DNA–Pkcs Function Regulated Specifically by Protein Phosphatase 5

DNA–PKcs function regulated specifically by protein phosphatase 5

Thomas Wechsler*, Benjamin P. C. Chen†, Ryan Harper*, Keiko Morotomi-Yano†, Betty C. B. Huang‡, Katheryn Meek§, James E. Cleaver¶, David J. Chen†ʈ, and Matthias Wabl*ʈ

*Department of Microbiology and Immunology and ¶UCSF Cancer Center, University of California, San Francisco, CA 94143; †Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; §Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824; and ‡Rigel Pharmaceuticals, Inc., 1180 Veterans Boulevard, South San Francisco, CA 94080

Contributed by James E. Cleaver, November 26, 2003 Unrepaired DNA double-strand breaks can lead to apoptosis or full-length human DNA–PKcs cDNA were cloned into the tumorigenesis. In mammals double-strand breaks are repaired matchmaker 3 (Clontech) bait plasmid pAS2-1. Then Saccha- mainly by nonhomologous end-joining mediated by the DNA–PK romyces cerevisiae strain AH109 was cotransfected with one of complex. The core protein of this complex, DNA–PKcs, is a DNA- the three cDNA libraries contained in plasmid pACT2 and yeast dependent serine͞threonine kinase that phosphorylates protein cells were selected on AdeϪ HisϪ TrpϪ LeuϪ -plates. The pACT2 targets as well as itself. Although the (auto)phosphorylation ac- plasmid DNA was reisolated from the selected yeast clones, and tivity has been shown to be essential for repair of both random the encoding cDNA was amplified by PCR and sequenced. double-strand breaks and induced breaks at the immunoglobulin locus, the corresponding phosphatase has been elusive. In fact, to Cell Lines, Cell Culture, and Irradiation Treatments. For our exper- date, none of the putative phosphatases in DNA double-strand iments, we used human HeLa cells and the Chinese hamster break repair has been identified. Here we show that protein ovary cell line V3 (DNA–PKcs mutant) transfected with empty phosphatase 5 interacts with DNA–PKcs and dephosphorylates expression vector (V3-JM) or vector with full-length human with surprising specificity at least two functional sites. Cells with DNA–PKcs (V3-F18), both under neomycin selection (11). V3 either hypo- or hyperphosphorylation of DNA–PKcs at these sites cells were grown in 5% CO at 37°C in ␣-DMEM supplemented show increased radiation sensitivity. 2 with 10% FBS, 1% penicillin͞streptomycin, and 200 ␮g͞ml neomycin for selection. HeLa cells were grown in DMEM nrepaired DNA double-strand breaks (DSB) either trigger supplemented with 10% FBS, 1% penicillin͞streptomycin, and Uapoptosis or lead to genomic rearrangements, which in turn 1% glutamax 1 (Invitrogen) in 5% CO2 at 37°C. For irradiation can lead to cancer. To fix DSBs, mammalian cells have at least experiments, cells were trypsinized, put into cell culture medium, two competing DSB repair pathways (1), which work either via and placed in a ␥-irradiator (288 rad͞min). Afterward the cells homologous recombination or, most frequently, via nonhomolo- were recovered in the incubator for graded time periods and then gous end-joining (NHEJ). NHEJ is mediated by the DNA–PK lysed. complex, which is composed of the proteins DNA-dependent CELL BIOLOGY protein kinase catalytic subunit (DNA–PKcs), the heterodimer GST Pull-Down. cDNA encoding protein phosphatase 5 (PP5) Ku70͞80, XRCC4, and ligase IV (2). Further functional inter- TPR domain (amino acid residues 1–150) was cloned into action partners include the Werner protein (3) and the endo͞ exonuclease Artemis (4). The importance of the serine͞ pQE80 vector (Qiagen, Valencia, CA) and cDNAs encoding threonine kinase activity of DNA–PKcs for NHEJ was DNA–PKcs regions (amino acid residues 1,878–2,261 and 2,500– demonstrated in severe combined immunodeficient (SCID) 2,700, respectively) were cloned into pGEX-KG vector. Cell mice (5), in which a mutated DNA–PKcs lacks this activity and lysates containing GST or GST–DNA–PKcs fusion proteins thus fails to repair DSBs induced by the immunoglobulin and T were incubated with Glutathione Sepharose 4B beads (Amer- cell receptor gene rearrangement processes (6, 7). As a conse- sham Biosciences) at 4°C for 1 h and washed three times with ⅐ ͞ ͞ quence, T and B cell development are impaired, and radiation GST binding buffer (20 mM Tris HCl, pH 7.5 150 mM NaCl sensitivity is increased (8). The kinase activity of DNA–PKcs is 0.1% Nonidet P-40). His-PP5 TRP fusion protein (in GST triggered by binding to the open DNA ends of the damaged site, binding buffer plus 1 mg͞ml BSA) was added to the glutathione recruited by the heterodimer Ku70͞80 (2). Then DNA–PKcs beads and incubated at 4°C for additional 2 h, followed by three phosphorylates itself on a cluster of six well conserved sites, from washes with GST binding buffer. The pulled-down His-PP5 TRP the threonine at position 2609 (T2609) to T2647, and then was subjected to SDS͞PAGE and Western blotting using anti- colocalizes at DNA damage foci with other repair proteins such His horseradish peroxidase-conjugated antibody (Qiagen). as H2AX and 53BP1 (9). When this step is blocked by mutations of these autophosphorylation sites, DNA end processing is Retroviral Constructs and Transduction. Full-length human PP5 was blocked and radiation sensitivity is increased (9, 10). cloned into the retroviral vector CRU5–IRES–GFP (IRES, Although the phosphorylation activity has been studied in internal ribosome entry site). Dominant negative PP5.GFP was quite some detail, little is known about the corresponding a fusion protein between human PP5 lacking the last 110 aa and dephosphorylation. Obviously, by the end of the repair process, GFP. Amphotropic Phoenix packaging cells were transfected either the phosphorylated protein has to be degraded or the with 2 ␮g of plasmid DNA by using Fugene 6 transfection phosphate group at T2609 or other sites has to be removed. If so, reagent (Roche Diagnostics). After 2 days, cultural supernatant it is not clear whether the dephosphorylation marks the end of containing the retrovirus were used to infect HeLa cells by the repair process or an intermediate step. The lack of infor- spin-infection at 300 ϫ g for1hwithpolybrene (5 ␮g͞ml). After mation is partly due to the fact that the putative protein phosphatase responsible for the dephosphorylation is unknown. Here we set out to identify this enzyme. Abbreviations: PP5, protein phosphatase 5; NHEJ, nonhomologous end-joining; DSB, double-strand breaks; FSC, forward scatter; SSC, side scatter; siRNA, small interfering RNA. Materials and Methods ʈTo whom correspondence may be addressed. E-mail: [email protected] or Yeast Two-Hybrid Screen. Fragments 7–9 (amino acid residues [email protected]. 1,885–2,780) and 9–11 (amino acid residues 2,458–3,364) from © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307765100 PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1247–1252 Downloaded by guest on September 27, 2021 Fig. 1. Binding of PP5 to DNA–PKcs. (A) Schematic representation of human DNA–PKcs and fragments used in yeast two-hybrid (7–9 and 9–11) and GST pull-down experiments. The numbers correspond to amino acid residues. (B) Schematic representation of human PP5 and fragments isolated by yeast two-hybrid screen from the corresponding libraries. The dashed line indicates that the fragments are in fact longer, but sequencing ended at the amino acid residue indicated. (C) Pull-down of GST-fusion proteins. (Upper) His-tagged TPR domain of PP5 (lane 1; input) was coimmunoprecipitated with the GST-DNA–PKcs fragment 2,500–2,700 with wild-type threonine (lane 4), aspartic acid (lane 5) at position 2,609, and fragment 1,878–2,267 (lane 6), beads only (lane 2), or GST only (lane 3), and visualized with anti His-horseradish peroxidase-conjugated antibody. (Lower) Pull-down input controls of GST-DNA–PKcs peptides were stained with Commassie blue.

3 days, HeLa cells were sorted for GFP-positive subclones by triplicates in six-well plates. Irradiated cells were collected after fluorescence-activated cell sorting (FACS). Subclones were 72 h and 120 h, and undamaged cells were collected after 96 h. grown up and tested for stable GFP expression by flow cytom- For this the supernatant, presumably containing the less viable etry. Expression of PP5 and PP5.GFP fusion protein was con- or dead cells, the PBS wash fraction and the trypsinized adherent firmed by Western blotting of whole cell and nuclear extracts. cells were pooled and spun down. Then they were resuspended in PBS containing 1% FBS and analyzed by flow cytometry. Cell Extracts and Immunoblotting. Nuclear extracts (P10 fraction) were prepared as described (12). Protein concentration was Results and Discussion measured by the Bradford assay, and equal protein amounts PP5 Is a Binding Partner of DNA–PKcs. We performed a yeast (20–60 ␮g) were separated by SDS͞8% PAGE. The proteins two-hybrid screen with two overlapping cDNA fragments of the were transferred by Western blotting onto a nitrocellulose human DNA–PKcs as baits, of Ϸ3 kb length each (fragments 7–9 membrane (Bio-Rad) and incubated with antibody 18-2 to and 9–11, Fig. 1A). The screened cDNA libraries originated from DNA–PKcs (Neomarkers, Fremont, CA), antibody to pT2609, the cell line 18-81, an Abelson virus-transformed mouse line of antibody to pS2056 site, antibody to DNA polymerase ␦ (Trans- the B lymphocyte lineage that hypermutates and switches its Ig duction Laboratories, Lexington, KY), antibody to PP5 (Trans- heavy chain gene constitutively (13); from human activated B duction Laboratories), or antibody to ATM pS1981 (Rockland, cells, which undergo Ig class switching; and from human brain. Gilbertsville, PA). Secondary antibodies were horseradish per- Among the cDNAs encoding putative interaction partners there oxidase-conjugated rabbit anti-mouse and goat anti-rabbit an- were five (four mouse and one human) that encoded different tibodies, respectively (Southern Biotechnology Associates). peptides derived from the N terminus of PP5 (Fig. 1B). All these cDNA fragments were fused to the GAL4 activation domain in Small interfering RNA (siRNA) Inhibition. siRNA duplexes were frame. Sequence analysis showed that all hits contained a synthesized by Dharmacon. The coding strand for PP5 was minimal region of the amino acid positions 1–89 of PP5, which AACCCCCGGCTGATGGAGCTC. Transfection of HeLa with is remarkable, because the 5Ј terminus of transcripts is usually siRNA duplexes was carried out by using Oligofectamine (In- underrepresented in cDNA libraries. This PP5 region contains vitrogen) according to manufacturer’s instruction. Forty-eight TPR motifs that have been implicated in protein–protein inter- hours after initial transfection, the HeLa cells were divided into actions (14). The PP5 peptides were recovered four times by two cultures and subjected to 10-Gy irradiation or mock treat- DNA–PKcs fragment 9–11 and once by fragment 7–9 (Fig. 1A). ment at 72 h. Thirty minutes after irradiation, the HeLa were The region covering these fragments stretches from amino acid harvested for Western blotting analysis. positions 2,458 to 2,780 and contains the major autophosphor- ylation site cluster that is phosphorylated in response to DNA Flow Cytometry Survival Assay. A total of 100,000 cells each of damage (9, 10). We therefore considered PP5 a candidate for an various subclones were irradiated and immediately seeded in enzyme, that dephosphorylates DNA–PKcs.

1248 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307765100 Wechsler et al. Downloaded by guest on September 27, 2021 Fig. 2. Phosphorylation status of DNA–PKcs after irradiation in PP5 overexpressing clones. PP5.C4 and PP5.C13 subclones were irradiated with IR (11.5 Gy) and recovered for 30–410 min. The undamaged control (UD) is shown on the left. Nuclear extracts were prepared, and 20–60 ␮g of protein was applied to a SDS gel. After Western blotting, the membrane was stained with rabbit antibody to DNA–PKcs pT2609 (A), rabbit antibody to DNA–PKcs pS2056 (B), or rabbit antibody to ATM pS1981 (C). For loading controls the same membranes were stripped and incubated with antibody to total DNA–PKcs or to DNA polymerase ␦. PP5 overexpresssion was visualized by anti-PP5 antibody. * and ** denote phosphorylated DNA–PKcs degradation products. CELL BIOLOGY PP5 is a ubiquitously expressed serine͞threonine protein wild-type PP5 in HeLa cells by using a retroviral vector. We then phosphatase that is present in both cytoplasm and nucleus (15, compared the phosphorylation level of T2609 in transduced 16). It dephosphorylates the proapoptotic protein ASK1 (17) subclones with the one in untransduced HeLa cells, with or and interacts with a variety of proteins including the Hsp90–GR without irradiation. When the cells were irradiated, we moni- (18) and Hsp90–HRI (19) heterocomplexes, CDC16͞27 of the tored phosphorylation after 30-, 60-, 120-, 240-, and 410-min anaphase-promoting complex (20), the A-subunit of PP2A (21), recovery time. To this end, we prepared nuclear extracts and ␣ ͞ ␣ hCRY2 (22), and the G 12 G 13 subunits of heterotrimeric G analyzed them by Western blotting with a polyclonal rabbit proteins (23). Furthermore PP5 can dephosphorylate the Alz- antibody that specifically reacts with pT2609 but not with heimer’s disease protein tau in vitro (24) and regulates p53 unphosphorylated T2609 (9) (Fig. 2A). Under these conditions, function (25). It is intriguing that PP5 is sensitive to okadaic acid the T2609 phosphorylation in the PP5 overexpressing subclones (15), considering that cells incubated with this compound have PP5.C4 and PP5.C13 was diminished, as compared to nontrans- decreased DNA–PKcs kinase activity (26). duced HeLa cells, whether undamaged or damaged by irradia- Perhaps because of short interaction time in vivo, we were tion. Furthermore, the dephosphorylation at T2609 occurred unable to coprecipitate PP5 and DNA–PKcs. Instead, to confirm much faster in clones PP5.C4 and PP5.C13, with the pT2609 and further map the interaction in vitro, we tried to precipitate signal returning to undamaged levels at 410 min. the PP5-TPR domain with two different peptides of DNA–PKcs Besides the 450-kDa band, which represents DNA–PKcs, we fused to GST. These peptides contained either the T2609 or also noticed two other bands stained by the antibody, at 240 kDa related sites (amino acids 2,500–2,700) or the serine at position 2,056 (S2056) (amino acids 1,878–2,261), which has been re- and 150 kDa respectively; these bands might result from degra- cently identified as an exclusive DNA–PKcs autophosphoryla- dation of DNA–PKcs. The 240-kDa fragment is probably due to tion site (B.P.C.C. and D.J.C., unpublished result) (Fig. 1A). the apoptotic cleavage at position 2,708–2,713 (27); we do not Whereas the T2609 fragment retrieved the PP5-TPR domain know anything about the identity of the 150-kDa band. At any very well, the S2056 fragment did not (Fig. 1C). When T2609 was rate, compared to HeLa control cells, the 240-kDa band is less changed to aspartic acid (T2609D) to mimic phosphorylation, intense in the PP5.C4 and PP5.C13 subclones; and the 150-kDa the interaction seemed to be somewhat weaker. These experi- band appears later after damage. ments suggested a strong interaction between PP5 and DNA– Apart from T2609, another functional DNA–PKcs phosphor- PKcs with the fragment spanning the 2609 site, and a much ylation site at S2056 has been recently defined (B.P.C.C. and weaker interaction, if any, with the fragment spanning the S2056 D.J.C., unpublished result). To find out whether this site is also site. regulated by PP5, we used a polyclonal rabbit antibody generated against this site, on the same nuclear extracts as before. Whereas Overexpression of PP5 Decreases Phosphorylation of DNA–PKcs but HeLa cells showed weak phosphorylation under undamaged Not of ATM. To determine the functional relevance of the conditions, it was absent in clones PP5.C4 and PP5.C13 and PP5͞DNA–PKcs interaction, we stably overexpressed human weaker in these clones at 30 min after irradiation (Fig. 2B). After

Wechsler et al. PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1249 Downloaded by guest on September 27, 2021 Fig. 3. Effect of dominant negative PP5.GFP on phosphorylation of DNA–PKcs. (A) Cellular localization in unfixed cells of either GFP in subclone PP5.C13 (Left) or PP5.GFP fusion protein in subclone PP5.C7 (Right). (B) The PP5.C7 subclone expressing dominant negative PP5.GFP was irradiated with 11.5 Gy and recovered for 30–410 min. Nuclear extracts, Western blotting, and staining with anti-pT2609 antibody were part of the experiment series shown in Fig. 2; the HeLa wild-type cells of Fig. 2A thus serve as control. * and ** denote phosphorylated DNA–PKcs degradation products. (C) Knock-down of PP5 by RNA interference. HeLa cells were transfected with siRNAs targeted against PP5. As control, HeLa cells were either mock treated or transfected with unspecific siRNAs. As before, nuclear extracts were prepared from undamaged cells and cells irradiated with 10 Gy after 30-min recovery time and stained with anti-pS2056 antibody and anti-PP5 antibody.

410 min, the S2056 was almost completely dephosphorylated in irradiation (undamaged) and after, the subclone PP5.C7 was PP5.C13 but not in HeLa and PP5.C4. Despite this difference, heavily phosphorylated at position T2609 at all time points (Fig. and the slight difference in the level of PP5 overexpression 3B). Even after 410 min, when the signal began to wane in the between subclones PP5.C4 and PP5.C13 (Fig. 2A), the observed untransduced HeLa cells (Fig. 2A, which was part of the same similarities of the phosphorylation patterns seem to exclude a experimental series and thus serves as a control), phosphoryla- site-specific effect of the retroviral vector integration. Therefore, tion remained strong. Apart from confirming that PP5 provides we conclude that PP5 is the phosphatase that is responsible for the dephosphorylation activity on DNA–PKcs, the findings in removing the phosphate group from T2609 and S2056 of DNA– clone PP5.C7 indicate that no other phosphatase can substitute PKcs after irradiation damage. However, ATM can also phos- for PP5 at T2609. In line with the phosphorylation patterns of phorylate DNA–PKcs at T2609 (B.P.C.C. and D.J.C., unpub- ATM in the PP5 overexpressing subclones, no pronounced lished result) (but not at S2056) and may be dephosphorylated differences occurred in the PP5.C7 clone (data not shown). But by PP5 as well. Because this, in part, may contribute indirectly obviously a possible regulation of ATM by PP5 needs a more to the phosphorylation differences, we tested our subclones for detailed investigation. ATM phosphorylation at the S1981 site (28). As we observed no Because phosphorylation levels at S2056 showed less pro- obvious differences (Fig. 2C), it seems that PP5 is fairly specific nounced differences between wild-type HeLa and PP5.C7 clone, for DNA–PKcs. we further assessed whether or not PP5 is the only phosphatase mediating the dephosphorylation of DNA–PKcs. We used spe- Dominant Negative and Decreased Expression of PP5 Increase Phos- cific siRNAs to knock down PP5 expression in HeLa cells and phorylation of DNA–PKcs. The import of PP5 into the nucleus irradiated or mock treated cells transfected with specific or apparently depends on its terminal 80 aa residues (29). One of the transduced HeLa subclones, PP5.C7, expressed a protein unspecific siRNAs and let them recover for 30 min. Although the that encodes the first 389 residues of PP5 fused to GFP, thus reduction in PP5 expression was mild, nuclear extracts stained lacking the sequence required for nuclear import. The PP5.GFP with antibody to the phosphorylated S2056 site (Fig. 3C) and also fusion protein had a molecular mass of Ϸ70 kDa, accumulated to pT2609 (data not shown) showed somewhat increased phos- in the cytoplasm (Fig. 3A), and could be detected by PP5 phorylation at both sites, under undamaged and damaged con- antibody (Fig. 3B). We reasoned that this fusion protein may act ditions. This points out that PP5 regulates both S2056 and T2609, as a dominant negative form. One possible way would be that but the effects of overexpression of wild-type and dominant PP5.GFP blocks the import mechanism while still binding to negative PP5 were weaker for the S2056 site. These differences (some of) the proteins that are needed for this import; endog- can be explained by the different mechanisms of RNA interfer- enous PP5 import would be competed out in this way. Another ence and dominant negative effects and may underscore the possibility came to mind when we considered the surprising importance of nuclear import of PP5 for its regulation. Consid- finding that the fusion protein was not completely denatured by ering the multitude of PP5 substrates, it is possible that more alcohol, as it still was fluorescent (not shown). Thus, it may be efficient elimination of the enzyme cannot be achieved without that the fusion protein does not fold correctly for the import, but arresting or killing the cells. Thus, selection against efficient still takes away the protein(s) required for it. Both before siRNA treatment may occur in the cultures. At any rate, we

1250 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307765100 Wechsler et al. Downloaded by guest on September 27, 2021 Fig. 4. Survival of irradiated cell lines, assayed by flow cytometry. (A) DNA–PKcs-deficient Chinese hamster ovary V3 cells transfected with either empty vector (V3-JM) or DNA–PKcs expressing vector (V3-F18) irradiated with 4 Gy. (B) HeLa subclones irradiated with 4 Gy and analyzed after 72 h and 120 h. (C and D) HeLa subclones were irradiated with 11.5 Gy and analyzed after 72 h and 120 h. FSC, forward scatter; SSC, side scatter. For all cell lines, the percentage of viable cells represented in the lower right quadrant of FSC͞SSC as shown in C was plotted compared to undamaged (UD) cultures.

conclude that PP5 cannot be replaced completely by another another way to measure the viability of the transduced cells, we phosphatase at the two phosphorylation sites investigated here. determined their ability to express GFP, with similar results. When we determined the numbers of GFP-positive cells in the Both Hypo- and Hyperphosphorylation Increase Radiation Sensitivity. very same experiments, they were in line with the survival rates From the experiments described above, it seems to be clear that calculated by counts in the FSC͞SSC (data not shown). Because PP5 can dephosphorylate DNA–PKcs. But does this enzymatic overexpression of PP5 protects cells against apoptosis after CELL BIOLOGY activity have a biological consequence? This seemed likely to us, oxidative stress mediated by the dephosphorylation of ASK1 as the expression of DNA–PKcs with mutations in the major (17), it was surprising that both the overexpression of wild-type autophosphorylation cluster (including T2609) are defective in PP5 and the expression of a dominant negative mutant lead to NHEJ (9, 10). Consequently, all of the transduced clones de- increased radiation sensitivity. A model for DNA–PKcs auto- scribed above PP5.C4 and PP5.C13, and PP5.C7, ought to show phosphorylation suggests at least two clusters of phosphoryla- increased radiation sensitivity. To address this question, we used tion sites, which are used in specific ways to regulated DNA– flow cytometry analysis, which allows quantitation of viable and PKcs function. Whereas the major cluster around the T2609 site dividing cells, as they appear in defined quadrants of the forward is crucial for end processing, other sites might be essential for (FSC) and side scatter (SSC) profiles. We determined the kinase disassembly (30). Thus, impaired phosphorylation or number of these cells per 10,000 events in the cytometer after premature dephosphorylation caused by PP5 overexpression irradiation with either 4 or 11.5 Gy at 72 and 120 h. Although the might block end processing and lead to radiation sensitivity. protocol does not provide absolute numbers of cell survivors, it Classically, autophosphorylation of DNA–PKcs has been linked does give ratios of live and dividing cells over all events that can to kinase dissociation and inactivation (30). Dephosphorylation be compared between cultures. To ensure that such an assay increases kinase activity in vitro; it has been postulated that this reasonably reflects cell viability, we compared DNA–PKcs de- may allow re-use of DNA–PKcs in subsequent repair events in ficient Chinese hamster ovary cells containing an empty vector vivo. Thus, in the absence of efficient dephosphorylation, caused (V3-JM) or reconstituted with a DNA–PKcs-expressing vector by less PP5 enzyme, radiation sensitivity may also be increased. (V3-F18) (9) 72 and 120 h after irradiation with 4 Gy. As However, we know that the initiation of repair process was expected, the DNA–PKcs-positive cells clearly had a higher intact, because damage foci formation of DNA–PKcs phosphor- number of viable and dividing cells per culture than the DNA– ylated at pT2609 seemed to be normal and stable in all subclones PKcs-deficient culture at both time points (Fig. 4A). Next, we (data not shown). Thus, downstream events must be responsible irradiated our transduced HeLa subclones and three different for decreased survival. GFP only controls (GFP.C1, GFP.C2, and GFP.C3) with either 4 or 11.5 Gy. After irradiation with 4 Gy, there was no difference Conclusion between the HeLa and the GFP controls (Fig. 4B). In contrast, Although numerous protein kinases are implicated in DSB subclones PP5.C4 and PP5.C13 were more radiosensitive, repair pathways, so far not a single protein phosphatase could whereas subclone PP5.C7 was most sensitive. To investigate the be shown to directly regulate DSB repair proteins (26). Here effects of a higher dose, we irradiated the cells with 11.5 Gy (Fig. we demonstrated that PP5 interacts with DNA–PKcs and 4 C and D). The differences are easily seen in the FSC͞SSC dephosphorylates it. We showed dephosphorylation mainly for panels (Fig. 4C), which after 5 days showed a distinct population the pT2609 site, and, to some extent, also for S2056; but, of of viable and dividing cells for HeLa and GFP.C1, whereas this course, other sites recently described in this region of DNA– was not the case for the cells with aberrant PP5 expression. As PKcs (10) may also be regulated by PP5. Nor is it excluded that

Wechsler et al. PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1251 Downloaded by guest on September 27, 2021 PP5 affects other protein targets; indeed, it has been shown We thank Cliff Wang and Christoph Baumann for discussion. This work that PP5 regulates p53 (25). However, in the experiments on was supported by National Institutes of Health Grant AG20684 and the p53, depletion of PP5 resulted in G1 growth arrest, whereas our Sandler Family Supporting Foundation (to M.W.) and National Insti- HeLa cells expressing the dominant negative PP5 divide tutes of Environmental Health Sciences Grant 1 R01 ES8061 (to J.E.C). normally. Only in response to irradiation is there a difference The work from D.J.C.’s laboratory was supported by National Institutes of Health Grant CA50519, CA86936, and PO1-CA92584. T.W. is a in survival, caused either by growth arrest or apoptosis (31). recipient of a Boehringer-Ingelheim Fonds fellowship and is very The dominant negative PP5 clone is also more sensitive to grateful for a stay in Dr. S. P. Jackson’s laboratory at The Wellcome phleomycin (data not shown), a radiomimetic chemical related Trust͞Cancer Research UK Institute of Cancer and Developmental to cancer therapeutics. This may make the nuclear import Biology in Cambridge, United Kingdom. This work has been performed mechanism of PP5 an attractive drug target for enhancing the as part of requirement for the Ph.D. degree at the Ludwig-Maximilians- efficiency of radiotherapy in cancer. University, Munich (supervisor Dr. Elisabeth Weiss).

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