-revised manuscript- Zyxin is a critical regulator of the ... · 18.01.2011 · treatment, HIPK2 forms a complex with the tumor suppressor p53 and phosphorylates p53 at Ser46 and

-revised manuscript-

Zyxin is a critical regulator of the apoptotic HIPK2-p53 signaling axis

Johanna Crone1, Carolina Glas1, Kathrin Schultheiss, Jutta Moehlenbrink,

Eva Krieghoff-Henning and Thomas G. Hofmann*

Cellular Senescence Group, Cell & Tumor Biology Program, Deutsches

Krebsforschungszentrum (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany

1 Johanna Crone and Carolina Glas contributed equally to the study

*Corresponding author:

Cellular Senescence Group A210, German Cancer Research Center (dkfz), Im Neuenheimer

Feld 280, 69120 Heidelberg, Germany, e-mail: [email protected]

total word count (excluding references): 3.965

abstract: 181 words

key words: DNA damage response, p53, HIPK2, Zyxin, Siah1

running title: Regulation of the HIPK2-p53 signaling axis by Zyxin

Precis: Findings identify Zyxin as novel regulator of DNA damage-induced cell death through

controlling the HIPK2-p53 signaling axis.

on March 28, 2021. © 2011 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 19, 2011; DOI: 10.1158/0008-5472.CAN-10-3486

http://cancerres.aacrjournals.org/

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ABSTRACT

HIPK2 activates the apoptotic arm of the DNA damage response by phosphorylating

tumor suppressor p53 at serine 46. Unstressed cells keep HIPK2 levels low through targeted

polyubiquitination and subsequent proteasomal degradation. Here we identify the LIM

domain protein Zyxin as a novel regulator of the HIPK2-p53 signaling axis in response to

DNA damage. Remarkably, depletion of endogenous Zyxin, which colocalizes with HIPK2 at

the cytoskeleton and in the cell nucleus, stimulates proteasome-dependent HIPK2

degradation. In contrast, ectopic expression of Zyxin stabilizes HIPK2, even upon enforced

expression of its ubiquitin ligase Siah-1. Consistently, Zyxin physically interacts with Siah-1,

and knock-down of Siah-1 rescues HIPK2 expression in Zyxin-depleted cancer cells.

Mechanistically, our data suggest that Zyxin regulates Siah-1 activity through interference

with Siah-1 dimerization. Furthermore, we show that endogenous Zyxin coaccumulates with

HIPK2 in response to DNA damage in cancer cells, and that depletion of endogenous Zyxin

results in reduced HIPK2 protein levels and compromises DNA damage-induced p53 Ser46

phosphorylation and caspase activation. These findings suggest an unforeseen role for Zyxin

in DNA damage-induced cell fate control through modulating the HIPK2-p53 signaling axis.

INTRODUCTION

The cellular DNA damage response (DDR) network constitutes an important anti-

tumor barrier which is inactivated during carcinogenesis (1). The DDR is a powerful cellular

response counteracting genomic instability through induction of cell cycle arrest and DNA

repair or cellular senescence and apoptosis (2, 3).

Homeodomain-interacting protein kinase 2 (HIPK2) is a critical regulator of cell fate

decisions during development and in response to genomic damage (3-5). In unstressed cells

HIPK2 levels are kept low through proteasome-dependent degradation which is facilitated by




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the ubiquitin ligases Siah-1 and WSB1 (4, 6-8). In response to DNA damage, HIPK2 is

stabilized and activated through the ATM/ATR pathway, which involves disruption of the

HIPK2-Siah-1 complex through ATM-mediated phosphorylation of Siah-1 (6, 9). If the

genome is severely damaged upon UV exposure, ionizing radiation or chemotherapeutic drug

treatment, HIPK2 forms a complex with the tumor suppressor p53 and phosphorylates p53 at

Ser46 and thereby stimulates expression of proapoptotic target genes (9-12). In response to

UV damage, HIPK2 is corecruited by the promyelocytic leukemia protein (PML) along with

p53 to PML nuclear bodies (10, 11, 13), which are nuclear multiprotein domains playing a

critical role in the regulation of p53 activity and p53-mediated cell fate upon DNA damage

(14-17).

The LIM (Lin-11, Isl-1 and Mec3) domain protein Zyxin localizes to focal adhesion

sites and the actin cytoskeleton where it interacts with a number of cytoskeletal proteins and

signaling components, thereby regulating actin cytoskeleton dynamics and cell motility and

migration (18-20). Blockage of Crm1-dependent nuclear export with leptomycin B (LMB)

leads to nuclear accumulation of Zyxin (21, 22). Consistent with these findings, Zyxin was

found to shuttle between the cytoplasm and the cell nucleus and to play a role in

mechanotransduction and atrial natriuretic peptide signalling. Interestingly, Zyxin has also

been implicated in cell death regulation (19, 23-26), but its role in apoptosis regulation is

controversial.

Intriguingly, both HIPK2 and Zyxin have been reported to exert tumor suppressive

functions: HIPK2 expression is reduced in breast and thyroid carcinoma (27, 28), it is

inactivated through mislocalization by the proto-oncogene HMGA1 (28) and is functionally

compromised by mutation in acute myeloid leukemia (AML) and myelodysplastic syndrome

(29). Moreover, there is evidence that in mice, HIPK2 acts as haplo-insufficient tumor

suppressor in the skin (30). For Zyxin, reduced expression has been associated with bladder

cancer progression (31), and Zyxin was shown to act as a tumor suppressor in Ewing sarcoma




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(32) and in prostate cancer cells (33). Although these findings indicate a tumor suppressive

activity of Zyxin, the signaling routes and molecular mechanisms linking Zyxin to tumor

suppression remain largely unclear.

In this study, we have identified Zyxin as a critical regulator for the p53 Ser46 kinase

HIPK2, which induces apoptosis in response to DNA damage. We demonstrate that Zyxin

expression is important to maintain HIPK2 protein stability, since Zyxin depletion results in

proteasome-dependent HIPK2 degradation. HIPK2 expression in Zyxin-depleted cells is

rescued by proteasome inhibition or by codepletion of the ubiquitin ligase Siah-1.

Furthermore, ectopically expressed Zyxin stabilizes HIPK2 and efficiently protects HIPK2

from Siah-1-mediated degradation, presumably by interfering with Siah-1 homodimerization.

Depletion of endogenous Zyxin in human hepatocellular carcinoma cells inhibits DNA

damage-induced p53 Ser46 phosphorylation and caspase-dependent PARP cleavage, a

hallmark of apoptotic cell death. These results suggest Zyxin as a critical regulator of DNA

damage-induced cell death induction through regulation of the HIPK2-p53 signaling axis, and

thereby propose a potential molecular basis for the tumor suppressive activity of Zyxin.

MATERIALS AND METHODS

Cell culture and transfections. U2OS, HepG2, MFC7 and 293T cells (all directly obtained

from ATCC) were maintained in DMEM/10 % FCS/1% (w/v) penicillin/ streptomycin/ 20

mM HEPES. Transient transfections were done using standard calcium phosphate

precipitation, Lipofectamine 2000 or HiPerFect (Qiagen, Hilden, Germany).

Expression plasmids and antibodies. HIPK2 and Siah1 expression vectors and antibodies

were described previously (10, 34, 43). Zyxin expression vectors were generated by standard

PCR reactions and were all verified by DNA sequencing by the DKFZ core facility. Zyxin

deletions contained the following regions: dLIM AAs 1-380, LIM1-3 AAs 380-572, LIM1/2




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AAs380-504 and LIM2/3 AAs 444-572. The rabbit zyxin antibodies were generated by

immunising rabbits with the following KLH-coupled peptide: TLKEVEELEQLTQQLM

(AA352-367 of human Zyxin). Antibodies were affinity purified against the peptide prior to

use. Other antibodies used were flag M2 (Sigma), Myc 9E10 (Santa Cruz), HA 3F10 (Roche),

HA 12CA5 (Roche), PARP (Pharmingen), Ser46 (Cell Signaling), p53 DO-I (Santa Cruz),

GAPDH V18 (Santa Cruz), EGFP mixture of clones 7.1 and 13.1 (Roche) and actin C4 (MP

Biomedical).

RT-PCR and oligos. Semi-quantitative RT-PCR against HIPK2, Zyxin and Siah was

performed using total RNA extracted with the RNA extraction kit from Qiagen and the

following oligonucleotides: Siah1 RT For 5` cctgtaaatggcaaggctctc 3`, Siah1 RT Rev 5`

ctagtctttgacaccagcattg 3`; HIPK2 RT For 5` ggcctcacatgtgcaagttttc 3`, HIPK2 RT Rev 5`

ttggtaggtatcaaggaggctc 3`; Actin RT For 5` cctcgcctttgccgatcc 3`, Actin RT Rev 5`

ggatcttcatgaggtagtcagtc 3`; Zyxin RT For 5` gttccacatcgcctgctt c 3`, Zyxin RT Rev 5` cagcca

ttgtcatctgcctc 3`

DNA damage induction. For UV irradiation, cell culture medium was removed and cells

were exposed to the indicated UV-C dosages using a Stratalinker 1800 (Stratagene). After

addition of fresh medium, cells were allowed to recover for the indicated time periods. For

Adriamycin treatments, the cells were incubated with fresh culture medium supplemented

with 1µg/ml Adriamycin for the indicated time periods.

Immunofluorescence microscopy. Cells were washed in PBS. For endogenous protein

stainings, preextraction was performed by 30 min incubation in CSK buffer (100mM NaCl,

300 mM sucrose, 3 mM MgCl2 and 10 mM PIPES pH6.8), 2 min incubation in CSK/0.5%

Triton-X-100 and a brief wash in CSK buffer. Cells were fixed with PBS/4%

paraformaldehyde for 30 min at room temperature. Immunofluorescence stainings were




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performed as published previously (44). The following primary antibodies were used: mouse

anti Flag (M2, Sigma), rabbit anti HIPK2 (10), goat anti Zyxin (N-19, Santa Cruz), mouse

anti c-Myc 9E10 (Santa Cruz), rat anti HA (3F10, Roche). The secondary antibodies we used

were Alexa-488-coupled goat anti mouse, Alexa-488-coupled goat anti rabbit, Alexa-568-

coupled donkey anti goat, Alexa-594-coupled donkey anti rat and Alexa-594-coupled goat

anti rabbit (Molecular Probes). Cells were examined using a confocal laser scanning

microscope (FluoView1000, Olympus) with a 60x oil objective using the sequential scanning

mode. All images were collected and processed using the FluoView Software (Olympus) and

sized in Adobe Photoshop 7.0.

RNA interference. For RNA interference the following targeting sequence was inserted into

the pSUPER vector to knock-down human Zyxin: 5`- GAAGGTGAGCAGTATTGAT -3`, or

the following dsRNAs synthesised by Dharmacon were used: 5´-

GTGTTACAAGTGTGAGGAC-3´ or 5´-GCCCAAAGTGAATCCCTT-3´. The following

target sequences were also used: HIPK2 5'-CAC CTA CGA GGT CTT AGA G-3'` and Siah-1

5´-GATAGGAACACGCAAGCAA-3´. For the control experiments the following sequence

targeting GL2 firefly luciferase 5´-CGTACGCGGAATACTTCGA-3´ was used (44) either as

dsRNA oligo or in pSUPER. All siRNA sequences were verified to confirm their specificity

to the respective target mRNA. Stable knock-downs were done by calcium phosphate

precipitation of pSUPER-shGL2 and pSUPER-shZyxin along with pWZL-neo into HepG2

cells and subsequent selection of G418-resistant cell pools.

Immunoprecipitation and immunoblotting. For coimmunoprecipitation experiments, cells

were lysed in buffer containing 150 mM NaCl, 50 mM HEPES pH 7.5, 5 mM EDTA, 0.5 %

NP-40 and protease inhibitors (Complete w/o EDTA, Roche, Mg132, Biomol, and PMSF,

Applichem) or 250 mM NaCl, 20 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% NP-40, 25 mM




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NaF, 10% Glycerol and protease inhibitors. For the HIPK2- Zyxin coimmunoprecipitation,

cells were lysed in buffer containing 50 mM Hepes, pH 7.4, 150 mM NaCl, 10% Glycerol,

1% Triton-X-100, 1,5 mM MgCl2, 1 mM EGTA, and protease inhibitors. Mouse Flag (M2),

mouse Myc or mouse HA antibodies were used with protein-A/G-coupled sepharose beads

(Santa Cruz). After incubation at 4°C on a rotating wheel the beads were washed 3 times in

NP-40 buffer (150 mM NaCl, 50 mM Hepes pH 7.5, 5 mM EDTA, 0.5 %, NP40). For total

cell lysates, cells were either lysed in buffer containing 20 mM Tris/HCl pH7.4, 1% NP-40,

150 mM NaCl, 5 mM EDTA, 10% Glycerol, 25 mM NaF and 1% SDS and DNA was sheared

by sonication, or cells were lysed in the same buffer containing only 0.1% SDS without

sonication. Immunoblotting was done as decribed (10). Quantifications were done using the

ImageJ software.

GST protein purification, in vitro translations and GST pulldowns. GST proteins were

purified from BL21 by standard protocols. For Siah and LIM constructs, bacteria were

incubated in the presence of 0.5 mM Zinc. HIPK2 and Zyxin were in vitro translated using the

TNT coupled Reticulocyte Lysate System (Promega). Pulldowns were incubated for 4 – 6 h

on a rotating wheel at 4°C in in-vitro interaction buffer (0.05% NP-40, 1mM Na3VO4 and 1

mM PMSF in PBS), washed 3 x 10 min in the same buffer, denatured, resolved by SDS page

and gels were subsequently Coomassie-stained and dried and the radioactive signal was

detected either by film or by phosphoimager.

RESULTS

Zyxin interacts with HIPK2

Using the N-terminal part of HIPK2 (encoding amino acids 1-565) as bait in a yeast

two-hybrid screen (34) against a fetal human brain cDNA library, we identified two identical

cDNA clones encoding the C-terminal region of Zyxin harbouring its LIM domains (Fig. 1




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A). This interaction was confirmed in vitro and further mapped to LIM domains 1 and 2 of

Zyxin and the N-terminal 188 amino acids of HIPK2 (Fig. 1 B-C). The interaction between

Zyxin and HIPK2 could also be detected in intact cells by coimmunoprecipitation

experiments (Fig.1D), which suggests that HIPK2 and Zyxin interact in vivo. These data

indicate interaction of HIPK2 and Zyxin in vitro and in vivo (a schematic view is shown in

Fig. 1E). We failed to demonstrate interaction of endogenous HIPK2 and Zyxin proteins,

which is likely due to complex formation of both factors at particular multiprotein domains,

such as cytoskeletal structures and nuclear bodies (Fig. 1E), which are very hard to solubilize

under conditions which preserve protein-protein interactions. In fact, immunofluorescence

stainings of endogenous HIPK2 and endogenous Zyxin showed colocalization of both factors

at cell edges (most likely representing focal contacts) and in a subset of HIPK2-containing

nuclear bodies.

Zyxin regulates HIPK2 protein levels

Therefore, we proceeded to analyze the functional interaction between HIPK2 and

Zyxin. Intriguingly, acute knock-down of endogenous Zyxin in various cancer cell lines

strongly reduced HIPK2 steady-state protein levels in unstressed cells, whereas HIPK2

mRNA levels remained unchanged (Fig.2 A-B). Stable Zyxin knockdown with a pSuper

construct containing an independent Zyxin target sequence also showed striking reduction of

the HIPK2 levels (Fig. 2 C). Using a gain of function approach, we found that ectopic

expression of Myc-tagged Zyxin leads to HIPK2 accumulation in a dose-dependent manner.

Interestingly, a Zyxin protein that was predominantly targeted to the nucleus by addition of

the SV40 NLS and point mutation of the internal NES sequence (Nuc-Zyxin) was even

slightly more efficient in stabilizing ectopically expressed HIPK2 than the wild type form of

Zyxin (Fig. 2 D-E), hinting at a role for nuclear Zyxin in this process. Taken together, these

results indicate that Zyxin regulates HIPK2 stability.




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Zyxin inhibits HIPK2 degradation in a Siah-1-dependent fashion

We and others have recently shown that HIPK2 steady-state levels are largely

controlled by regulated proteasomal degradation (6-9). Therefore, we examined whether

reduced HIPK2 protein levels in Zyxin-depleted cells originate from enhanced HIPK2 protein

destabilization. Effective proteasome inhibition (as evident by accumulation of the

proteasome target p53) with the proteasome inhibitor MG-132 fully rescued HIPK2

expression (Fig. 3A), indicating that Zyxin depletion indeed results in increased HIPK2

degradation.

One important mediator of HIPK2 break-down is the E3 ubiquitin ligase Siah-1 (6, 8,

35). Since Zyxin depletion results in proteasome-dependent HIPK2 degradation, we

investigated if Zyxin protects HIPK2 from degradation by antagonizing Siah-1 function. As

expected, Siah-1 expression resulted in efficient HIPK2 degradation (Fig. 3B). Remarkably,

coexpression of Zyxin rescued HIPK2 levels even in presence of its ubiquitin ligase (Fig. 3B).

These findings indicate that Zyxin expression protects HIPK2 from Siah-1-mediated

degradation.

Next we investigated whether knock-down of Zyxin may lead to Siah-1-dependent

HIPK2 degradation. To this end, we knocked down Siah-1 by RNAi in shZyxin cells.

Strikingly, knockdown of Siah-1 specifically rescued endogenous HIPK2 expression in

shZyxin cells (Fig. 3C). Taken together, these findings suggest that depletion of Zyxin results

in Siah-1-dependent HIPK2 degradation.

Zyxin colocalizes and physically interacts with Siah-1

Because of the functional interaction of Zyxin with Siah-1, we next asked whether

both molecules physically interact. To this end, we performed coimmunoprecipitation

analyses from cell lysates expressing Myc-Zyxin and HA-Siah-1. Interestingly, ligase-




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deficient Siah-1 (Siah-1C44S) was readily coimmunoprecipitated with Zyxin, in contrast to

wildtype Siah-1 (Fig. 4A). Nuc-Zyxin showed a slightly increased binding to Siah-1 in

comparison to the wildtype form (Fig. 4B). Similar results were obtained by reciprocal

immunoprecipitation analyses (Fig. 4C). Consistent with these findings, indirect

immunofluorescence staining and confocal microscopy revealed that ligase-deficient Siah-1

as well as wildtype Siah-1 readily colocalized with Nuc-Zyxin (Fig. 4E,F). Siah-1C44S and

wildtype Siah-1 showed a comparable subcellular distribution (Fig. 4D). Furthermore, also

wildtyp Zyxin and wildtype Siah-1 colocalized in the cytoplasm (Fig. 4G), however, to a

lesser extent than wildtype Zyxin and Nuc-Siah-1 (Fig. 4H). Thus, these findings suggest that

Siah-1 and Zyxin form a protein complex, which may occur preferentially in the cell nucleus.

To examine whether Zyxin and Siah-1 may interact directly, we performed GST

pulldown assays. In vitro translated 35S-labelled Zyxin was efficiently pulled down by GST-

Siah1 (Figure 4I), suggesting that the interaction between both proteins is indeed direct.

Further mapping experiments indicated that the RING domain of Siah-1 (AAs 1-80) mediates

the interaction with Zyxin (Fig. 4J). Taken together, these results indicate that Zyxin

physically interacts with Siah-1.

Zyxin interferes with Siah-1 homodimerization

Since Siah-1 forms dimers via its substrate binding domain (36, 37), we next assessed

whether Zyxin regulates Siah-1 homodimerization. To this end we coexpressed wild type,

ligase-proficient HA-Siah-1 and Flag-Siah-1 proteins in presence or absence of Myc- (Nuc-)

Zyxin and investigated Siah-1 homodimerization by coimmunoprecipitation analyses. In the

absence of Zyxin, HA-Siah-1 efficiently coprecipitated with Flag-Siah-1 (Fig. 5A). In

contrast, in presence of Zyxin the homodimerization of Siah-1 was significantly reduced (Fig.

5A), and again, Nuc-Zyxin showed a slightly stronger effect. A comparable interfering effect

of Zyxin on Siah-1C44S homodimer formation was observed (Fig. 5B). Taken together, these




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data suggest that Zyxin may modulate HIPK2 stability through interference with the assembly

of functional active Siah-1 dimers.

Zyxin accumulates upon DNA damage induced by UV and Adriamycin treatment

To get insight into the functional role of endogenously expressed Zyxin in the DNA

damage response, we examined Zyxin protein levels in response to DNA damage in human

HepG2 hepatoma cells. Interestingly, lethal UV damage (70 J/m2) (6) resulted in clear Zyxin

accumulation (Fig. 6A). In addition, Zyxin accumulation correlated with HIPK2 stabilization

and p53 Ser46 phosphorylation (Fig. 6A). Sublethal UV damage (20 J/m2) (6), however,

resulted in weak and transient Zyxin accumulation (Fig. 6A). Furthermore, also cells treated

with a lethal dose of the DNA-damaging chemotherapeutic drug Adriamycin showed elevated

Zyxin levels (Fig. 6B), indicating that Zyxin accumulates in response to severe DNA damage,

and that Zyxin accumulation correlates with HIPK2 stabilization and p53 Ser46

phosphorylation.

We also addressed the subcellular localization of endogenous Zyxin in U2OS and

HepG2 cells before and after UV treatment using indirect immunofluorescence staining and

confocal microscopy. Zyxin predominantly resided in the cytoplasm in unstressed cells. Most

cells only showed weak nuclear staining of Zyxin, and no significant changes in the

subcellular distribution of Zyxin were detectable after DNA damage (data not shown).

Zyxin depletion inhibits DNA damage-induced p53 Ser46 phosphorylation and apoptotic

caspase activation

To examine the functional relevance of Zyxin in DNA damage-induced HIPK2

stabilization and p53 Ser46 phosphorylation, we used HepG2 cells stably depleted for Zyxin

(shZyxin) and control cells (shGL2). Cells were exposed to UV light, a well established

HIPK2-activating stimulus (10, 11), and subsequently analysed by immunoblotting. In




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accordance with published data (6, 8), HIPK2 was stabilized upon UV and a robust p53 Ser46

phosphorylation was evident in the shGL2 control cell pool (Fig. 6D). In contrast, Zyxin-

depleted cells showed a clear reduction in UV-induced HIPK2 stabilization and p53 Ser46

phosphorylation (Fig. 6D). Zyxin-depleted cells showed no signs of caspase activation upon

UV damage, whereas the control cell pool showed characteristic caspase-dependent cleavage

of the caspase substrate protein PARP (Fig. 6D). Taken together, these findings demonstrate a

role of Zyxin in the regulation of DNA damage-induced p53 Ser46 phosphorylation and

apoptotic caspase activation.

DISCUSSION

The Ser/Thr protein kinase HIPK2 is an emerging regulator of cell fate decisions in

development, morphogenesis and genotoxic stress (3-5). In order to activate the cell death

response upon chromosomal damage, HIPK2 stimulates various downstream signaling

pathways most prominently the tumor suppressor p53. HIPK2-mediated phosphorylation of

p53 at Ser46 potentiates the activation of proapoptotic p53 target genes, which finally leads to

cell death (10, 11). Activation of HIPK2 in response to genotoxic stress is facilitated through

the checkpoint kinases ATM and ATR which phosphorylation-dependently inhibit HIPK2

proteolysis by the ubiquitin ligase Siah-1 through site-specific phosphorylation of Siah-1 (6).

Our findings here demonstrate that the LIM domain protein and focal adhesion

component Zyxin is essential for HIPK2 stabilization, p53 Ser46 phosphorylation and

caspase-activation in response to DNA damage. In addition, our data indicate that Zyxin is

also essential to maintain the low level steady-state HIPK2 protein expression in unstressed

cells, which suggests that HIPK2 may fulfil important, but so far only little investigated

functions in cells in the absence of genotoxic stress. However, the functional relevance of

HIPK2-Zyxin interaction in unstressed cells and DNA-damaged cells remain to be clarified

and may pave the way to uncover novel cellular functions of HIPK2 and Zyxin. Moreover,




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potential functional interplay between HIPK2 and Zyxin in response to DNA damage remains

to be elucidated in the future.

Previous reports have linked Zyxin to apoptosis regulation and indicate both pro- and

anti-apoptotic activities of Zyxin, suggesting tissue and cell type specific as well as stimulus-

dependent functions of Zyxin (23, 25, 26). For example, Zyxin has been shown to exert

oncogenic activities by facilitating cell migration in melanoma cells (38, 39), but it can also

act as a tumor suppressor in Ewing carcinoma and prostate cancer cells (32, 33). Similar

oncogenic versus tumor suppressive activities have been attributed to the ubiquitin ligase

Siah-1 (40, 41). Since our results here demonstrate that Zyxin and Siah-1 interact and,

furthermore, that Zyxin modulates Siah-1 dimerization and thereby presumably its activity, it

is tempting to speculate that increased Zyxin levels - such as in case of DNA damage -

contribute to uncoupling the ubiquitin ligase Siah-1 from its substrates. As Zyxin potently

regulates HIPK2 stability in a Siah-1-dependent fashion, we propose that Zyxin may control

HIPK2 stability through regulation of Siah-1 availability. The resulting stabilization of the

Siah-1 target protein HIPK2 then alters the fate of the cells subjected to DNA damage by

activating the apoptotic program.

Remarkably, in intact cells exclusively the ligase-deficient Siah-1 point mutant but not

the ligase-proficient wildtype form of Siah-1 could readily be shown to interact with Zyxin.

This may suggest that the ligase activity of Siah-1 interferes with Zyxin binding, possibly

through interaction with the E2 enzyme or Siah-1 autoubiquitination. Nontheless, the

inhibiting effect of Zyxin on the dimerization of the wild type Siah-1 proteins is quite

pronounced, arguing that a weak and transient interaction between both factors may occur.

This interpretation is further supported by our finding that wildtype Siah1-1 and Zyxin show

partial colocalization. Although our data clearly indicate that Zyxin regulates HIPK2 stability

through interference with Siah-1 function, it remains to be determined whether direct binding




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of Zyxin to Siah-1 and/or HIPK2 is required for the observed protection of HIPK2 from Siah-

1-mediated breakdown.

Taken together, our findings here indicate a novel function for Zyxin in the regulation

of the p53 response and cell fate decision in response to DNA damage. Based on our

observations, it appears conceivable to speculate that downregulation of Zyxin, such as

described in a subset of human breast cancers (42), might influence the responsiveness to

DNA-damaging cancer treatments such as radio- and chemotherapy.

ACKNOWLEDGEMENTS

We are grateful to Tilman Polonio-Avalon for generation of Siah-1 constructs, the

DKFZ core facilities for DNA sequencing and to the members of the Hofmann laboratory for

helpful comments and discussions.

GRANT SUPPORT

This study was supported by funds from the Deutsche Forschungsgemeinschaft, the

Deutsche Krebshilfe, the Helmholtz-Gemeinschaft and the Baden-Württemberg Stiftung (all

to T.G.H).

REFERENCES

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6. Winter M, Sombroek D, Dauth I, Moehlenbrink J, Scheuermann K, Crone J, et al. Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR. Nat Cell Biol 2008;10:812-24. 7. Choi DW, Seo YM, Kim EA, Sung KS, Ahn JW, Park SJ, et al. Ubiquitination and degradation of homeodomain-interacting protein kinase 2 by WD40 repeat/SOCS box protein WSB-1. J Biol Chem 2008;283:4682-9. 8. Kim SY, Choi DW, Kim EA, Choi CY. Stabilization of HIPK2 by escape from proteasomal degradation mediated by the E3 ubiquitin ligase Siah1. Cancer Letters 2009;279:177-84. 9. Dauth I, Kruger J, Hofmann TG. Homeodomain-Interacting Protein Kinase 2 Is the Ionizing Radiation-Activated p53 Serine 46 Kinase and Is Regulated by ATM. Cancer Res 2007;67:2274-9. 10. Hofmann TG, Moller A, Sirma H, Zentgraf H, Taya Y, Droge W, et al. Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2. Nat Cell Biol 2002;4:1-10. 11. D'Orazi G, Cecchinelli B, Bruno T, Manni I, Higashimoto Y, Saito S, et al. Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. Nat Cell Biol 2002;4:11-9. 12. Di Stefano V, Rinaldo C, Sacchi A, Soddu S, D'Orazi G. Homeodomain-interacting protein kinase-2 activity and p53 phosphorylation are critical events for cisplatin-mediated apoptosis. Exp Cell Res 2004;293:311-20. 13. Moller A, Sirma H, Hofmann TG, Rueffer S, Klimczak E, Droge W, et al. PML is required for homeodomain-interacting protein kinase 2 (HIPK2)-mediated p53 phosphorylation and cell cycle arrest but is dispensable for the formation of HIPK domains. Cancer Res 2003;63:4310-4. 14. Hofmann TG, Will H. Body language: the function of PML nuclear bodies in apoptosis regulation. Cell Death Differ 2003;10:1290-9. 15. Bernardi R, Pandolfi PP. Role of PML and the PML-nuclear body in the control of programmed cell death. Oncogene 2003;22:9048-57. 16. Krieghoff-Henning E, Hofmann TG. Role of nuclear bodies in apoptosis signalling. Biochim Biophys Acta 2008;1783:2185-94. 17. Dellaire G, Bazett-Jones DP. Beyond repair foci: subnuclear domains and the cellular response to DNA damage. Cell Cycle 2007;6:1864-72. 18. Kadrmas JL, Beckerle MC. The LIM domain: from the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol 2004;5:920-31. 19. Nix DA, Fradelizi J, Bockholt S, Menichi B, Louvard D, Friederich E, et al. Targeting of zyxin to sites of actin membrane interaction and to the nucleus. J Biol Chem 2001;276:34759-67. 20. Hoffman LM, Jensen CC, Kloeker S, Wang CL, Yoshigi M, Beckerle MC. Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling. J Cell Biol 2006;172:771-82. 21. Nix DA, Beckerle MC. Nuclear-cytoplasmic shuttling of the focal contact protein, zyxin: a potential mechanism for communication between sites of cell adhesion and the nucleus. J Cell Biol 1997;138:1139-47. 22. Hervy M, Hoffman L, Beckerle MC. From the membrane to the nucleus and back again: bifunctional focal adhesion proteins. Curr Opin Cell Biol 2006;18:524-32. 23. Kato T, Muraski J, Chen Y, Tsujita Y, Wall J, Glembitski CC, et al. Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent nuclear accumulation of zyxin and Akt. J Clin Invest 2005;115:2716-30.




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24. Yoshigi M, Hoffman LM, Jensen CC, Yost HJ, Beckerle MC. Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement. J Cell Biol 2005;171:209-15. 25. Cerisano V, Aalto Y, Perdichizzi S, Bernard G, Manara MC, Benini S, et al. Molecular mechanisms of CD99-induced caspase-independent cell death and cell-cell adhesion in Ewing's sarcoma cells: actin and zyxin as key intracellular mediators. Oncogene 2004;23:5664-74. 26. Chan CB, Liu X, Tang X, Fu H, Ye K. Akt phosphorylation of zyxin mediates its interaction with acinus-S and prevents acinus-triggered chromatin condensation. Cell Death Differ 2007;14:1688-99. 27. Pierantoni GM, Fedele M, Pentimalli F, Benvenuto G, Pero R, Viglietto G, et al. High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth. Oncogene 2001;20:6132-41. 28. Pierantoni GM, Rinaldo C, Mottolese M, Di Benedetto A, Esposito F, Soddu S, et al. High-mobility group A1 inhibits p53 by cytoplasmic relocalization of its proapoptotic activator HIPK2. J Clin Invest 2007;117:693-702. 29. Li XL, Arai Y, Harada H, Shima Y, Yoshida H, Rokudai S, et al. Mutations of the HIPK2 gene in acute myeloid leukemia and myelodysplastic syndrome impair AML1- and p53-mediated transcription. Oncogene 2007;26:7231-9. 30. Wei G, Ku S, Ma GK, Saito S, Tang AA, Zhang J, et al. HIPK2 represses beta-catenin-mediated transcription, epidermal stem cell expansion, and skin tumorigenesis. Proc Natl Acad Sci U S A 2007;104:13040-5. 31. Sanchez-Carbayo M, Socci ND, Charytonowicz E, Lu M, Prystowsy M, Childs G, et al. Molecular profiling of bladder cancer using cDNA microarrays: defining histogenesis and biological phenotypes. Cancer Res 2002;62:6973-80. 32. Amsellem V, Kryszke MH, Hervy M, Subra F, Athman R, Leh H, et al. The actin cytoskeleton-associated protein Zyxin acts as a tumor suppressor in Ewing tumor cells. Exp Cell Res 2005;304:443-56. 33. Yu YP, Luo JH. Myopodin-mediated suppression of prostate cancer cell migration involves interaction with zyxin. Cancer Res 2006;66:7414-9. 34. Hofmann TG, Jaffray E, Stollberg N, Hay RT, Will H. Regulation of homeodomain-interacting protein kinase 2 (HIPK2) effector function through dynamic small ubiquitin-related modifier-1 (SUMO-1) modification. J Biol Chem 2005;280:29224-32. 35. Moehlenbrink J, Bitomsky N, Hofmann TG. Hypoxia suppresses chemotherapeutic drug-induced p53 Serine 46 phosphorylation by triggering HIPK2 degradation. Cancer Letters 2009;292:119-24. 36. Matsuzawa S, Li C, Ni CZ, Takayama S, Reed JC, Ely KR. Structural analysis of Siah1 and its interactions with Siah-interacting protein (SIP). J Biol Chem 2003;278:1837-40. 37. Polekhina G, House CM, Traficante N, Mackay JP, Relaix, F, Sassoon, DA, et al. Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling. Nat Struct Biol 2002;9:68-75. 38. van der Gaag EJ, Leccia MT, Dekker SK, Jalbert NL, Amodeo DM, Byers HR. Role of Zyxin in differential cell spreading and proliferation of melanoma cells and melanocytes. The Journal of investigative dermatology 2002;118:246-54. 39. Drees BE, Andrews KM, Beckerle MC. Molecular dissection of zyxin function reveals its involvement in cell motility. J Cell Biol 1999;147:1549-60. 40. Telerman A, Amson R. The molecular programme of tumour reversion: the steps beyond malignant transformation. Nat Rev Cancer 2009;9:206-16. 41. House CM, Moller A, Bowtell DD. Siah proteins: novel drug targets in the Ras and hypoxia pathways. Cancer Res 2009;69:8835-8.




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42. Wang W, Huper G, Guo Y, Murphy SK, Olson JA, Jr., Marks JR. Analysis of methylation-sensitive transcriptome identifies GADD45a as a frequently methylated gene in breast cancer. Oncogene 2005;24:2705-14. 43. Hofmann TG, Stollberg N, Schmitz ML, Will H. HIPK2 Regulates Transforming Growth Factor-beta-Induced c-Jun NH(2)-Terminal Kinase Activation and Apoptosis in Human Hepatoma Cells. Cancer Res 2003;63:8271-7. 44. Milovic-Holm K, Krieghoff E, Jensen K, Will H, Hofmann TG. FLASH links the CD95 signaling pathway to the cell nucleus and nuclear bodies. EMBO J 2007;26:391-401. FIGURE LEGENDS

Figure 1. Zyxin and HIPK2 interact and colocalize. (A) Schematic representation of the

Zyxin fragment recovered in a Yeast two Hybrid screen with the HIPK2 N-terminus as bait.

(B-C) GST pulldown assays with the indicated GST fusions (B: HIPK2 fragments, C: Zyxin

fragments) and 35S-labeled, in vitro translated proteins as indicated (D) Western Blot showing

precipitated Flag-HIPK2 and coprecipitated Myc-tagged Zyxin. Expression levels in the

lysates are shown below (E) Schematic representation of the HIPK2/Zyxin interaction

domains deduced from the pulldown assays. (F) Confocal images of immunofluorescence

stainings of endogenous HIPK2 (green) and Zyxin (red) in pre-extracted U2OS cells.

Colocalization is shown in yellow. DNA was counterstained with Hoechst (last panels).

Figure 2. Zyxin regulates HIPK2 stability. (A, B) Western Blot of U2OS (A) or MCF7 (B)

cells transfected with the indicated siRNA oligos, probed with the indicated antibodies. Actin

serves a loading control. In A, knockdown efficiency and HIPK2 mRNA levels are controlled

by RT-PCR (lower panel). (C) Western Blot (upper panel) of HepG2 cell pools stably

transfected with the indicated shRNA plasmids. Zyxin and HIPK2 mRNA levels are

controlled by RT-PCR (lower panel). (D) HT1080 cells stained for exogenously expressed,

Myc-tagged wild type Zyxin or nuclear-targeted (Nuc-) Zyxin. The cartoons show the

respective constructs with mutated residues depicted in red. (E) Cotransfection of Flag-




18

HIPK2 and increasing amounts of wild type or Nuc-Zyxin in 293T cells. GFP serves as

transfection control.

Figure 3. Zyxin interferes with proteasome-dependent HIPK2 degradation.

(A) U2OS cells transfected with control or Zyxin siRNA and treated with the proteasome

inhibitor MG132 (20 µM) for 12 hours as indicated. p53 serves as positive control for

proteasome inhibition, actin is used as loading control. (B) Western Blot of H1299 cells

transiently transfected with the indicated constructs. (C) Western Blot (upper panel) of stably

Zyxin-depleted HepG2 cell pools additionally transfected with control or Siah-1 siRNA.

Actin serves as loading control. Siah-1 knockdown was determined by RT-PCR (lower

panel).

Figure 4. Zyxin colocalizes and interacts with Siah-1. (A-C) Western Blots of

Coimmunoprecipitations of exogenously expressed Myc-Zyxin or Myc-Nuc-Zyxin and HA-

Siah-1 or ligase-deficient HA-Siah1C44S from 293T cells probed with the antibodies indicated.

(D-H) Confocal images of U2OS cells expressing the indicated Zyxin (green) and Siah-1

(red) proteins. Colocalization is shown in yellow. (I, J) GST pulldown assays were performed

with the indicated GST-Siah-1 fusion proteins and 35S-labeled in vitro translated Zyxin.

Figure 5 Zyxin regulates Siah-1 homodimerization. (A, B) Western Blot of a

coimmunoprecipitation experiment. 293T cells were cotransfected with (A) HA- and Flag-

Siah-1 or (B) HA- and Flag-Siah-1C44S in the presence of the indicated Zyxin constructs and

Flag-Siah-1 was precipitated from the lysates. Quantification of the ratio of coprecipitated

HA-Siah-1 to precipitated Flag-Siah-1 was performed using the ImageJ software.




19

Figure 6. Zyxin regulates DNA damage-induced p53 Ser46 phosphorylation (A, B)

Western Blots of HepG2 treated with the indicated doses of UV (A) or Adriamycin (B),

probed with the indicated antibodies. (C) Western Blot of stably control- or Zyxin-depleted

HepG2 cell pools treated with lethal UV doses, probed with the indicated antibodies. Note the

increase in Zyxin levels after DNA damage induction. Caspase-mediated PARP cleavage was

used as an indicator of apoptosis induction.




Kinase domain 11911

Kinase domain 5651Bait:

HIPK2

Isolated clone

A

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t

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in Y2H screen

-1-1

88

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9-5

20

Inpu

t

B

WB: 250-

Flag-HIPK2 - +Myc-Zyxin + +

autoradio-gram

Coomassiestaining

100 -

GS

T

GS

T

GS

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10%

HIPK2

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33-

24

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130

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95

-

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Pro LIM2LIM3 Kinase domain1 572 11911

EHIPK2 Zyxin

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Merge/DNAMergeHIPK2 Zyxin

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Crone et al., Figure 1




A B C

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yx

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D

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+ - +- + +- - -

+ + +

-+++

++++- -

short exposure

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WB: Flag


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GFP

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shZyxin

siL

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iah-

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yxin

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y

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Siah-1 1-80

Crone et al., revised Figure 4




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Flag-Siah-1+

--

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A

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shGL2 shZyxin

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ated

6h 16h

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actin

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actin

Crone et al revised Figure 6

GAPDH

HepG2

HepG2





Published OnlineFirst January 19, 2011.Cancer Res Johanna Crone, Carolina Glas, Kathrin Schultheiss, et al. axisZyxin is a critical regulator of the apoptotic HIPK2-p53 signaling

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