γ1 accumulation at the expense of protein kinase A (PKA) activity is known to play a Journal of Proteome Research • Vol. 6, No. 5, 2007 2029
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Figure 3. Expression of nuclear PLC-γ1 puncta in HCT116 nuclei. The first three panels show immunofluorescent staining of PML (green), PLC-γ1 (red), and DAPI (blue) in HCT116 nuclei. The distribution of PLC-γ1 (red) in IMR90 fibroblasts is shown in the far right panel. The scale bars denote 10 µm.
role in the maintenance of the transformed phenotype of promyelocytic leukemia cells such as the NB4 cell line.25-27 In NB4 cells, PML protein is sequestered by a PML-RAR alpha fusion protein which results in its microspeckled rather than punctate distribution. To test for any association between PML/ PML fusion proteins and PLC-γ1, we immunoprecipitated PML from NB4 lysates and performed Western blots with an antiPLC-γ1 antiserum. These experiments revealed that PLC-γ1 was not found in a complex with PML in NB4 cells (Figure 2). PML and PLC-γ1 Form Nuclear Puncta. PLC-γ1 is a eukaryotic, ubiquitously expressed isoform of the PLC-γ subtype of the phosphoinositide-specific PLC isozymes.28-30 PLC-γ is involved in cellular proliferation and differentiation, and its enzymatic activity, the hydrolysis of PtdIns(4,5)P2 to diacylglyercol and inositol 1,4,5-trisphosphate, is up-regulated by a variety of growth factors and hormones.28-30 PLC-γ1 does not express any recognizable nuclear localization signal and is generally resident in the cytoplasm from where it shuttles to various membranes following the receipt of mitogenic stimuli. However there have been reports of nuclear PLC-γ1,31-34 and expression in this compartment has been linked to cellular transformation.31,32 Dual immunostains of PLC-γ1 and PML were conducted to assess their distribution in HCT116 nuclei. Interestingly, we found that PLC-γ1 forms prominent nuclear puncta of similar magnitude to PML-NDs in HCT116 nuclei (Figure 3). To our knowledge, this is the first description of punctate nuclear PLC-γ1. PLC- γ1 puncta were frequently juxtaposed with PML-NDs, though did not co-localize. The close apposition of nuclear structures is thought to facilitate their exchange of nucleoproteins and is a newly recognized paradigm for the co-regulation of nuclear domains.35 A good example of this is the apposition of PML-NDs with ionizing radiation induced foci following genotoxic damage.16,36 Elegant examples of PML-ND juxtaposition but not co-localization can also be found with splicing domains enriched with SC35 as well as coiled bodies and have been used to back a model of specific nuclear deposition sites for effector function.37 Collectively, our immunoprecipitation and immunofluorescence data would suggest that both PML and PLC-γ1 may associate with common scaffold elements. The nuclear punctate distribution of PLC-γ1 in HCT116 nuclei contrasts markedly with the vesicular staining of PLC-γ1 seen in normal diploid fibroblasts (Figure 3). In these cells, the staining was largely cytoplasmic and vesicular and indicated PLC-γ1 localized to either the Golgi or endoplasmic reticulum, which is reminiscent of Ras induced PLC-γ1 translocation.38 We considered the possibility that glucose deprivation may induce the translocation of PLC-γ1 between cellular compartments in a similar fashion to that seen with DAXX.39 However, 2030
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modification of serum and glucose levels in our culture media did not generate reproducibly altered staining patterns for PLCγ1. Size-Exclusion Chromatography Shows Comigration of PLC-γ1 and PML. To confirm the expression of nuclear PLCγ1 puncta, we prepared nuclear extracts of HCT116 nuclei that were size-fractionated using a Sephacryl 500 column. Eluted fractions were immunoblotted for PML, PLC-γ1, and a cognate PML-ND cargo protein, DAXX. As we have previously found, PML elution occurred across a broad swathe of fractions, with size estimates ranging from 50 kDa to megaDaltons. This is consistent with the preservation of fragmented PML-NDs and monomeric PML. PLC-γ1 and DAXX elution occurred in the fractions with peak PML concentration and multiple PML isoforms40 (Figure 4). The MS identification of PLC-γ1 as a nuclear PML-ND resident protein was initially surprising given that PLC-γ1 activity is customarily associated with a cytoplasmic location, concordant with mitogenic signaling via receptor tyrosine kinases. However, several reports have suggested that PLC-γ1 and, indeed, many of the enzymes responsible for inositiol lipid metabolism, can locate in the nucleus as well as the cytoplasm.31-34 Molecular details of one nuclear PLC-γ1 activity have recently emerged from a study of nerve growth factor (NGF) signaling34 in which NGF was found to stimulate translocation of PLC-γ1 from the cytoplasm to nucleus. PLCγ1 was shown to act as a guanine nucleotide exchange factor (GEF) for phosphatidyl-inositol 3′ phosphate enhancer (PIKE), a small nuclear GTPase that stimulates PI-3K activity. Interestingly, PLC-γ1 has also been shown to be required for survival following various stresses,41-43 a role that appears analogous to that of PML-NDs in mediating intranuclear stress responses. A more general role for nuclear PLC-γ1 in mitogenic signaling is suggested from a study using pancreatic tissue from PTEN/ PML null mice.44 PTEN is a phosphatidylinositol phosphatase (and tumor suppressor) that inhibits the mitogenic PI-3K/AKT (PKB) signaling pathway. Loss of PTEN in tumors leads to an accumulation of pAKT at the plasma membrane. Many targets of pAKT are nuclear; however, and in this respect, it is provocative that pAKT was recently found to be sequestered in PML-NDs. This sequestration was proposed to be a method whereby mitogenic signaling could be inactivated.44 Our finding that PLC-γ1 associates with PML-NDs would support the notion that PML-NDs can suppress nuclear mitogenic stimuli by sequestering multiple effector proteins. The identification of prominent nuclear puncta of PLC-γ1 was completely unexpected, and preliminary attempts to discover the stimuli that underlie this distribution are unclear. We can rule out serum or glucose deprivation effects. It is also unlikely that background DNA damage is responsible for nuclear PLC-γ1 accumulation, as HCT116 cells detect endogenous DNA damage
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Figure 4. Evidence for high molecular weight nuclear complexes containing PLC-γ1, PML, and DAXX. Fractions eluted from the sizeexclusion matrix were blotted for PML (upper bands), PLC-γ1 (middle bands), and DAXX (lower bands). The void volume and sizeexclusion limit is indicated. Thyroglobulin (667 kDa) eluted in fractions B2/3, ferritin (440 kDa) in fraction B5, and aldolase (168 kDa) in fractions B6/7.
primarily at the G2 phase, which would represent a small fraction of the cells in culture at any one time. At least two (and probably more) pathways are over-active in HCT116 cells. One involves PLC-γ1 and the other, ERK. Either of these may contribute to the nuclear deposition of PLC-γ1. The finding that PLC-γ1 coprecipitates with PML, but not vice-versa, suggests that only a fraction of the pool of PLC-γ1 stably associates with PML. This is consistent with the immunohistochemistry, which shows the majority of PLC-γ1 juxtaposing rather than completely co-localizing with PML-NDs. The association of PML and PLC-γ1 is also likely to be cell type dependent, much like the pAKT/PML association. The PLC-γ1 mitogenic pathway is known to be highly active in colon carcinoma cells,45 just as the AKT pathway is overactive in pancreatic tissue. Additionally, overactive PLC-γ1 signaling promotes metastasis.46 In each case, a high nucleoplasmic concentration of AKT or PLC-γ1 effectors could lead to their association with the PML-ND scaffold. Likewise, the background signaling events in different cell types is likely to influence the distribution of either protein. In the case of normal fibroblasts, the predominant expression pattern is cytoplasmic and vesicular, though preliminary gel filtration data suggests that nuclear PLC-γ1 is also expressed in these cells. Collectively, these data indicate that a subset of the nuclear pool of PLC-γ1 is recruited to PML-NDs where it constitutes a legitimate PML-ND cargo protein. The comigration of PLC-γ1 with fractions containing peak concentrations of PML in sizeexclusion chromatography also agrees with the immunohistochemical observation of PLC-γ1 puncta of similar size to PMLNDs.
Conclusion These experiments are the first to isolate a novel PML interacting partner by identification of its peptide fingerprint. Almost all protein associations with the PML-ND have been made with known candidates following affinity capture of ectopic overexpressed proteins. Attempts to identify PML-ND cargo proteins without bias have generally failed because of the lability of the bonds between PML-NDs and their cargo proteins. Our identification of a lipid modifying protein bound
to PML is particularly interesting given the increasing prominence of phosphoinositides in nuclear signaling pathways as well as gene expression.47 The position of the PML-ND in regulating the stress-induced transcriptome would be entirely compatible with its interaction with signaling proteins, traditionally found in the cytoplasm, that are now found to have nuclear activities.
Acknowledgment. This work was supported by a Cambridge BBSRC Case Studentship 00/A2/G/06975 (C. L. Dovey) and BBSRC project grant BBC5098231 (A. H. Wyllie, T. Rich, B. J. Ferguson). References (1) Dundr, M.; Misteli, T. Biochem. J. 2001, 356, 297-310. (2) Gorski, S. A.; Dundr, M.; Misteli, T. Curr. Opin. Cell. Biol. 2006, 18, 284-290. (3) Visintin, R.; Amon, A. Curr. Opin. Cell. Biol. 2000, 12, 752. (4) Dundr, M.; Misteli, T.; Olson, M. O. J. Cell. Biol. 2000, 150, 433446. (5) Andersen, J. S.; Lyon, C. E.; Fox, A. H.; Leung, A. K. L.; Lam, Y. W.; Steen, H.; Mann, M.; Lamond, A. I. Curr. Biol. 2002, 12, 1-11. (6) Pandey, A.; Andersen, J. S.; Mann, M. Sci. STKE 2000, 37, PL1. (7) Cho, S.; Park, S. G.; Lee, D. H.; Park, B. C. J. Biochem. Mol. Biol. 2004, 37, 45-52. (8) Wang, Y.; Cortez, D.; Yazdi, P.; Neff, N.; Elledge; S. J.; Qin, J. Genes Dev. 2000, 14, 927-939. (9) Kischkel, F. C.; Hellbardt, S.; Behrmann, I.; Germer, M.; Pawlita, M.; Krammer, P. H.; Peter, M. E. EMBO J. 1995, 14, 5579-5588. (10) Cain, K.; Brown, D. G.; Langlais, C.; Cohen, G. M. J. Biol. Chem. 1999, 274, 22686-22692. (11) Borden, K. L. Mol. Cell. Biol. 2002, 15, 5259-5269. (12) Kentsis A., Borden, K. L. Curr. Protein Pept. Sci. 2000, 1, 49-73. (13) Dellaire, G.; Bazett-Jones, D. P. BioEssays 2004, 9, 963-977. (14) Eskiw, C. H.; Dellaire, G.; Mymryk, J. S.; Bazett-Jones, D. P. J. Cell Sci. 2003, 116, 4455-4466. (15) Dellaire, G.; Ching, R. W.; Ahmed, K.; Jalali, F.; Tse, K. C.; Bristow, R. G.; Bazett-Jones, D. P. J. Cell Biol. 2006, 175, 55-66. (16) Varadaraj, A.; Dovey, C.; Laredj, L.; Ferguson, B.; Alexander, C.; Lubben, N.; Wyllie, A. H.; Rich, T. J. Pathology 2007, 211, 471480. (17) Wang, Z. G.; Ruggero, D.; Ronchetti, S.; Zhong, S.; Gaboli, M.; Rivi, R.; Pandolfi, P. P. Nat. Genet. 1998, 20, 266-272. (18) Gurrieri, C.; Capodieci, P.; Bernardi, R.; Scaglioni, P. P.; Nafa, K.; Rush, L. J.; Verbel, D. A.; Cordon-Cardo, C.; Pandolfi, P. P. J. Natl. Cancer. Inst. 2004, 96, 269-279. (19) Matunis, M. J.; Zhang, X. D.; Ellis, N. A. Dev. Cell. 2006, 11, 596597.
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