Molecular Determinants of PDLIM2 in Suppressing HTLV-I Tax-Mediated Tumorigenesis

Oncogene (2010) 29, 6499–6507 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc SHORT COMMUNICATION Molecular determinants of PDLIM2 in suppressing HTLV-I Tax-mediated tumorigenesis

JFu1,2, P Yan1,2,SLi1,2,ZQu1,2 and G Xiao1,2

1University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA and 2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Human T-cell leukemia virus type I (HTLV-I) encodes a virus replication but also required to immortalize many Tax oncoprotein that has crucial roles in both virus different cells including human primary T cells (Pozzatti replication and cell transformation. Our recent studies et al., 1990; Tanaka et al., 1990; Grassmann et al., 1992). suggest that the counterbalance between HTLV-I/Tax In addition, Tax-transformed cells induce tumors when and PDZ-LIM domain-containing protein PDLIM2 may introduced into nude or severe combined immuno- determine the outcome of HTLV-I infection. Although deficiency mice (Pozzatti et al., 1990; Oka et al., 1992). HTLV-I represses PDLIM2 epigenetically and specifi- More importantly, the HTLV-I genome without Tax cally in transformed cells, PDLIM2 shuttles Tax into the loses its original transforming ability (Yamaoka et al., nuclear matrix for ubiquitination-mediated proteasomal 1992), whereas Tax transgenic mice develop various degradation, thereby suppressing the transforming tumors depending on the type of the promoters used to ability of HTLV-I. Here, we have further shown that drive Tax expression (Nerenberg et al., 1987; Grossman PDLIM2 binds to Tax directly, which was mediated by et al., 1995; Peebles et al., 1995). Notably, Tax- a putative a-helixmotifofPDLIM2ataminoacids immortalized lymphocytes in vitro and Tax-mediated 236–254. Consistently, selective disruption of this short- T-cell lymphoma in animal resemble closely the helix crippled PDLIM2 in shutting Tax to the nuclear phenotype of HTLV-I-transformed T cells and HTLV- matrix for ubiquitination-mediated degradation, therefore, I-induced adult T-cell leukemia, respectively (Akagi PDLIM2 lost the ability in tumor suppression. Although the et al., 1995; Kwon et al., 2005; Hasegawa et al., 2006). C-terminal LIM domain of PDLIM2 was not required for The Tax oncoprotein exerts its oncogenic role largely Tax binding, it was important for PDLIM2 to interact with through deregulation of cellular transcription factors that the nuclear matrix. Accordingly, the LIM domain was are critical for cell growth and division, such as nuclear essential for PDLIM2-mediated Tax repression. On the factor-kB (Sun and Yamaoka, 2005; Grassmann et al., contrary, the N-terminal PDZ domain of PDLIM2 was 2005). In the cytoplasm, Tax recruits the inhibitor of dispensable for all these events, although the PDZ domain nuclear factor-kB kinase complex into specific perinuclear was involved in PDLIM2 binding to cytoskeleton. These structures for inhibitor of nuclear factor-kBkinase studies dissect functional sequences within PDLIM2 and activation, resulting in degradation of inhibitor of nuclear their distinct roles in Tax regulation. factor-kB and subsequent nuclear translocation of nuclear Oncogene (2010) 29, 6499–6507; doi:10.1038/onc.2010.374; factor-kB factors including p65, the prototypic member of published online 13 September 2010 nuclear factor-kB(Xiaoet al., 2006). In the nucleus, Tax recruits p65 and other cellular transcriptional components Keywords: HTLV-I; Tax; PDLIM2; protein shuttling; into interchromatin granules to form discrete transcrip- protein degradation; tumorigenesis tional hot spots named ‘Tax nuclear bodies’ or ‘Tax nuclear foci’ (Semmes and Jeang, 1996; Bex et al., 1997). Although the mechanisms by which Tax hijacks cellular signaling for its oncogenic action have been Introduction extensively investigated, the molecular studies on how HTLV-I/Tax is regulated by cellular factors are still Human T-cell leukemia virus type I (HTLV-I) is an lacking. Recently, we have identified PDLIM2, a newly oncogenic retrovirus etiologically associated with adult identified PDZ–LIM domain-containing protein with T-cell leukemia (Gallo, 2005; Takatsuki, 2005; Yoshida, ubiquitination-promoting activity, as a potent inhibitor 2005). The virus encodes a 40-kDa regulatory protein of Tax. In addition to promoting Tax polyubiquitina- named Tax. The Tax protein is not only required for tion, PDLIM2 also associates with and shuttles Tax from its functional sites including Tax nuclear foci into Correspondence: Dr G Xiao, Department of Microbiology and the nuclear matrix for proteasomal degradation (Yan Molecular Genetics, Hillman Cancer Center Research Pavilion, et al., 2009a). Accordingly, PDLIM2 coexpression University of Pittsburgh Cancer Institute, 5117 Centre Avenue, prevents downstream signaling and subsequent tumor- Pittsburgh, PA 15213, USA. igenicity of Tax. Interestingly, PDLIM2 is conversely E-mail: [email protected] Received 25 June 2009; revised 30 June 2010; accepted 1 July 2010; repressed by HTLV-I, which involves the epigenetic published online 13 September 2010 regulation but independent of the Tax oncoprotein Molecular determinants of PDLIM2 on Tax inhibition JFuet al 6500 (Yan et al., 2009b). More importantly, reconstitution of Interestingly, the amino acids 243–253 are located PDLIM2 into HTLV-I-transformed T cells blocks their within a putative a-helix motif of PDLIM2 at tumorigenicity in mice. These findings suggest that the amino acids 236–254 (http://www.predictprotein.org; counterbalance between PDLIM2 and HTLV-I/Tax Figure 1b). Of note, our previous studies suggested may determine the outcome of viral infection. Thus, it that short helix is the preferential structure for Tax is very interesting and important to address the binding (Xiao et al., 2000). To test whether the short molecular actions of PDLIM2 on Tax suppression. a-helix is responsible for the PDLIM2–Tax interaction, we disrupted the helix by smaller internal deletions or point substitutions with prolines. As shown in Figure 1g, disruption of the short helix, by its middle Results and discussion part small deletion (D241–245 or D246–250) or leucine to proline substitutions (LL241/242PP), completely Amino acids 236–254, but not the PDZ and LIM domains abolished the PDLIM2–Tax interaction, although of PDLIM2, are required for Tax interaction mutations of the N- or C-terminal of the short helix Although our previous studies showed that PDLIM2 is able (D236–240 or D251–255, or EE249/250PP) already led to to associate with Tax in vivo (Yan et al., 2009a), it remains large loss of the interaction. Together, these studies unknown whether the association is direct and independent clearly suggested that the putative a-helix motif of of other cellular proteins. To address the important issue, PDLIM2 is the Tax-binding motif essential for the we examined whether PDLIM2 and Tax proteins purified PDLIM2–Tax interaction. from bacteria bind to each other using the in vitro co- immunoprecipitation assay. As shown in Figure 1a, purified Different sequences within Tax are involved in PDLIM2 Glutathione S-Transferase (GST)-Tax could not be pulled interaction down by anti-Myc antibody. In the presence of Myc- To identify the sequences with Tax involved in PDLIM2 PDLIM2, however, GST-Tax was efficiently pulled down binding, we took advantage of the Tax small deletion by the anti-Myc antibody. These in vitro binding assays mutants that systematically cover the whole sequences indicated that PDLIM2 can directly interact with Tax. of Tax (Figure 2a; Fryrear et al., 2009). Interestingly, To define the sequences within PDLIM2 that are all those mutants still retained the ability of PDLIM2 responsible for its interaction with Tax, we initially binding, same as the wild-type Tax (Figure 2b), tested the role of the N-terminal PDZ domain of suggesting that different sequences within Tax can PDLIM2 in Tax binding (Figure 1b), as it is known complementarily interact with PDLIM2. This data are that Tax contains a PDZ domain-binding motif (PBM) consistent with the fact that Tax is an intrinsically at the C-terminus (Rousset et al., 1998). Surprisingly, disordered protein, which contains typical disordered mutation of the PBM motif within Tax (Tax DPBM) regions that exist dynamically for protein interaction failed to block the Tax–PDLIM2 interaction (Radivojac et al., 2007; Boxus et al., 2008). (Figure 1c). This result suggested that the PDLIM2– Tax interaction is not mediated by Tax PBM and PDLIM2 PDZ. In further support of this, deletion of The Tax-binding motif and LIM domain, but not the PDZ the PDZ domain of PDLIM2 (DPDZ) did not affect its domain of PDLIM2, are required for promoting Tax interaction with Tax either (Figure 1d). Similarly, polyubiquitination and degradation deletion of the C-terminal LIM domain (DLIM) had The major function of PDLIM2 in Tax repression is to no effect on Tax binding. In fact, simultaneous deletion promote Tax polyubiquitination and subsequently of both PDZ and LIM domains (DPDZ/LIM) did not proteasomal degradation (Yan et al., 2009a). Thus, affect the PDLIM2–Tax interaction much. In sharp we next examined the role of the different function contrast, deletion of the sequences between the PDZ and sequences of PDLIM2 in Tax polyubiquitination LIM domains (D79–278) prevented the PDLIM2–Tax and degradation. Consistent with previous studies interaction. Thus, the sequences between the PDZ and (Yan et al., 2009a), PDLIM2 strongly promoted Tax LIM domains contain Tax-binding motif. polyubiquitination (Figure 3a). In correspondence To pinpoint the sequences within PDLIM2 required with the ubiquitin E3 liagase function of the LIM for Tax binding, we generated various small internal domain of PDLIM2 (Tanaka et al., 2007), the DLIM deletion mutants of PDLIM2 (Figure 1b). Interestingly, or DPDZ/LIM mutant of PDLIM2 failed to promote internal deletion of amino acids 243–253 (D243–253) Tax polyubiquitination. Similarly, all PDLIM2 mutants caused complete loss of the PDLIM2–Tax interaction defective in Tax binding such as the D79–278, D243–253, (Figure 1e). The loss of Tax binding seemed not because LL241/242PP and EE249/250PP mutants lost their of overall structure disruption of PDLIM2, because ability in promoting Tax polyubiquitination. On the deletions of the sequences ahead of or behind the motif other hand, the PDLIM2 DPDZ and D195–207 mutants, (D195–207 and D258–278) did not affect the PDLIM2– which were capable of binding to Tax, still retained the Tax interaction. Most importantly, deletion of this motif ability in promoting Tax polyubiquitination. These did not affect PDLIM2 binding to p65 (Figure 1f), results suggested that through its middle Tax-binding another target of PDLIM2 (Tanaka et al., 2007). These motif, PDLIM2 physically interacts with Tax, which in results indicated that the amino acids 243–253 are turn allows the C-terminal LIM domain to promote required specifically for the Tax binding. polyubiquitination of Tax.

Oncogene Molecular determinants of PDLIM2 on Tax inhibition JFuet al 6501 12 a b PDZ LIM IP-IB GST-Tax PDLIM2 WT

GST-Tax 236 SSSFRLLQEALEAEERGGT 254 Input Myc-PDLIM2 PDLIM2 PDZ + + GST-Tax PDLIM2 LIM - + Myc-PDLIM2

PDLIM2 PDZ/LIM c 123 IP-IB Myc-PDLIM2 PDLIM2 79-278

Myc-PDLIM2 PDLIM2 195-207 Input Tax PDLIM2 243-253 + + + Myc-PDLIM2 PDLIM2 258-278 - + - Tax WT --+TaxPBM 1 78 279 349 amino acid

d 123 4 56 e 1234 56 IP-IB Tax IP-IB Tax

Tax Tax

Input PDLIM2 Input PDLIM2 & & mutants mutants

+ + + + + + Tax + + + + + + Tax r r 3 8 o T Z M M 8 o T 07 5 78 7 ct W D I I 27 ct W 2 2 2 2 e P L /L - Myc-PDLIM2 e - - - - Myc-PDLIM2 V Z 9 V 5 43 8 79 D 7 19 2 5 P 2

f 12 3 g 1 2 3 4 5 6 7 8 IP-IB HA-p65 IP-IB Tax

HA-p65 Tax Input Input WT Myc-PDLIM2 243-253 & mutants + + + HA-p65 + + + + + + + + Tax r 0 5 0 5 P P r 3 to T 4 4 5 5 P P to T 5 c W -2 -2 -2 2 2 0 c W 2 e 6 1 6 1- 4 5 e 3- Myc-PDLIM2 V 3 4 4 5 /2 /2 Myc-PDLIM2 V 4 2 2 2 2 41 9 2 2 24 L E L E Figure 1 Amino acids 236–254, a putative short a-helix, of PDLIM2 are required for Tax binding. (a) Direct binding of PDLIM2 to Tax. GST-Tax and His-Myc-PDLIM2 proteins puriﬁed from Escherichia coli were mixed together and incubated at 4 1C for 2 h, followed by immunoprecipitation (IP) using anti-Myc antibody and immunoblotting (IB) using anti-Tax antibodies. As a negative control, the same amount of puriﬁed GST-Tax protein in the absence of His-Myc-PDLIM proteins was included in the parallel experiment. The inputs of GST-Tax and his-Myc-PDLIM2 were analyzed by IB using anti-Tax and anti-Myc antibodies, respectively. Both anti-Tax and anti-Myc antibodies were generated from hybridomas as described before (Qu et al., 2004; Qing et al., 2005). (b) Schematic representation of PDLIM2 and its mutants used in this study. Amino acid 236–254, a putative short a-helix that acts as the Tax-binding motif of PDLIM2, are also shown. (c) Dispensable role of Tax PBM in PDLIM2 binding. A total of 293 cells were transfected with Myc-PDLIM2 alone or together with Tax WT or Tax EV352/353AS, the DPBM disruption mutant). Whole-cell lysates from the transfected cells were subjected to IP using anti-Tax antibody and IB using anti-Myc antibody. The inputs of Myc- PDLIM2, Tax and Tax DPBM mutant were analyzed by direct IB. (d) Requirement of the middle region between the PDZ and LIM domains for PDLIM2 binding to Tax. Whole-cell lysates from 293 cells transfected with Tax alone or together with the indicated Myc-PDLIM2 constructs were analyzed by IP using anti-Myc antibody and IB using anti-Tax antibody. The inputs of Tax and Myc-PDLIM2 constructs were analyzed by IB. (e) Requirement of amino acids 243–253 for PDLIM2 binding to Tax. Whole-cell lysates from 293 cells transfected with Tax alone or together with the indicated PDLIM2 internal deletion constructs were analyzed by IP using anti-Myc antibody and IB using anti-Tax antibody. (f) Dispensable role of amino acids 243–253 of PDLIM2 in p65 binding. Whole-cell lysates from 293 cells transfected with HA-p65 alone or together with Myc-PDLIM2 (WT) or its amino acids 243–253 deletion mutant (D243–253) were analyzed by IP using anti-Myc antibody and IB using Hemagglutinin-Horseradish Peroxidase (HA-HRP) antibody. (g) Requirement of amino acids 236–254 for PDLIM2 binding to Tax. Whole-cell lysates from 293 cells transfected with Tax alone or together with the indicated PDLIM2 constructs were analyzed by IP using anti-Myc antibody and IB using anti-Tax antibody.

Oncogene Molecular determinants of PDLIM2 on Tax inhibition JFuet al 6502 a PDLIM2 DLIM, as this HTLV-I-transformed T cell Tax WT line expresses a modest level of endogenous PDLIM2 et al. 1 353 amino acid (Yan , 2009a). Consistently, Tax protein underwent a modest proteasomal degradation, which was signiﬁ- Tax 1-29 cantly accelerated by stable expression of exogenous Tax 30-52 PDLIM2 (Figure 3c). More importantly, the Tax degradation was completely blocked by expression of Tax 53-99 PDLIM2 DLIM. These results indicated that PDLIM2 Tax 100-150 DLIM functions as a dominant-negative version of PDLIM2, further suggesting the important roles of Tax 151-202 the LIM domain and Tax-binding motif in PDLIM2- Tax 203-254 mediated degradation of Tax.

Tax 255-289

Tax 290-322 The Tax-binding motif and LIM domain, but not the PDZ domain of PDLIM2, are required for Tax subcellular Tax 323-353 redistribution In addition to promoting Tax polyubiquitination, b 1 2 3 4 5 6 7 8 9 10 11 PDLIM2 shuttles Tax from its functional sites such as Myc-PDLIM2 perinuclear locations and Tax nuclear foci into the IP-IB nuclear matrix for proteasomal degradation (Yan et al., Tax & mutants * IgG 2009a). Our previous studies indicated that the Tax- Input Myc-PDLIM2 shuttling function of PDLIM2 can be visualized by the +++++ + +++ ++Myc-PDLIM2 indirect immunofluorescence staining to detect dimin- r 9 2 9 0 2 4 9 2 3 to T 2 5 9 5 0 5 8 2 5 ishment or disappearance of Tax perinuclear aggregates c W - - - 1 -2 -2 2 -3 -3 e 1 30 53 - 1 3 - 0 3 Tax V 00 5 0 5 9 2 1 1 2 25 2 3 and nuclear foci (Yan et al., 2009a). In line with previous studies (Semmes and Jeang, 1996; Bex et al., Figure 2 Multiple sequences within Tax are involved in PDLIM2 1997; Yan et al., 2009a), Tax strongly localized to the binding. (a) Schematic representation of Tax-GFP and its mutants. Of note, the fused green fluorescent protein (GFP) weren’t shown. perinuclear aggregates and discrete nuclear bodies (b) Interaction between PDLIM2 and Tax or Tax mutants. Whole- (Figure 4a, panel 1 from left) when Tax was expressed cell lysates from 293 cells transfected with the indicated cons- in Hela cells, which lack expression of endogenous tructs were analyzed by IP using anti-GFP antibody and IB PDLIM2 (Yan et al., 2009a). Consistent with our using anti-Myc antibody. The same membrane was stripped and re-immunoblotted with the anti-GFP antibody. The input of previous studies (Yan et al., 2009a), coexpression of Myc-PDLIM2 was also shown. IgG, immunoglobulin G. PDLIM2 resulted in significantly less staining of Tax proteins and almost complete disappearance of Tax perinuclear aggregates and nuclear bodies (panel 2). Highly consistent with their dispensable role in When the proteasome was inhibited, the disappearance promoting Tax ubiquitination, the PDZ domain and of Tax staining in the perinuclear aggregates and nuclear amino acids 195–207 as well as amino acids 258–278 are bodies was associated with increased Tax amount in the not involved in PDLIM2-stimulated Tax proteasomal nuclear matrix (Yan et al., 2009a). Although it lost the degradation, because PDLIM2 mutants deleting these cytoskeleton-binding ability, the PDZ deletion mutant sequences, like the wild type PDLIM2, were able to could still shuttle Tax (panel 3), similar to the PDLIM2 promote Tax degradation in our pulse-chase assays wild type. In contrast, the LIM deletion mutant largely (Figure 3b). In sharp contrast, disruption of the LIM lost the ability in Tax shuttling, as evidenced by normal domain (DLIM) or the Tax-binding motif (D243–253, expression of Tax in perinuclear aggregates and nuclear LL241/242/PP, EE249/250PP) resulted in the disability bodies (panel 4). In high agreement with their abilities of PDLIM2 in promoting Tax proteasomal degrada- in Tax binding, the amino acids 195–207 or 258–278 tion. It should be noted that the functional differences of deletion mutant behaved like wild-type PDLIM2 (panels the PDLIM2 mutants were not because of their different 5 and 7) whereas the amino acids 243–253 deletion expressions, as their expression levels were comparable mutant as well as the LL241/242PP and EE249/250PP (Figures 3a and b, lower panels). Thus, both Tax- mutants of PDLIM2 lost the Tax-shuttling ability binding motif and LIM domain of PDLIM2 are crucial (panels 6, 8 and 9). Of note, all the mutations except for promoting Tax polyubiquitination and subsequent the PDZ deletion did not affect expression pattern of proteasome-mediated degradation, although they have PDLIM2, particularly the cytoskeleton binding under distinct roles in these events. the immunofluorescence staining, suggesting no overall As PDLIM2 DLIM mutant binds to Tax, but does structure alterations of the PDLIM2 mutants. These not induce Tax degradation, it will be interesting to results suggested that the Tax-shuttling function of examine whether this mutant can function as a PDLIM2 depends on the Tax-binding motif and LIM dominant-negative form of PDLIM2. To address this domain. important issue, we generated C8166 cells stably As the nuclear matrix is nuclear framework through- expressing an empty vector, PDLIM2 wild type or out the nucleoplasm, the expression of Tax and

Oncogene Molecular determinants of PDLIM2 on Tax inhibition JFuet al 6503 a 1 2 3 4 5 6 7 8 9 10 11 c 250 123456789 100 Ubi- Tax 75 Tax 50 PDLIM2 37 & kDa Mutants Tax Lamin B

0 3 3 0 3 3 0 3 3 (h) Chase PDLIM2 & --+--+--+MG132 mutants C8166 + + + + + + + + + + + HA-Ubi - ++++++++++ Tax r o T Z IM M 07 3 8 P P ct W D I 2 5 7 P P e P L /L - -2 -2 2 0 V Z 95 3 9 4 5 Myc-PDLIM2 D 1 24 7 /2 /2 P 1 49 24 2 L E L E b 1 2 3 4 5 6 7 8 9101112131415161718192021222324252627

Tax

PDLIM2 & mutants

Lamin B 033033033 03303 3033 0 33 033033 (h) Chase MG132 Vector WT PDZ LIM 195-207 243-253 258-278 LL241/242PP EE249/250PP PDLIM2 Tax Figure 3 Tax-binding motif and LIM domain are required for PDLIM2-promoted Tax polyubiquitination and degradation. (a) Effect of different sequences within PDLIM2 on the promotion of Tax polyubiquitination. In all, 293 cells transfected with the indicated constructs were treated with 2.5 mM proteasome inhibitor MG132 for 16 h, followed by nuclear extraction as described before (Yan et al., 2009a), and IP using anti-Tax antibody and IB using HA-HRP antibody. The inputs of Myc-PDLIM2 and Tax were analyzed by IB. (b) Effect of different sequences within PDLIM2 on the promotion of Tax degradation. A total of 293 cells transfected with the indicated constructs were mock-treated or treated with 10 mM protein synthesis inhibitor cycloheximide in the presence or absence of 10 mM MG132 for 3 h. The insoluble nuclear fractions were then used for IB as described before (Yan et al., 2009a). (c) Dominant-negative function of PDLIM2 DLIM mutant. HTLV-I-transformed T-cell line C8166 stably expressing PDLIM2, PDLIM2 DLIM or an empty vector was used for the pulse-chase assays as described in b.

PDLIM2 in these two distinct subcellular locations fractions, suggesting that PDLIM2 shuttles Tax into cannot be distinguished by the immunofluorescence the nuclear matrix for proteasomal degradation (Yan staining. To further confirm the role of different et al., 2009a). Similar to the PDLIM2 wild type, sequences within PDLIM2 in shuttling Tax into the PDLM2 mutants deleting the PDZ domain (DPDZ), nuclear matrix, we also performed the subcellular amino acids 195–207 (D195–207) and 258–278 (D258– fraction assay using 293 cells, which express undetect- 278) were able to shuttle Tax into the nuclear matrix. able endogenous PDLIM2 protein (Yan et al., 2009a). In contrast, PDLIM2 mutants deleting the LIM domain As shown in Figure 4b, Tax was expressed in the (DLIM and DPDZ/LM) and mutants disrupting the cytoplasm, soluble nuclear fraction (nucleoplasm) and Tax-binding motif (D79–278, D243–253, LL241/242PP insoluble nuclear fraction (nuclear matrix) in the and EE249/250PP) lost this ability. Together with the absence of PDLIM2. It should be noted that Tax immunofluoresence studies, these results suggested that proteins in the nuclear matrix were resistant to the the Tax-binding motif and LIM domain, but not the proteasome-mediated degradation in the absence of PDZ domain, are essential for PDLIM2-mediated PDLIM2, similar to those in the cytoplasm and shuttling of Tax into the nuclear matrix. nucleoplasm (Figure 3b; Yan et al., 2009a). In agree- Recent studies suggest that PDLIM2 is expressed in ment with our previous studies (Figure3b; Yan et al., both cytoplasm and nucleus with the ability to interact 2009a), coexpression of PDLIM2 resulted in significant with cytoskeleton (Torrado et al., 2004; Loughran increase in Tax’s ratio in the nuclear matrix with et al., 2005; Tanaka et al., 2005; Yan et al., 2009a, b). decrease in Tax total protein (Figures 4b and c). However, it still remains largely unknown how PDLIM2 Proteasome inhibition would increase Tax expression is expressed in the distinct subcellular compartments only in the nuclear matrix, but not other cellular and the role of this different subcellular expression in

Oncogene Molecular determinants of PDLIM2 on Tax inhibition JFuet al 6504 PDLIM2 functions. To address this issue, we initially expression patterns of PDLIM2 and Tax. Consistently, examined the effect of treatment of leptomycin (LMB), LMB did not interfere with PDLIM2-mediated Tax a speciﬁc inhibitor of chromosome region maintenance-1 nuclear degradation. The failure of LMB was not owing (CRM1)-dependent nuclear export, on the subcellular to its inefﬁciency in CRM1 inhibition, because inhibitor expression of PDLIM2. As shown in Supplementary of nuclear factor-kBa nuclear export was blocked in the Figure 1, LMB treatment did not affect subcellular same LMB-treated cells. These data suggested that the

a Tax Vector PDLIM2 WT PDZ LIM 195-207 243-253 258-278 LL241/242PP EE249/250PP Nucleus Merge PDLIM2 Tax

b 1 2 3 4 5 6 7 8 9 10 11 c Cytoplasm Soluble nucleus Insoluble nucleus 100 Tax 80

PDLIM2 60 & mutants

Cytoplasm 40

Hsp90 20 Ratio of Tax in different

subcellular localizations (%) 0 Tax ++++++++++Tax r o T Z M M 8 7 53 78 P P ct D I I 7 20 2 2 P P e W L /L 2 - - - 2 50 V P Z 9- 5 3 8 4 2 D 7 9 24 25 /2 / Myc-PDLIM2 PDLIM2 P 1 1 49 24 2 L E & L E mutants d Cytoplasm Soluble nucleus Insoluble nucleus Sp-1 100 Soluble nuclear fraction 80 Tax 60

PDLIM2 40 & mutants 20

LaminB Ratio of PDLIM2 in different subcellular localizations (%) 0 + + + + + + + + + + Tax Insoluble nuclear fraction +++++++++ ++Tax 7 3 8 P P T Z IM IM 8 0 5 7 P P r W D L L 27 2 -2 -2 2 0 o T Z M M 8 7 3 8 P P / - 5- 3 8 4 5 t I I 7 0 5 7 P P Z 9 9 4 5 2 2 c W D L /L 2 -2 -2 -2 P 0 D 7 1 2 2 1/ / Myc-PDLIM2 e P Z - 5 3 8 42 5 P 4 49 V D 79 9 4 5 2 2 Myc-PDLIM2 2 2 P 1 2 2 / 9/ L E 1 4 L E 24 2 L E L E

Oncogene Molecular determinants of PDLIM2 on Tax inhibition JFuet al 6505 nuclear exports of both PDLIM2 and Tax are CRM1- PDLIM2 in its subcellular expression. In association inpendent. To deﬁne the mechanism of PDLIM2 with its inability to bind to cytoskeleton (Torrado et al., shuttling between the cytoplasm and the nucleus, we 2004; Loughran et al., 2005; also see Figure 4a), the then examined the role of distinct sequences within PDZ domain deletion mutant showed an increased

a 123456789 b 2.5 Vector Tax/PDLIM2 LL241/242PP Tax Tax/Vector Tax/PDLIM2 EE249/250PP 2 Tax/PDLIM2 1 0.75 0.72 0.92 0.95 0.71 0.96 0.93 Tax/PDLIM2 PDZ PDLIM2 1.5 Tax/PDLIM2 LIM & Tax/PDLIM2 243-253 mutants Tax/PDLIM2 258-278 1 Hsp90 OD 560/670 nm - + + + + + + + + Tax 0.5 r r o to T Z M 3 8 P P t c W D I 25 27 P P ec e P L - - 2 0 Myc-PDLIM2 V V 3 8 24 5 24 25 / /2 0 41 9 2 24 Days 1 2 3 4 5 6 7 L E L E

c 120 100 80 60 **p=0.0089 40 **p=0.0077 **p=0.0062 20

Colony formation (%) 0 pCLXSN Tax Tax Tax Tax Tax Tax Tax Tax pMX pMX WT PDZ LIM 243-253 258-278 LL241/242PP EE249/250PP

Myc-PDLIM2

d 120 100 80 60 **p=0.0089 **p=0.01 40 **p=0.0096 20 Tumor weight (mg) 0 pCLXSN Tax Tax Tax Tax Tax Tax Tax Tax pMX pMX WT PDZ LIM 243-253 258-278 LL241/242PP EE249/250PP

Myc-PDLIM2 Figure 5 Tax-binding motif and LIM domain of PDLIM2 are required for suppressing Tax-mediated tumorigenesis. (a) Generation of Rat-1 cells stably expressing both Tax and Myc-PDLIM2 constructs. The pCLXSN-Tax Rat-1 stable cells we generated before (Yan et al., 2009a) were re-infected with pMX-Myc-PDLIM2 constructs, followed by selection with both G418 and puromycin. Expression levels of Tax and Myc-PDLIM2 constructs in the stable cells were examined by direct IB using the whole-cell lysates. The expression of endogenous Hsp90 proteins was used as a loading control. The Tax and Hsp90 bands were also quantitated by densitometry and their ratios were indicated. (b) Growth curve of the Rat-1 stable cell lines. The cells cultured under normal growth medium were subjected to 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide assay to determine viable cells. (c) Effect of different sequences within PDLIM2 on Tax-mediated in vitro anchorage-independent growth. The indicated Rat-1 stable cells were plated in soft agar for colony formation. Colony numbers were counted at day 21 after plating. The data presents the percentile of colony formation compared with the cells expressing Tax alone (set as 100). Error bars indicate s.d. (n ¼ 3). (d) Effect of different sequences within PDLIM2 on Tax-mediated tumor growth in mice. The indicated Rat-1 stable cells were subcutaneously inoculated into severe combined immunodeﬁciency mice. The mice were killed at day 14 after inoculation and tumor weights were measured. The data presented are the mean±s.d. (nX3).

Figure 4 Tax-binding motif and LIM domain of PDLIM2 are required for shuttling Tax to the nuclear matrix. (a) Effect of different sequences within PDLIM2 on Tax subcellular distribution. Hela cells transfected with Tax alone or together with different PDLIM2 mutants were analyzed by the indirect immunofluorescence assays (Qing et al., 2007). Tax, PDLIM2 WT and its mutants, and nuclei are shown in green, red and blue, respectively. Original magnification, Â 400. (b) Effect of different sequences within PDLIM2 on Tax subcellular expression. In all, 293 cells were transfected with Tax alone or together with the indicated PDLIM2 constructs, followed by cell fractions and IB to detect expression levels of the indicated proteins. The detailed protocol for cytoplasmic fraction, soluble nuclear fraction and insoluble nuclear fraction was described previously (Yan et al., 2009a). (c) Quantitation of Tax protein in different subcellular fractions. The Tax bands in b were quantitated by densitometry and the ratio of Tax in the cytoplasm, soluble nuclear fraction and insoluble nuclear fraction were presented in percentile. (d) Effect of different sequences within PDLIM2 on its subcellular expression. The PDLIM2 and its mutants in b were also quantitated by densitometry and the ratio of their expression levels in the cytoplasm, soluble nuclear fraction and insoluble nuclear fraction was presented in percentile. A full colour version of this figure is available at the Oncogene journal online.

Oncogene Molecular determinants of PDLIM2 on Tax inhibition JFuet al 6506 expression in the cytoplasm (Figure 4d). Interestingly, this suppression function of PDLIM2 on Tax-mediated the PDZ domain deletion mutant also showed a tumorigenesis. On the contrary, deletion of the PDZ decreased expression in the nucleoplasm, but no obvious domain (DPDZ) or amino acids 258–278 (D258–278) effect in the nuclear matrix expression, suggesting an had no statistically significant effect on the suppression additional role of the PDZ domain in PDLIM2 of Tax-mediated transformation. These in vitro results nucleoplasmic expression. Although the LIM domain were further substantiated by our in vivo studies. As was not required for PDLIM2 binding to cytoskeleton shown in Figure 5d, the LIM domain and Tax-binding (Loughran et al., 2005; Figure 4a), it was involved in motif, but not the PDZ domain or amino acids 258–278, PDLIM2 expression in the nuclear matrix, as deletion were required to suppress Tax-mediated tumorigenesis of this important domain led to significant decrease in in severe combined immunodeficiency mice. the nuclear matrix, but increase in the cytoplasm and In conclusion, we have dissected different functional nucleoplasm of PDLIM2 protein (Figure 4d). This sequences within PDLIM2. We have shown that a finding explained why the LIM domain is required for putative short a-helix of PDLIM2 at amino acids PDLIM2-mediated Tax shuttling into the nuclear 236–254 functioned as the selective Tax-binding motif, matrix, although it is not required for PDLIM2 binding therefore required for Tax subcellular redistribution into to Tax. Consistent with these results, deletion of both the nuclear matrix, polyubiquitination, degradation and PDZ and LIM domains led to almost exclusive expres- eventual suppression of Tax tumorigenicity. Interest- sion of PDLIM2 in the cytoplasm, whereas deletion of ingly, the C-terminal LIM domain and the N-terminal the entire region between the PDZ and LIM domains PDZ domain were respectively involved in PDLIM2 led to dramatic increase in the nuclear matrix with binding to the nuclear matrix and cytoskeleton, significant decrease in the nucleoplasm of PDLIM2 although both domains were dispensable for Tax protein. On the other hand, disruptions of the Tax- binding. In further support of the fact that PDLIM2 binding motif or small internal deletions of the middle targeted Tax into the nuclear matrix for proteasomal region of PDLIM2 had no effect on its subcellular degradation, the LIM domain, but not the PDZ distribution. These studies suggested that different domain, was required for PDLIM2-mediated Tax domains of PDLIM2 have distinct roles in PDLIM2 suppression. These studies thus provide important subcellular expression and Tax shuttling. insights into the molecular actions of PDLIM2 on Tax regulation. These studies also have the general signifi- Both Tax-binding motif and LIM domain, but not cance in cancer biology and treatment, given our recent the PDZ domain of PDLIM2, are involved in findings linking PDLIM2 epigenetic repression to PDLIM2-mediated suppression of Tax tumorigenicity pathogenesis of different cancers such as breast cancer To investigate the significance of these important and colon cancer (Qu et al., 2010a, b). domains in PDLIM-mediated suppression of Tax tumorigenicity, we generated Rat-1 fibroblasts stably expressing Tax alone or together with different PDLIM2 mutants (Figure 5a). Although expression of Conflict of interest Tax alone or together with PDLIM2 or PDLIM2 mutants had no effect on the growth of Rat-1 cells in The authors declare no conflict of interest. normal culture condition (Figure 5b); Tax could promote colony formation of Rat-1 cells in soft agar (Figure 5c). Consistent with our previous studies (Yan Acknowledgements et al., 2009a), PDLIM2 was able to prevent the Tax- mediated anchorage-independent growth of Rat-1 cells. We thank N Raab-Traub for Rat-1 cells, OJ Semmes for Tax- Highly consistent with their roles in Tax shuttling and GFP mutants, and NIH AIDS Research & Reference Reagent proteasomal degradation, deletion of the LIM domain Program for various reagents. This study was supported by (DLIM) or disruption of Tax-binding motif (D243–253, NIH/NCI Grant R01 CA116616 and ACS Grant RSG-06-066- LL241/242PP and EE249/250PP) resulted in loss of 01-MGO to G Xiao.

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