Receptor proteins associated with the plasma membranes are under constant flux by receptor-mediated endocytosis. Some receptor proteins are continuously recycled to the plasma mem-brane,1 and others are sorted to the lysosomes where protein degradation takes place.2 For lysosomal degradation, ubiquiti-nation serves as a targeting signal for these proteins’ insertion into the multivesicular bodies (MVBs) within the lumen of late endosomes, which either mature to lysosomes or fuse with lyso-somes.3,4 The formation and sorting of MVBs is tightly regulated by the coordinated action of endosomal sorting complex required for transport (ESCRT) machinery that is composed of ESCRT complexes (0–III), and their associated proteins. There have
Chromatin modifying protein 1A (Chmp1A) is a member of the Endosormal sorting complex required for transport (eSCRt)-III family whose overexpression induces growth inhibition, chromatin condensation and p53 phosphorylation. p53 is a substrate for Ataxia telangiectasia mutated (AtM), which can be activated upon chromatin condensation. thus, we propose that Chmp1A regulates AtM, and the nuclear localization signal (NLS) is required for AtM activation. our data demonstrated that overexpression of full-length Chmp1A induced an increase in active, phosphorylated AtM in the nucleus, where they co-localized. It also induced an increase in phospho-p53 in the nucleus, and in vitro AtM kinase and p53 reporter activities. the intensity of phospho-p53 closely followed that of ectopically induced full-length Chmp1A, suggesting a tight correlation between Chmp1A overexpression and p53 phosphorylation. on the other hand, Chmp1A depletion (reported to promote cell growth) had minor effects on phospho-AtM and p53 expression compared with control, which had very little expression of these proteins. NLS-deleted cells showed uniform cytoplasmic-Chmp1A expression and acted like shRNA-expressing cells (cell growth promotion and minimal effect on AtM), demonstrating the significance of NLS on AtM activation and growth inhibition. C-deleted Chmp1A, detected in the cytoplasm at the enlarged vesicles, increased phospho-AtM and p53, and inhibited growth; yet it had no effect on in vitro AtM kinase or p53 reporter activities, suggesting that the C-domain is not required for AtM activation. Finally, AtM inactivation considerably reduced Chmp1A mediated growth inhibition and phosphorylation of p53, showing that Chmp1A regulates tumor growth partly through AtM signaling.
Chromatin modifying protein 1A (Chmp1A) of the endosomal sorting complex required
for transport (ESCRT)-III family activates ataxia telangiectasia mutated (ATM)
for PanC-1 cell growth inhibitionSumanth Manohar,2,† Matthew Harlow,1,† Hahn Nguyen,1 Jing Li,1 Gerald R. Hankins2 and Maiyon park1,*
1Department of Biochemistry and Microbiology; Joan C. edwards School of Medicine; Marshall University; Huntington, WV USA; 2Department of Biology; West Virginia State University; Institute, WV USA
†these authors contributed equally to this work.
Keywords: Chmp1A, ATM, ESCRT-III, P53, PanC-1
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been great advances in the understanding of the structures,5-10 protein-protein interactions of ESCRT complexes in their assem-bly and disassembly during MVB biogenesis,11-13 and the sort-ing of MVBs for protein degradation.14-17 ESCRT machinery has also been implicated in enveloped virus budding18-21 and cytoki-nesis21-23 since these processes utilize similar mechanisms to those used for MVB formation.
A few components of ESCRT have been linked to can-cer development through the regulation of receptor activity.24 Knockdown of tumor susceptibility gene 101 (TSG101, yeast Vps23, ESCRT-I) induces an oncogenic transformation in fibro-blasts and metastatic tumors in nude mice.25 TSG101 inter-acts with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) to control epidermal growth factor receptor (EGFR).26 In addition, TSG101 interacts with Hepatocellular
of chromatin modification in ATM activation. When activated by auto-phosphorylation at serine 1981 (S1981) in human,42 ATM phosphorylates serine (S) or threonine (T) within SQ/TQ motifs found in substrates, such as P53, checkpoint kinase1 and 2 (Chk1 and Chk2), CyclinD1, breast cancer susceptibility protein-1 (BRCA1) and histone variant H2AX.44,45 ATM was shown to stabilize and activate p53 by phosphorylation at serine 15 (S15).46-48 Since Chmp1A overexpression induces chromatin condensation and p53 phosphorylation at serine 15 we propose that Chmp1A regulates tumor cell growth by the control of a nuclear ATM signal.
Results
Full-length Chmp1A overexpression induces an increase in phospho-ATM and p53 in the nucleus. To test whether Chmp1A regulates ATM, we induced overexpression of Chmp1A (full-length), obtained the nuclear and cytoplasmic fractions, and examined ATM activation. We induced overex-pression of full-length Chmp1A with 1,000 ng/ml of dox treat-ment based on our previous report.40 Since ATM is activated when it is phosphorylated at serine 1981 (pATM-S1981) we used this phospho-specific antibody to examine ATM activa-tion. As shown in Figure 1A, Chmp1A was robustly induced in the nucleus upon dox supplementation. In addition, pATM-S1981 protein level was mainly increased in the nuclear fraction compared with control. Total ATM protein level was very low regardless of Chmp1A overexpression. When activated, ATM has been shown to phosphorylate p53 at serine 15. Previously we have shown that Chmp1A overexpression increased phos-pho-p53 at serine 15 (pP53-S15) in whole cell extracts.40 In this paper we re-examined pP53-S15 expression using nuclear and cytoplasmic fractions. As shown, pP53-S15 was significantly increased in the nuclear fraction in the cells overexpressing Chmp1A.
Immunocytochemistry substantiated the protein gel blot data. Top parts in Figure 1B show 2 cells lacking ATM expression (red), one of each without or with Chmp1A ectopic expression (green). Ectopically expressed Chmp1A (green) and pATM-S1981 (red) protein were detected as thick rod-like struc-tures in the nucleus potentially at the places of condensed chro-matin (inset in the bottom parts, Fig. 1B). The pATM-S1981 expression was evident only when Chmp1A was ectopically induced; compare no-dox (middle parts) to dox-treated (bottom parts). As for phospho-P53, Chmp1A overexpression induced an accumulation of pP53-S15 in the nucleus, whose intensity closely reflected that of Chmp1A ectopic expression (Fig. 1C). Although Chmp1A and pP53-S15 were both detected in the nucleus, they exhibited distinct expression patterns: a thick rod-like appearance for Chmp1A (also shown in Fig. 1B) verses an even distribution for pP53-S15. We examined pATM and pp53 expression over a period of 4 d and found that Chmp1A modu-lated their expression mostly 1 d after dox supplement (only day 1 shown). Collectively our data demonstrated that overexpres-sion of full-length Chmp1A induced an activation of ATM and an increase in phospho-p53 in the nucleus.
Carcinoma Related Protein 1 (HCRP1, yeast Vps37A, ESCRT- 1) whose depletion causes an inhibition of EGFR receptor down-regulation.27 In Drosophila melanogaster, Vps25 (a member of ESCRT-II) mutant cells undergo autonomous and non-autono-mous over-proliferation resulting from the increased activity of Notch receptors.28 Collectively these reports demonstrate that ESCRT components function in tumor development by the reg-ulation of receptors for mitogenic signaling.
ESCRT-III and associated proteins localize in the cytoplasm as monomers, which oligomerize to form complexes at the mem-brane of late endosomes.29,30 ESCRT-III proteins consist of basic and acidic residues at the N- and C-half, respectively. A few members are also expressed in the nucleus and basic N-halves are sufficient for their nuclear localization.30 The C-terminal half contains a MIT-interacting motif (MIM, approximately 10 amino acids long) at its extreme end. MIM is required for the interaction with proteins containing a microtubule interact-ing and trafficking (MIT) domain, which is found in Vacuolar protein sorting 4 (Vps4) AAA-ATPase.30-32 Vps4 associates with the ESCRT-III protein complex through MIM domains, which facilitates Vps4 ATP hydrolysis and disassembly of the ESCRT-III complex during MVB formation.9,33,34 Deletion of MIM and flanking sequences of ESCRT-III promotes complex formation and membrane association, which are visualized as enlarged endosomes in the cytoplasm.30,31
Chromatin modifying protein 1A (Chmp1A, yeast Did2/Vps 46-1), which also stands for Charged multivesicular body protein 1A, is a member of the ESCRT-III complex. Human Chmp1A also consists of a basic N-half and a MIM-containing acidic C-half, and is implicated in multivesicular body forma-tion.8,13,32,35-38 In addition, Chmp1A was shown to interact with a Polycomb-group (PcG) protein, BMI1 and regulate chroma-tin structure and cell cycle progression.39 We provided the first evidence that Chmp1A functions as a tumor suppressor, espe-cially in the pancreas. Evidence of Chmp1A as a tumor sup-pressor includes: reduced/mis-localized expression of Chmp1A in various human pancreatic tumors relative to normal pancre-atic tissue; decreased growth in cell culture and of xenograft tumors upon Chmp1A overexpression in human pancreatic ductal tumor (PanC-1) cells; accumulation of phospho-p53 (pP53) upon Chmp1A overexpression; and growth promotion of PanC-1 cells and conversion of non-tumorigenic human embry-onic kidney cells to cells capable of forming xenograt tumors in athymic mice by stable knock-down of Chmp1A.40 Furthermore, Chmp1A protein localizes to the nucleus in all-trans retinoic acid (ATRA)-responsive PanC-1 cells upon ATRA treatment,41 dem-onstrating the significance of nuclear localization of Chmp1A in the mediation of ATRA signaling.
Ataxia telangiectasia mutated (ATM) plays a crucial role in the signal transduction networks that regulate cell cycle progres-sion. ATM acts in response to DNA double-stranded breaks and activates cell cycle checkpoints.42 ATM also participates in cellular pathways that are not directly linked to DNA damage. Inhibitors of histone deacetylases (HDACs), such as trichostatin A or chloroquine were shown to activate ATM kinase activity independently of DNA damage,43 indicating the significance
1,500 ng/ml or 1,000 ng/ml of dox to induce overexpression of NLS- or C-deletion of Chmp1A, respectively.
Using these stable clones we performed cell growth assays. As previously shown,40 full-length Chmp1A overexpressing PanC-1 cells (solid black line) showed growth inhibition compared with control (broken black line) (Fig. 2C). PanC-1 cells containing NLS-deleted Chmp1A plasmids (broken red line) showed signifi-cant growth promotion compared with cells carrying full-length plasmids over a period of 4 d in the absence of dox (broken black line). Upon dox-treatment (solid red line), cell growth was inhib-ited until day 2.5 compared with no-dox control but the growth was promoted and became similar to control from day 3 on. In the presence or absence of dox, cells carrying NLS-deleted plasmids
Chmp1A silencing has little effect on phospho-ATM and P53. In a previous report we have shown that shRNA-medi-ated stable knockdown of Chmp1A induces growth promo-tion of PanC-1 cells compared with non-silencing (NS) control shRNA.40 We used these cells and examined whether Chmp1A silencing had any effect on phospho-ATM and P53. As shown in Figure 1D, Chmp1A expression was knocked down signifi-cantly by stable introduction of Chmp1A specific shRNA, com-pared with control. pATM-S1981 was almost absent in the cells expressing control shRNA and had no change upon Chmp1A depletion over a period of 4 d. However, a small amount of pP53-S15 expression was detected in the control, which was reduced slightly by Chmp1A silencing on day 1 only. Since Chmp1A depletion had a minor effect on phospho-ATM and p53 we mainly used overexpression to examine the effect of Chmp1A on ATM signaling.
Nuclear localization signal (NLS) of Chmp1A is required for growth inhibition. As shown in Supplemental Figure 1A, Chmp1A contains a NLS at the N terminus and a MIM at the C terminus. Since ATM is a nuclear signal, we made a NLS-deletion construct of Chmp1A (Fig. 2A), and investi-gated whether NLS is required for growth inhibition. A num-ber of ESCRT-III members including Chmp1A localize to the nucleus and cytoplasm. C-terminal deleted proteins localize exclusively in the cytoplasm at the enlarged vesicles although they contain a basic N-half, which has been shown to be suf-ficient for nuclear localization.30 Thus we made a C-deletion by removing MIM at the C-terminus to use as a control for NLS-deletion (Fig. 2A). Although we deleted mainly MIM from the C terminus we refer it as C-deletion in this paper. Dox-inducible stable clones of PanC-1 cells were generated to induce overex-pression of NLS- or C-deletion of Chmp1A. NLS- or C-deleted Chmp1A protein expression was greatly induced at 1,500 ng/ml or 1,000 ng/ml of dox supplement (Fig. 2B). Thus we used
Figure 1. Chmp1A overexpression induces an increase in phospho-AtM and p53, but Chmp1A silencing had little effect on phospho-AtM and p53. (A) Induction of phospho-AtM and p53 upon overexpression of full-length Chmp1A. pAtM-S1985 and pp53-S15 expression was in-creased upon Chmp1A overexpression especially in the nuclear fraction. Chmp1A was also detected strongly in the nuclear fraction upon dox supplementation. Lamin B1 was used for nuclear marker and Gapdh for loading control. (B and C) ectopically expressed Chmp1A co-localized with pAtM-S1981 as dense rod-like structures in the nucleus. one day after the induction of Chmp1A overexpression, the cells were processed for immunocytochemistry with indicated antibodies. pAtM-S1981 was detected only in the cells expressing Chmp1A ectopically. Co-localiza-tion of 2 proteins was visualized as shown in the inset. total AtM ex-pression was low in the presence or absence of Chmp1A overexpression (B). pp53-S15 showed even nuclear expression but its intensity reflected Chmp1A ectopic expression (C). ND; no-dox, D; dox, Blue; nucleus, Red; pp53 or pAtM, Yellow; co-localization. (D) Chmp1A silencing had little effect on phospho-AtM and p53. Chmp1A expression was visible in con-trol cells. Upon Chmp1A silencing, Chmp1A expression was significantly reduced over a period of 4 d. pAtM-S1981 and pp53-S15 were very low in the cells expressing control shRNA. Upon Chmp1A silencing, pAtM-S1981 remained low and pp53-S15 showed slight reduction only on day 1 compared with control. NS; non-silencing shRNA, S; Chmp1A silencing shRNA.
solid to broken green line). Collectively, removal of NLS but not C-terminal domain abolished the growth inhibitory function of full-length Chmp1A, demonstrating the significance of NLS in the suppression of tumor growth.
Nuclear localization signal (NLS) but not C-domain of Chmp1A is required for ATM activation. The above data clearly demonstrate the requirement for NLS of Chmp1A on growth inhi-bition. Next we examined whether NLS- or C-deletion of Chmp1A had any effect on pATM-S1981 and pP53-S15. Overexpression of NLS-deleted Chmp1A was induced significantly on day 2. As in full-length control, pATM or pp53 expression was low in the absence of dox. However, unlike full-length Chmp1A, pATM or pp53 was not increased with the overexpression of NLS-deletion over a period of 4 d (Fig. 3A). As for C-deletion, overexpression was induced significantly from day 2 on, most robustly on day 2. The pATM-S1981 protein was detected noticeably from day 1 even in the absence of dox. With overexpression of C-deletion, pATM-S1981 showed a considerable increase on day 3. The pP53-S15 expression was evident on day 2 in the control and increased strongly with dox supplement (Fig. 3B). Compared with full-length or NLS-deletion, C-deletion showed significant increase of pATM-S1981 and pP53-S15 expression in the presence or absence of dox (compare Fig. 3B to 1A or 3A). However, it took longer to induce an increase of pP53-S15 (day 2) or pATM-S1981 (day 3) with overexpression of C-deletion compared with full-length Chmp1A, which induced pP53-S15 (Figs. 1A and 4B) and pATM (Fig. 1A) on day 1.
Immunocytochemstry found both NLS- and C-deleted Chmp1A proteins in the cytoplasm but with distinct expression patterns: even (NLS-deletion) verses localized (C-deletion) dis-tribution (compare Fig. 3Ca–d or Sup. Fig. 1Bb–f). Consistent with protein gel blot analyses, cells overexpressing NLS-deleted Chmp1A showed very little pATM-S1981 (Fig. 3Cb) or pP53-S15 (Sup. Fig. 1B) protein expression. The localized expression of C-deleted Chmp1A in the cytoplasm presumably represents its expression at the enlarged vesicles/endosomes (white arrow in Sup. Fig. 1Bh) as reported for various ESCRT-III members.8,30 Unlike NLS-deletion, pATM-S1981 was detected strongly in the nucleus and cytoplasm of the cells overexpressing C-deleted Chmp1A (Fig. 3Ce). In the control cells, pATM-S1981 expres-sion was seen in the cytoplasm (data not shown). Although both full-length and C-deleted Chmp1A induced an increase of pATM-S1981 in the nucleus, the pattern was different for each: dense dotted pattern for full-length verses uniformly distributed pattern for C-deletion. In addition, pP53-S15 was detected in the nucleus of the cells overexpressing C-deleted Chmp1A (Sup. Fig 1Bg, h). Compared with full-length or NLS-deletion, C-deletion exhibited substantial levels of pATM-S1981 (for 4 d) and pP53-S15 (day 2) regardless of dox supplementation. Collectively, the results indicate that NLS but not C-domain is required for the induction of phospho-ATM and P53. The data also suggest that C-deletion-mediated growth inhibition is not due to ATM activa-tion but probably due to stabilization of proteins such as ATM and P53.
Overexpression of full-length but not NLS- or C-deleted Chmp1A induces an increase in p53 reporter and in vitro
grew more quickly than cells carrying full-length Chmp1A plas-mids (compare red to black lines). On the other hand, cells car-rying C-deleted plasmids showed minor growth elevation at the beginning but rapid inhibition afterward (broken green line) compared with full-length (broken black line) in the absence of dox (Fig. 2C), indicating a leak. Additional slight growth inhibi-tion was introduced by the overexpression of C-deletion (compare
Figure 2. Generation of stable clones to overexpress NLS- or C-deleted Chmp1A followed by cell growth analyses. (A) Schematic diagram illus-trating deletion constructs of Chmp1A. NLS- or C-domain is highlighted as green or yellow. Amino acids 20 through 36 were removed for NLS-deletion and 186 through 196 for C-deletion. (B) Dose dependent induc-tion of NLS- or C-deletion of Chmp1A. overexpression of NLS-deletion was induced the most at 1,500 ng/ml and C-deletion at 1,000 ng/ml of dox treatment. Gapdh was used as loading control. (C) opposing effects on tumor cell growth were seen with NLS-deletion (promotion) and C-deletion (inhibition). In the absence of dox, NLS-deletion showed robust growth promotion (red broken line) compared with full-length (black broken line). Cells overexpressing NLS-deleted Chmp1A showed temporary growth inhibition until day 2 but similar growth rate from day 3 compared with no-dox control. C-deleted Chmp1A induced gradual growth inhibition (green broken line) compared with full-length (black broken line) in the absence of dox from day 2.5 on. In the presence of dox (green solid line), the cells showed further growth inhibition compared with no-dox control (green broken line), but a simi-lar growth pace compared with full-length overexpressing cells (black solid line). the data was obtained from 3 independent experiments. error bars represent SeM.
tumor cell growth (compare broken gray to solid black line in Fig. 4A). Cells treated with dox plus ATM kinase inhibitor showed considerable growth promotion compared with cells treated with dox only (compare broken to solid black line), yet exhibited growth inhibition compared with no-dox treated con-trol (compare gray to black broken line). Our in vitro ATM kinase assays demonstrate that Chmp1A mediated activation of ATM induces phosphorylation of rp53 at S15. Thus we examined
ATM kinase activities. Next we tested whether the Chmp1A mediated increase in p53 phosphorylation is transmitted to P53-regulated signal transduction. We investigated this possibility by measuring p53 reporter activity in HEK 293T cells. As shown in Figure 3D, overexpression of full-length Chmp1A increased p53 reporter activity compared with control GFP. Interestingly, full-length Chmp1A increased p53 reporter activity the most when 25 ng or 150 ng was transfected, with p values of 0.0017 and 0.021 compared with control. Next we examined whether NLS- or C-deletion of Chmp1A had any effect on p53 reporter activity. Unlike full-length, these deletions had a negative effect on reporter activity when compared with control, which was set as 1 (Fig. 3D). Since serine 15 of p53 is a substrate for ATM kinase,43 Chmp1A-mediated activation of ATM could potentially lead to p53 phosphorylation. To examine this, we performed in vitro ATM kinase assays using recombinant p53 protein (rP53) as substrate and measured the phosphorylation level of rp53 at S15. As shown in Figure 3E, overexpression of full-length Chmp1A considerably increased the phosphorylation level of rp53 at S15 compared with control. However, overexpression of NLS- or C-deletion did not change the phosphorylation status of rp53 compared with control. Therefore, these results further confirm that Chmp1A, through its nuclear localization, regulates ATM signaling, which leads to p53 activation.
Inhibitor-mediated inactivation of ATM kinase causes par-tial attenuation in Chmp1A-mediated growth inhibition and p53 phosphorylation. To test whether Chmp1A-mediated ATM activation is linked to growth inhibition we inactivated ATM kinase with Ku-55933, a specific inhibitor of ATM kinase. Based on a previous report,49 we used Ku-55933 at 10 μMol concen-tration. ATM kinase inhibitor alone had little effect on PanC-1 cell growth for up to 3 d (data not shown) and was not included in the following assays. As reported previously and shown ear-lier in Figure 2, overexpression of full-length Chmp1A inhibited
Figure 3. NLS- but not C-domain of Chmp1A is required for AtM activa-tion. (A) overexpression of NLS-deleted Chmp1A had no effect on pAtM or pp53. NLS-deleted Chmp1A overexpression was detected mainly on day 2. pAtM-S1981 or pp53-S15 expression was very low in the absence (ND) or presence (D) of dox. (B) overexpression of C-deleted Chmp1A induced an increase in pAtM-S1981 and pp53-S15. C-deleted Chmp1A overexpression was observed on day 2, 3 and 4, but most strongly on day 2. pAtM-S1981 and pp53-S15 expression was increased on day 3 and 2, respectively. (C) ectopically induced NLS- or C-deleted Chmp1A was detected in the cytoplasm and showed no co-localization with pAtM-S1981. top part, NLS-deletion and bottom part, C-deletion of Chmp1A with dox supplement. Note that very low pAtM-S1981 is de-tected in the NLS-deletion on day 2 (b), but is strong in the nucleus and cytoplasm of the C-deletion on day 2 (e). NLS-deletion of Chmp1A was evenly distributed in the cytoplasm but C-deletion was localized to the vesicle structures in the cytoplasm. (D) overexpression of full-length, but not NLS- or C-deletion of Chmp1A induces p53 reporter activation in HeK 293t cells. p53 reporter activities were obtained by dividing the experimental values with control GFp after normalized for renilla activi-ties. p53 fold activity was calculated by setting control GFp as 1. error bars represent SeM. (e) Full-length Chmp1A overexpression increased in vitro AtM kinase activity. overexpression of full-length Chmp1A increased phosphorylation of rp53 at S15. However, overexpression of NLS- or C-deleted Chmp1A had no effect on rp53 phosophorylation. rp53 expression was examined as input control.
diminished protein expression of total and phospho-p53 on day 1 (Fig. 4B). Chmp1A overexpression had little effect on total and phospho-p53 on day 2, in the presence or absence of ATM kinase inhibitor. Unexpectedly, ATM kinase inhibitor treatment led to a decrease of ectopic Chmp1A expression mostly on day 1.
Discussion
In this paper we provide supporting evidence that Chmp1A reg-ulates tumor growth through ATM signaling and that nuclear localization of Chmp1A is required for growth inhibition and ATM activation. At first, we showed that Chmp1A overexpress-ing cells induced ATM activation by examining phosphorylation of ATM at serine 1981 (pATM-S1981), an indication of activa-tion.42 Protein gel blot analyses showed that overexpression of full-length Chmp1A had an impact on phospho-ATM (pATM) and phospho-p53 (pP53) 1 d after dox treatment. pATM-S1981 expression was almost absent in the control but increased con-siderably with Chmp1A overexpression, especially in the nuclear fraction. We previously reported that Chmp1A overexpression induced an increase in pP53-S15 in whole cell extracts of PanC-1 cells.40 In this study, we additionally showed that pP53-S15 expression was increased, primarily in the nuclear fraction. Total ATM was low in the control and Chmp1A overexpression did not change its level. As expected based on previous reports, Chmp1A protein level was significantly reduced by shRNA-mediated silencing of Chmp1A compared with non-silencing control shRNA. Similar to the no-dox control for the overexpression sys-tem, pATM-S1981 was almost absent in cells expressing control shRNA. Neither did Chmp1A silencing produce any changes in pATM-S1981 protein level. In addition, control shRNA express-ing cells showed very little pP53-S15 over a 4 d period. The pP53-S15 expression was slightly reduced with Chmp1A depletion on day 1 only. This demonstrates the minimal effect of Chmp1A knockdown on ATM signaling; thus, we focused on Chmp1A overexpressing cells for the rest of the studies.
To substantiate protein gel blot data, we studied sub-cellular localization of pATM and pP53. Consistently, pATM-S1981 and pP53-S15 were almost absent in the control, but strongly induced by Chmp1A overexpression. Although both pATM-S1981 and pP53-S15 were detected in the nucleus they displayed distinct expression patterns. Chmp1A and pATM-S1981 were co-expressed as thick rod-like structures, presumably at the con-densed chromatin, based on previous report from Hollenberg’s lab, who demonstrated that ectopically induced Chmp1A locally increased nuclear DNA concentration through chromatin con-densation.39 As for pP53-S15, it exhibited uniform expression in the nucleus and did not entirely co-localize with Chmp1A. But the intensity of pP53-S15 reflected that of ectopically induced Chmp1A expression. Taken together, our data demonstrate that Chmp1A overexpression induced an increase in pATM-S1981, which co-localized with ectopically induced Chmp1A, poten-tially at the condensed chromatin. Although pP53-S15 did not exhibit co-localization with Chmp1A, the results indicate a tight correlation between Chmp1A overexpression and p53 phosphorylation.
whether inhibition of ATM activity produced an opposite effect on p53 phosphorylation in the presence of Chmp1A overex-pression. As shown earlier in Figure 1A, Chmp1A overexpress-ing cells induced an accumulation of total and phospho-p53 on day 1. However, addition of ATM kinase inhibitor significantly
Figure 4. AtM kinase inhibitor diminished Chmp1A-mediated induction in growth inhibition and p53 phosphorylation. (A) AtM kinase inhibitor partially abolished the growth inhibition produced by Chmp1A overex-pression. Upon dox supplement full-length Chmp1A induced growth inhibition; compare black solid line to gray broken line, no-dox control. In the presence of AtM kinase inhibitor, Chmp1A overexpressing cells showed considerable growth promotion; compare broken (dox plus AtM inhibitor) to solid black (dox alone) line. (B) AtM kinase inhibitor reduced Chmp1A-mediated increase in pp53. Notice the reduction of pp53-S15 and total p53 expression by AtM inhibitor treatment in Chmp1A overexpressing cells, especially on day 1 (top part). Neither Chmp1A overexpression nor AtM kinase inhibitor had much effect on pp53-S15 or total p53 on day 2 (bottom part). In addition, AtM kinase inhibitor induced a decreased expression of Chmp1A, especially on day 1. Lamin B1 was used for nuclear marker and Gapdh for loading control. ND: no-dox, D: dox, D/Ai: dox plus AtM kinase inhibitor.
Chmp1A silencing as shown in Figure 1D. These data clearly demonstrate the significance of nuclear localization of Chmp1A on tumor growth inhibition and ATM activation. Hollenberg’s lab identified Chmp1A as a binding partner of BMI1 (Polycomb-group, PcGtranscription repressoe) and showed that ectopically induced Chmp1A recruited the PcG complex to condensed chro-matin.39 Thus, it is likely that the nuclear localized Chmp1A is responsible for interacting/recruiting the PcG complex to con-densed chromatin, which is closely associated with ATM activa-tion. Without being physically present in the nucleus, Chmp1A is not able to induce condensation of chromatin where the PcG complex is recruited. Subsequently Chmp1A can’t activate ATM to inhibit pancreatic tumor cell growth. Besides NLS, Chmp1A contains a coiled-coil domain, which overlaps with NLS (see Supplemental A). Since the coiled-coil domain mediates protein-protein interactions, it is equally possible that the NLS-deleted protein may prevent Chmp1A from interacting with proteins reg-ulating ATM or other cell cycle signaling pathways. Mutational analyses specific for the coiled-coil domain of Chmp1A will address whether protein-protein interaction(s) is required for the inhibition of tumor growth.
When ATM is activated in response to DNA damage, H2AX is phosphorylated at serine 139 (called γH2AX), and forms foci at DNA double-strand breaks (DSBs).42,50 Since Chmp1A induces ATM activation, it is possible that the dense structures shown in
Previously we have shown that Chmp1A was recruited to the nucleus upon ATRA treatment in ATRA-responsive pancreatic tumor cells. With the same treatment, however, Chmp1A was localized at the membrane in ATRA non-responsive cells,41 dem-onstrating the significance of nuclear localization for the media-tion of the ATRA signal. NLS-predicting PSORT program identified a bipartite NLS in Chmp1A and Hollenberg’s lab also reported the presence of a NLS in Chmp1A protein.39 Several additional members of ESCRT-III are expressed in the nucleus but the program did not identify NLS in these members. To determine the effect of nuclear localization on growth and ATM signaling, we made a NLS-deletion construct of Chmp1A. Upon dox-treatment, NLS-deleted Chmp1A was detected exclusively in the cytoplasm, demonstrating that the NLS domain is required for the protein to enter the nucleus. As a control we generated a C-deletion construct of Chmp1A by removing 11 amino acids from the C-terminus for the following reasons. The C-terminus of ESCRT-III is associated with proper endosomal sorting of pro-teins for lysosomal degradation. When their C-terminal domain is deleted, these proteins are detected in the cytoplasm at the enlarged vesicles/endosomes.8,30 C-deletion of Chmp1A exhib-ited a similar cytoplasmic expression pattern although it contains an intact NLS. Hence this C-deletion of Chmp1A was used to determine the specific effect of NLS on ATM activation and growth inhibition.
Cells transformed with NLS-deleted Chmp1A showed steady and robust growth promotion compared with full-length con-trol in the absence of dox. With overexpression of NLS-deletion, growth was inhibited compared with the no-dox control at the beginning, but promoted rapidly and became similar to control. Overall, NLS-deletion showed significant growth promotion compared with full-length Chmp1A, regardless of dox-treat-ment. The growth promotion observed with NLS-deleted Chmp1A in the absence of dox might be due to a leak from the Tet-on system used for conditional overexpression. In cells car-rying NLS-deleted Chmp1A plasmids, pATM-S1981 or pP53-S15 were hardly detected in the presence or absence of dox, even with much longer exposure of film in protein gel blot analyses. This indicates that NLS-deleted Chmp1A had negligible effect on ATM signaling over a period of 4 d although overexpression of NLS-deletion was induced considerably on day 2. To validate we fixed cells and processed for immunocytochemistry 1 and 2 d after dox-treatment. Although NLS-deleted Chmp1A was clearly expressed in the cytoplasm, only background levels of pATM-S1981 or pP53-S15 were detected as shown in Figure 3C and Supplemental Figure 1B.
Overexpression of NLS-deleted Chmp1A showed an effect on growth and ATM signaling comparable to Chmp1A silencing. On day 4, cells expressing Chmp1A shRNA40 or NLS-deletion (Fig. 2C) showed an approximately doubled number of cells compared with shRNA or full-length control, respectively. pATM-S1981 or pP53-S15 expression was nearly absent in the cells carrying NLS-deleted Chmp1A plasmids in the absence of dox, and showed very little change upon dox supplementation (Fig. 3A). Similarly, pATM-S1981 and pP53-S15 protein levels were low in the cells expressing control shRNA and were not changed much with
Figure 5. Working model showing the significance of NLS of Chmp1A on AtM activation and tumor cell growth inhibition. overexpression of full-length Chmp1A induces activation of AtM and p53 phosphory-lation, leading to tumor cell growth inhibition. our data suggests a potential positive feedback loop (shown as reverse arrows with broken line), which needs future investigation. overexpression of NLS-deleted Chmp1A had no effect on AtM or p53 and induces promotion of tumor growth. overexpression of C-terminal deleted Chmp1A induces stabi-lization/activation of AtM and p53, leading to tumor growth inhibi-tion. Stabilization of protein is presumably caused by the inhibition of protein degradation. Solid black arrows: positive effect; broken black arrows: positive effect hypothesized based on present study, but needs to be determined; broken gray arrows: no or negative effect based on present study; ┴: inhibitory effect.
of proteins such as ATM or P53. We showed the effect of full-length, NLS- or C-deletion of Chmp1A on protein expression of pATM and pP53. Full-length Chmp1A increased phospho-ATM and p53 on day 1 (Figs. 1A and 4B) and C-deletion increased pATM on day 3 and pp53 on day 2 (Fig. 3B). With protein gel blot analyses we observed that the necessary film exposure was shortest with C-deletion, followed by full-length and longest with NLS-deletion. In immunocytochemistry, pATM-S1981 was strongly detected in the nucleus and cytoplasm, and pP53-S15 was increased in the nucleus compared with control on day 2 and 3 (Fig. 3C). These data demonstrate that pATM-S1981 and pP53-S15 expression were much more robust in cells carrying C-deleted Chmp1A in the presence or absence of dox compared with full-length or NLS-deletion. As shown, C-deleted Chmp1A showed a delayed increase in both proteins compared with full-length. This delayed increase may be due to the time required for the accumulation of stabilized proteins through inhibition of protein degradation.
Next we investigated whether Chmp1A-mediated ATM activation is associated with growth inhibition by treating cells with a specific inhibitor of ATM kinase. As shown in Figure 2, overexpression of full-length Chmp1A induced growth inhibi-tion. In the presence of ATM kinase inhibitor, however, cells overexpressing full-length Chmp1A showed considerable growth promotion. Yet the growth was not fully promoted to the con-trol level, suggesting that ATM is partially responsible for the growth inhibition of Chmp1A. Protein gel blots were performed to examine whether p53 functions downstream of ATM. p53 and pP53-S15 expression were again increased with Chmp1A over-expression. Chmp1A-mediated increases in p53 and pP53-S15 were diminished to control levels by ATM kinase inhibitor treat-ment mainly one day after dox-treatment, roughly the same time as when Chmp1A regulated ATM. Unexpectedly, ATM kinase inhibitor treatment resulted in a decrease of Chmp1A expression, indicating a potential positive feed back loop between Chmp1A and ATM. In support of this suggestion, Chmp1A contains 2 potential phosphorylation sites (SQs) for ATM kinase at its C ter-minus (underlined in Sup. Fig. 1A). Further studies are required to determine whether Chmp1A is a functional substrate of ATM kinase and whether there is such a feedback loop between Chmp1A and ATM. Nonetheless these results demonstrate that Chmp1A regulates tumor cell growth partly through ATM. It also suggests the presence of other signaling pathways by which Chmp1A inhibits tumor cell growth.
In summary, we present the first evidence demonstrating that Chmp1A regulates ATM and its target, P53, in the nucleus to control PanC-1 cell growth. As shown in the working model (Fig. 5), overexpression of full-length Chmp1A induced ATM activation and tumor growth inhibition. When NLS was deleted, Chmp1A had no effect on both ATM and P53, and promoted tumor cell growth. This demonstrates that NLS of Chmp1A is essential for ATM activation and growth inhibition. However, C-deleted Chmp1A inhibits tumor cell growth compared with full-length, potentially through the stabilization of proteins such as ATM or P53. More questions need to be addressed including whether Chmp1A regulates tumors other than pancreatic tumors
the nuclei (Fig. 1B) could correspond to γH2AX foci. Therefore, we tested this possibility. The data indicate that Chmp1A/pATM-S1981 positive dots may not represent γH2AX associ-ated focus formation for the following reasons. First, γH2AX expression was increased upon overexpression of full-length Chmp1A on day 1 as in phospho-ATM. However, γH2AX but not pATM, was detected in the control cells carrying full-length Chmp1A plasmids in the absence of dox (Sup. Ca, b). Second, overexpression of NLS-deletion showed very little phospho-ATM in the presence or absence of dox-treatment. But, γH2AX was robustly increased with overexpression of NLS-deleted Chmp1A, especially on day 3 (Sup. Ca). Third, although γH2AX showed partial co-localization with DAPI-DNA in cells overexpressing full-length Chmp1A, it is relatively patchy compared with the dense pATM or Chmp1A expression pattern (compare Sup. Fig 1Cb with Fig. 1B).
We additionally examined the effect of Chmp1A on ATM signaling via in vitro ATM kinase assays using recombinant p53 (rP53) protein as substrate. Overexpression of full-length but not NLS-deleted Chmp1A induced phosphorylation of rp53 protein at serine 15. We also performed p53 reporter assays in HEK 293T cells. Full-length Chmp1A increased p53 reporter activity, although it did not show concentration dependant activation of p53 reporter for as yet unknown reasons. NLS-deletion had a negative effect on p53 reporter activity. We also tested whether Chmp1A activates p53 reporter activity in PanC-1 cells. Although full-length Chmp1A overexpression induced an increase in p53 reporter activity in HEK 293T cells, it had no effect on p53 reporter activity in PanC-1 cells (data not shown). This result is not so surprising since most pancreatic tumors acquire various p53 mutations and PanC-1 cells were shown to have only one copy of mutated P53.51 Nonetheless, these data provide supporting evidence that NLS of Chmp1A is required for ATM activation.
Cells transformed with C-deleted Chmp1A showed growth inhibition in the absence of dox, indicating a leak as in the NLS-deletion. In the presence of dox, C-deletion induced addi-tional growth inhibition, although slight. Upon addition of dox, C-deleted Chmp1A protein was visibly induced from day 2 (most robustly on day 2), when the growth inhibition began compared with full-length no-dox control. Control cells expressed a sub-stantial amount of pATM-S1981 over a 4 d period, and pP53-S15 on day 2. With dox-supplementation, these protein levels were increased further on day 3 and 2, respectively. The effect of C-deletion on pATM-S1981 and pP53-S15 was robust, yet delayed, compared with full-length, which showed an increase on day 1. In C-deleted Chmp1A overexpressing cells, pATM-S1981 was robustly detected in the nucleus and cytoplasm, and pP53-S15 was increased in the nucleus compared with control, on both day 2 and 3. However, overexpression of C-deleted Chmp1A had no effect on in vitro ATM kinase activity and a negative effect on p53 reporter activity. Since overexpression of C-deleted Chmp1A increased pATM-S1981 and pP53-S15 in protein gel blot analy-ses, this indicates that C-deletion inhibits tumor cell growth, but not via ATM activation. Rather, our data suggests that C-deleted Chmp1A induces growth inhibition through the stabilization
cells for 24 h and replaced with media containing FBS and puromycin.
p53 luciferase reporter assays. p53 luciferase reporter plasmid was obtained from Panomics. HEK 293T cells were transiently transfected with CMV6 plasmid containing full-length, NLS- or C-deletion constructs of Chmp1A. We used GFP expressing vector (under same CMV promoter as Chmp1A constructs) as a control for Chmp1A plasmids and empty vector under TK pro-moter for p53 reporter plasmid. The data on p53 control were not included in the graph since the values were very low com-pared with GFP control or experimental values. A total of 450 ng (Chmp1A, p53 reporter and control plasmid) plus 30 ng of renilla plasmid to monitor transfection efficiency were trans-fected into HEK 293T cells using Plus and Lipofectamine reagent (Invitrogen). Twenty-four hours later, cells were lysed with 200 ul of lysis buffer (Promega) and 50 ul of lysates were used to measure p53 luciferase activity using Dual Luciferase Assay Kit from Promega. Two independent experiments with triplicates produced similar results.
In vitro ATM kinase assays with immunoprecipitation. Stably transformed PanC-1 cells were supplemented with dox to induce Chmp1A overexpression. One day later, cells were lysed with kinase lysis buffer containing protease inhibitor cocktail and centrifuged to collect the supernatant (lysate). ATM protein was immunoprecipitated with polyclonal ATM antibody over-night at 4°C followed by incubation with agarose G beads for 2 h at 4°C. Immunoprecipitants were washed 3 times with lysis buf-fer and 3 times with kinase buffer (Cell Signaling Technology). For kinase assays, the agarose G beads coupled with immunopre-cipitated ATM were mixed with recombinant p53 protein (Active Motif) and 50 μM ATP in kinase buffer and incubated for 30 min at 30°C. The assays were terminated by the addition of 4x SDS sample buffer and the reaction products were analyzed by immunoblot with phospho-p53 (serine 15) followed by total P53. No-dox treated cells were included as control.
ATM kinase inhibitor assays. PanC-1 cells transformed with full-length Chmp1A plasmids were used in these assays. No-dox, dox, and dox plus ATM kinase inhibitor treated groups were included and ATM-kinase specific inhibitor only treated cells were used as negative control. Equal numbers of cells were seeded in 10 cm dishes, 3 plates per group, for a total 12 dishes per day. The following day, cells were replenished with media containing dox or ATM inhibitor alone, or dox plus ATM inhibitor, and kept in the dark since both dox and ATM inhibitor are light sensitive. From the next day on, cell numbers were counted at approxi-mately the same time of the day, over a period of 3 d. The media was replaced with new media containing the proper supplement on day 2.
Acknowledgments
We thank Margaret McFarland for valuable comments on this manuscript. We thank Marshall University School of Medicine Genomics Core for use of the Thermocycler, Nanodrop and automated DNA sequencer. This research is supported in part by NIH COBRE (RR020180-02) and WV INBRE (P20 RR-16477) grants and by NASA WV Space Grant Consortium.
and whether other ESCRT-III members function similarly in tumor development. The prognosis of pancreatic cancer patients is the lowest among all the cancer types. Our data provide inno-vative understanding of the molecular mechanisms of pancreatic cancer and would aid in the development of new therapeutics for pancreatic cancer patients.
Materials and Methods
Cell culture, antibodies and antibiotics. The detailed proce-dures on cell culture and working conditions of Chmp1A anti-body were described in our previous reports in reference 40. Other antibodies were purchased from commercial sources: rab-bit polyclonal antibodies against P53, phospho-p53 and mouse monoclonal antibody against Gapdh from Cell Signaling, Lamin B1 from Millipore, ATM from Calbiochem and phospho-ATM at serine 1981 from Rockland. Goat anti-rabbit and mouse HRP conjugated secondary antibody were purchased from Chemicon and Alexa Fluor conjugated secondary antibodies from Molecular Probes. Recombinant p53 protein was obtained from Active Motif. Antibiotics for generating and maintaining stable clones of PanC-1 cells including G418, hygromycin B, doxycy-cline and puromycin were purchased from Clontech and ATM kinase inhibitor KU-55933 from Calbiochem. 4–12% gradient gels were purchased from Lonza Biosciences to analyze ATM and phospho-ATM protein.
Generation of Chmp1A deletion constructs followed by stable clones expressing deletion constructs. NLS- or C-deleted Chmp1A constructs were ordered from Blue Heron Biotechnology (Bothell, WA) and sub-cloned into a tetracycline responsive elements (TRE) containing vector or CMV6 vector using restriction enzyme digestion followed by ligation. TRE-vectors containing NLS- or C-deleted Chmp1A plasmids were transfected into PanC-1 cells expressing regulatory plasmid and selected for stable clones following the protocols described previ-ously in reference 40.
Growth assays, protein gel blot and immunocytochemical anaysis. The detailed protocols on growth assays, protein gel blot and immunocytochemical analyses were described in our previous reports in reference 40 and 41. For nuclear and cyto-plasmic fractionation, the cell pellet was lysed with hypotonic buffer and the cell suspension was passed through a 20-gauge needle and centrifuged to collect supernatant (cytoplasm frac-tion). The pellet was re-suspended in nuclear lysis buffer, vor-texed and centrifuged to collect supernatant (nuclear fraction). For immunostaining, the cells were fixed one or 2 d after dox supplement as indicated and processed for staining following the protocol used previously in reference 40 and 41. For overexpres-sion studies, cells were either starved to synchronize cell cycle or not synchronized. Both methods produced similar results in growth and ATM signaling. Shown here are the data from non-synchronized cells. Using shRNA technology, we generated PanC-1 cells that stably knockdown Chmp1A. We used these cells and stably expressed shRNAs (control or Chmp1A specific) with puromycin following the protocols described in our pre-vious reports in reference 40 and 41. For silencing we starved
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