Molecular Characterization of Ring Finger Protein 11

Michael K. Connor,1 Peter B. Azmi,1,3 Venkateswaran Subramaniam,1 Hoaxia Li,c and Arun Seth1,2,3

1Molecular and Cellular Biology and 2Laboratory of Molecular Pathology, Sunnybrook and Women’s College Health Sciences Centre; and 3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Abstract proper RNF11 function by AKT may prove to be Ring finger proteins serve many vital functions within detrimental to patient outcomes, making RNF11 a the cell. We have identified RNF11, a novel 154-amino potential target for novel cancer therapeutics. acid ring finger–containing protein, which is elevated in (Mol Cancer Res 2005;3(8):453–61) breast cancer. Within its ring finger domain, RNF11 contains an AKT phosphorylation site (T135) that is Introduction situated within a 14-3-3 binding domain. In WM239 cells The identification and characterization of new proteins has with constitutively active AKT, RNF11 exhibits seven never been more prevalent than it is in today’s research distinct phosphopeptides as measured using environment. Many debilitating diseases, including cancer, two-dimensional phosphopeptide mapping. Upon rely on therapies that are ineffective at managing symptoms inhibition of the AKT pathway or mutation of T135, the and controlling disease progression. In addition, cancer phosphorylation at one of these sites is virtually provides an additional challenge in that tumors respond eliminated, suggesting that AKT may phosphorylate initially to treatments and often become resistant to therapy RNF11 at T135. Moreover, RNF11 is phosphorylated by and the disease continues its deadly progression (1-3). Thus, AKT in vitro and is recognized by phospho-AKT the identification of new therapeutic strategies and novel substrate antibodies. RNF11 shows enhanced binding to potential drug targets is essential if cancer management and 14-3-3 in WM239 cells compared with that seen in the treatment is to improve. To that end, we have identified that parental WM35 cells which have low AKT activity. ring finger protein 11 (RNF11), a 154–amino acid protein, is Furthermore, treatment of WM239 cells with LY294002 elevated in 90% of invasive ductal carcinomas of the breast reduces RNF11/14-3-3 interactions suggesting that (4, 5). RNF11 contains a PY motif in its NH terminus and a RNF11/14-3-3 binding is regulated by AKT. In addition, 2 ring finger domain near the COOH-terminal end (ref. 6; RNF11/14-3-3 binding is enhanced by constitutively Fig. 1). PY motifs are known to interact with WW-domains in active AKT and is diminished by dominant-negative partner proteins (7, 8). We have shown this for RNF11, which AKT. There is also reduced 14-3-3 binding to T135E interacts with the E3 ligases, Smurf2 and AIP4 (9, 10), both RNF11. RNF11 localization was altered from the of which are WW-containing proteins. In fact, the RNF11 PY cytoplasm to the nucleus by activated AKT. Thus, domain is identical to that found in Smad7, a protein that phosphorylation of RNF11 by AKT either causes its binds to Smurf2 and acts to inhibit the transforming growth nuclear localization or induces degradation of factor-h (TGF-h) signaling pathway by degrading the TGF-h cytoplasmic RNF11. In addition, T135E RNF11, which receptor (11) and preventing the activation of the receptor does not bind 14-3-3 and is not phosphorylated by AKT, activated Smads (2 and 3). This gives RNF11 a potentially causes a greater enhancement of transforming growth important role in the TGF-h signaling pathway. RNF11 may factor-B signaling than wild-type RNF11. It is clear compete with Smad7 for binding to Smurf2, thereby altering that RNF11 function, localization, and potentially, or possibly inhibiting Smad7 activity. In addition, ring finger degradation are regulated by AKT. Disregulation of domains are also important mediators of protein-protein interactions (12, 13), giving another potential region for the regulation of RNF11 function. However, the importance of Received 9/30/04; revised 6/14/05; accepted 7/14/05. this domain in mediating RNF11 function is as yet undefined. Grant support: Canadian Breast Cancer Research Initiative grant #012099 to AKT is a serine/threonine kinase that controls a signaling A. Seth and a postdoctoral fellowship from the U.S. Department of Defense Breast Cancer Research Program (DAMD17-02-1-0573) to M. Connor. Canadian pathway which plays an integral role in many cellular functions Foundation for Innovation. (14-16). AKT has also been shown to be important in breast The costs of publication of this article were defrayed in part by the payment of cancer. Activated AKT phosphorylates the cyclin-dependent page charges. This article must therefore be hereby marked advertisement in KIP1 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. kinase inhibitor p27 on threonine 157 and alters its Note: M.K. Connor is currently with the Department of Kinesiology and Health subcellular localization from the nucleus, where it acts to Science, York University, Toronto, Ontario, Canada. suppress the cell cycle and maintain cells in a quiescent state, to c Deceased Requests for reprints: Arun Seth, Sunnybrook and Women’s College Health the cytoplasm which causes disruption of the normal regulation Sciences Centre, 2075 Bayview Avenue, Room E-423B Toronto, Ontario, M4N of the cell cycle (17). This permits cell cycle entry and likely 3M5. Phone: 416-480-6100; Fax: 416-480-5703. E-mail: [email protected] Copyright D 2005 American Association for Cancer Research. plays a role in the transformation of normal cells to a malignant doi:10.1158/1541-7786.MCR-04-0166 phenotype. Elevated AKT levels are associated with poor

FIGURE 1. The RNF11 sequence contains multiple regulatory elements. The primary amino acid sequence of RNF11 showing potential regulatory regions.

patient prognosis in multiple cancers, including breast and between phosphorylated serine/threonine residues (24). One prostate cancer (17). In addition, AKT phosphorylation of the of the two optimal binding sequences of the 14-3-3 proteins, BAD protein prevents apoptosis by sequestering BAD away RxxS/T(p)xP, generates a sequence partially overlapping an from the antiapoptotic protein BCL-2. Phosphorylation of BAD optimal RxRxxS/T sequence, where the serine or threonine is by AKT promotes its interaction with 14-3-3 (18, 19). This phosphorylated by the AKT protein (15, 25, 26). This is the prevents the BAD-dependent regulation of BCL-2 activity case for RNF11, where we observed a potential AKT site at preventing the induction of apoptosis in response to cellular T135 nestled within a putative 14-3-3 binding domain. Thus, insults. This would make a cell with elevated AKT activity AKT-dependent 14-3-3 binding may also be important in susceptible to malignant transformation due to the altered mediating RNF11 localization and function, and may provide phosphorylation status of target proteins. insight into the relevance of elevations in RNF11 expression We have identified an optimal 14-3-3 binding sequence observed in breast cancer. within the RING finger domain of RNF11. 14-3-3 molecules This study was designed to characterize the function of the are a family of proteins that bind phosphorylated serine/ novel protein RNF11 which is elevated in cancer. We show that threonine residues within a consensus binding sequence (20). RNF11 is phosphorylated by AKT and that this phosphorylation Several members of this gene family are down-regulated in mediates RNF11 binding to 14-3-3. AKT phosphorylation also breast cancer and serve as molecular markers of potential seems to promote an alteration of the subcellular distribution of clinical interest (20, 21). The 14-3-3 proteins have roles in RNF11, possibly by the selective proteasomal degradation of signal transduction pathways that control cell cycle check- cytoplasmic RNF11. In support of this, mutant RNF11 that does points, mitogen-activated protein kinase activation, apoptosis, not bind 14-3-3 and is not phosphorylated by AKT enhances and programs of gene expression (22). Biochemical, structural, TGF-h signaling to a greater extent than does wild-type and genetic data have revealed some of the molecular basis of RNF11. 14-3-3 function resulting in a model in which 14-3-3 stabilizes conformations of bound ligands to promote their interactions with downstream targets, or facilitate their subsequent modifi- Results cation by kinases and phosphatases (23). Although just seven Phosphorylation of RNF11 by AKT In vitro genes form the 14-3-3 family, their function in signal Bioinformatical analyses of the RNF11 amino acid transduction has remained obscure due to the plethora of sequence identified a potential AKT phosphorylation site lo- interacting proteins. 14-3-3 binding has been shown to regulate cated at T135 located within a 14-3-3 binding domain (Fig. 1). its partners in a positive and/or negative fashion. Interaction To determine whether or not RNF11 was in fact phosphor- with the partner proteins is through specific interactions ylated by AKT, recombinant RNF11 protein was incubated

with recombinant constitutively active AKT and 32P-gATP ylation using this method, we did conclusively identify the (Fig. 2A). Under these conditions, a phosphorylated RNF11 presence of numerous RNF11 trypsin fragments in our sample band is clearly evident (Fig. 2A, lane 3). This band was not with high confidence (Fig. 2B), which were not present in the present when the reactions were conducted in the presence GSK3h sample. of a 100 molar excess of cold ATP or at 4jC (Fig. 2A, lanes 1 and 2). In addition, we used another recombinant protein, the In vivo Phosphorylation of RNF11 breast cancer–associated protein 2 (BCA2), in these experi- In order to validate our observed in vitro AKT phosphor- ments and observed phosphorylated bands that migrated with ylation of RNF11, we attempted to generate two-dimensional a much slower mobility as expected (lanes 4 and 5). As a phosphopeptide maps for RNF11. Wild-type RNF11 vectors positive control, reactions were conducted using recombinant were transfected into WM239 cells which possess constitutively GSK3h (Fig. 2A, lane 6). Because the migration of the elevated AKT activity. Upon incubation with 32P-orthophos- phosphorylated RNF11 band was similar to that of GSK3h, phate and subsequent trypsinization, RNF11 yielded seven we wanted to ensure that we were actually detecting distinct phospho fragments (Fig. 3A). This pattern was phosphorylated RNF11 in lane 3 and not detecting a reproducible and fragment 4 was the most intensely phosphor- contamination with GSK3h. A subset of reactions was carried ylated in the WM239 cells. In the presence of the phosphoi- out in parallel where one sample was incubated with 32P- nositide-3-kinase inhibitor, LY2940002, the pattern of RNF11 gATP and another was incubated with cold ATP. The radio- phosphorylation was altered. The resultant inhibition of AKT active sample was run on a gel (Fig. 2A), whereas the activity drastically reduced the intensity of, but did not entirely nonradioactive sample was subjected to MS/MS mass eliminate, fragment number 4 (Fig. 3B). When T135 was spectrometry. Although we were unable to detect phosphor- mutated to glutamate (T135E), fragment 4 was lost, in addition to fragments 1, 2, 3 and 5, however, fragments 6 and 7 were strongly labeled. Thus, it may be that the T135E mutation may cause changes that also affect phosphorylation of other sites. Nonetheless, the loss of spot 4 in Fig. 3C coupled with the reduction in intensity of fragment 4 in Fig. 3B is consistent with that fragment containing T135 and being phosphorylated by AKT. To further corroborate our findings, we evaluated the phosphorylation pattern of RNF11 in the parental WM35 cell line (Fig. 3D). In WM35 cells, there is a loss of two phosphorylation sites compared with the pattern observed in WM239 cells. Consistent with T135 being contained within fragment 4 and being phosphorylatable by AKT, spot 4 is not evident in the phosphopeptide map of RNF11 from WM35 cells (Fig. 3D versus Fig. 3A).

AKT-Mediated Binding of RNF11 to 14-3-3 RNF11 contains a perfect 14-3-3 binding site within its COOH-terminal region that contains a potential AKT phosphorylation site (T135; Fig. 1). AKT-mediated regulation of 14- 3-3 binding has been shown for other proteins (18, 19). We wanted to test whether 14-3-3 bound to RNF11 and if this binding was regulated by AKT. WM35 and WM239 cells were transfected with glutathione S-transferase (GST)-RNF11 expression vectors. Cell lysates were incubated with glutathione-linked sepharose beads and GST-RNF11 was isolated by centrifugation. When RNF11-associated proteins were probed with pan 14-3-3 antibodies, RNF11/14-3-3 association was evident (Fig. 4A). Binding of 14-3-3 to RNF11 in WM35 cells was easily detectable despite modest amounts of phosphorylated (active) AKT (Fig. 4B). However, when corrected for the FIGURE 2. RNF11 is phosphorylated by AKT. A. His-tagged RNF11 amount of RNF11 in the GST-pulldowns, the level of RNF11/ was amplified and isolated from bacteria using nickel columns. Purified 14-3-3 interaction was lower than in WM239 cells in the His-RNF11 was incubated with recombinant Akt and 32P-gATP (lane 3). As negative controls, reactions were run in the presence of excess cold presence of LY2940002. RNF11/14-3-3 binding was enhanced ATP (lane 1)orat4jC(lane 2). Similar experiments were conducted using upon activation of AKT in WM239 cells (Fig. 4A). When BCA2, a cancer protein that contains an AKT site within a 14-3-3 binding domain (lanes 3 and 4). GSK3h was run as a positive control (lane 6). corrected for the lower amount of RNF11 present in the GST- B. Parallel reactions with those in (A) were conducted and subjected to pulldowns from WM239 cells, 14-3-3/RNF11 binding was >6- MS/MS mass spectrometry to verify the presence of purified HIS-tagged fold higher when AKT was active. When the GST-pulldowns RNF11. Sequences of trypsin fragments that were identified as RNF11 are shown and their corresponding amino acid positions indicated with were probed with an antibody that was specific for phosphor- superscript numbers. ylated AKT substrates (Cell Signaling Technologies, Pickering,

FIGURE 3. RNF11 is phosphorylated at multiple sites. A. FLAG-RNF11 was labeled with 32P in WM239 cells, which possess elevated AKT activity, and subjected to digestion with trypsin. RNF11 fragments were then subjected to two-dimensional electrophoresis. B. RNF11 was prepared as in (A) except that cells were treated with LY294002 to inhibit AKT activity. C. Same as (A) except T135E mutant RNF11 was used instead of wild-type cells. D. Similar analyses were conducted using WM35 cells, the parental line of WM239, in which AKT activity is normal.

Ontario, Canada), there was positive staining which corre- RNF11 and 14-3-3 as indicated by the amount of 14-3-3 present sponded with GST-RNF11 and followed the pattern of AKT in FLAG-RNF11 immunoprecipitations (Fig. 5B). In contrast, activity (Fig. 4A). WM239 cells showed a much higher staining the presence of constitutively active AKT resulted in a much with the AKT substrate antibody than did WM35 cell lysates. higher amount of RNF11 bound to 14-3-3 than did dominant- This was consistent with a higher AKT activity in WM239 cells negative AKT, despite similar levels of RNF11 in each as indicated by increased levels of phosphorylated AKT in immunoprecipitation (Fig. 5B). To determine the role of T135 WM239 cells compared with WM35 cells (Fig. 4B). Treatment in this AKT-dependent RNF11/14-3-3 interaction, WM239 with LY2940002 reduced the levels of phosphorylated AKT cells were transfected with either wild-type or T135E FLAG- (Fig. 4B), which coincided with both a reduction in the amount RNF11 expression vectors in the presence or absence of of RNF11-associated 14-3-3 and a somewhat lower staining LY2940002. As shown previously (Fig. 4A), there was a strong with the AKT substrate antibody (Fig. 4A). Notably, association between wild-type RNF11 and 14-3-3 in WM239 LY2940002 treatment of WM239 cells failed to completely cells (Fig. 5C). In contrast, the interaction between T135E inhibit the phosphorylation and activation of AKT (Fig. 4B). RNF11 and 14-3-3 was virtually eliminated (Fig. 5C). This may explain the residual 14-3-3/RNF11 binding in Treatment with LY2940002 dramatically reduced wild-type LY2940002-treated WM239 cells (Fig. 4A) and the persistence RNF11/14-3-3 binding (Fig. 5C). Interestingly, it seems that of phosphorylation of RNF11 peptide fragment number 4 RNF11 binding to 14-3-3 requires not only the negative charge following LY2940002-mediated AKT inhibition (Fig. 3B). that phosphorylation confers to an amino acid, but also the To confirm that the results in Fig. 3 were directly an effect of presence of the phosphate group itself. Mutation of phosphor- AKT, we employed a slightly different approach. MCF-7 cells ylatable S or T to D or E is routinely used as a tool to mimic were transiently transfected with FLAG-RNF11 expression phosphorylation, as D and E substitutions will confer a negative vectors and cDNAs for either dominant-negative or constitu- charge at the substituted amino acid site. Thus, the lack of tively active AKT (Fig. 5). Equal amounts of constitutively binding of the T135E mutant RNF11 to 14-3-3 (Fig. 5C) active and dominant-negative AKT were expressed following suggests that charge alone is insufficient for 14-3-3 binding to transfection and these vectors had no effect on the levels of RNF11. FLAG-RNF11 expression (Fig. 5A). In addition, expression of these AKT expression vectors altered AKT activity. AKT AKT and RNF11 Localization activity, as measured using a phospho-GSK3h antibody, was 14-3-3 proteins are known to mediate cellular localization/ low in cells expressing dominant-negative AKT, whereas sequestration of their binding partners (27). To evaluate transfection with constitutively active AKT resulted in a much whether the observed AKT-mediated 14-3-3 binding to higher AKT activity (Fig. 5A). However, the presence of RNF11 affected RNF11 subcellular localization. MCF-7 cells dominant-negative AKT resulted in a very limited interaction of were transfected with wild-type RNF11 expression vectors in

the absence or presence of constitutively active AKT. Cells intermittently activated, expression of RNF11 is very high. In were fractionated into their nuclear and cytoplasmic compo- addition, when normal cells were treated with the protease nents using a digitonin-permeabilization technique (28). When inhibitor, MG132, no elevation in RNF11 protein levels was nuclear (N) and cytoplasmic (C) fractions were probed with evident (Fig. 6B). This suggested that there is another AKT- anti-FLAG antibodies, wild-type RNF11 was located predom- independent mechanism of RNF11 degradation. Because inantly in the cytoplasmic compartment (Fig. 6A). Upon RNF11 interacts with the E-3 ligase Smurf2, we expressed expression of constitutively active AKT, RNF11 subcellular RNF11 with Smurf2 and its binding partner Smad7 in HEK293 compartmentalization was altered, with the majority of the cells (Fig. 6C). Smurf2 expression alone (2 Ag) had no effect on protein being sequestered in the nuclear fraction (Fig. 6A). This RNF11 degradation (lanes 1-3). However, adding only a result may be due to either a translocation of RNF11 from the minimal amount of Smad7 (0.2 Ag) dramatically reduced the cytoplasm to the nucleus or an accelerated degradation of the level of RNF11 (lane 1 versus 7). Increasing Smad7 input cytoplasmic RNF11 fraction. further reduced RNF11 expression, with 4 Ag of Smad7 cDNA eliminating RNF11 expression entirely (lane 5). Surprisingly, Degradation of RNF11 Protein the expression of Smurf2 was also inhibited following the It was noticed that RNF11 expression was consistently lower addition of Smad7 (lane 1 versus lanes 5-7). This suggested the when vectors were transfected into WM239 cells compared formation of a tripartite RNF11/Smad7/Smurf2 complex which with WM35 cells (Fig. 4A). This suggested that the higher resulted in the degradation of both RNF11 and Smurf2. AKT activity in WM239 cells may be accelerating RNF11 degradation. In order to determine whether RNF11 proteolysis RNF11 Function in TGF-b Signaling was indeed elevated by AKT, WM239 cells were transfected In order to test the cellular impact of 14-3-3 binding to with wild-type and T135E RNF11 expression vectors (Fig. 6B). RNF11, we used a TGF-h-responsive system which we have Wild-type RNF11 was expressed at low levels, whereas the already shown that RNF11 can enhance TGF-h signaling and expression of T135E RNF11 was much greater. Thus, relieve Smurf2-mediated inhibition of TGF-h signaling (5). preventing phosphorylation of T135 may inhibit RNF11 Expression of Smurf2 represses TGF-h signaling as measured degradation. When ubiquitin-mediated proteolysis was using a TGF-h-responsive luciferase assay. The coexpression of inhibited by treatment of WM239 cells with the proteasome RNF11 with Smurf2 relieves Smurf2-mediated repression on inhibitor MG132 (MG) resulted in an increase in wild-type TGF-h signaling (Fig. 7, lanes 3 versus 4). When we RNF11, reaching levels approaching that of T135E RNF11 coexpressed the RNF11 mutant (T135E) with Smurf2, we (Fig. 6B). In contrast, in normal cells, where AKT is only found that the T135E mutant induces a moderately better relief of the Smurf2 inhibition of the 3TPLux reporter (Fig. 7, lanes 4 versus 5). This trend is more pronounced when we look at the effects of the expression of RNF11 or the T135E mutant on TGF-h signaling in the absence of Smurf2. There is a significant 35% increase in TGF-h signaling (P = 0.007) when T135E RNF11 is expressed compared with wild-type RNF11 (Fig. 7, lanes 6 versus 7).

Discussion The initial observation that RNF11 was elevated in 90% of invasive ductal carcinomas suggested that RNF11 may be important in cell transformation. Ring finger–containing proteins, such as CBL, MDM2, and ROC1, are involved in the ubiquitin-mediated proteolysis pathway (13, 29-34). This pathway has been implicated as a mechanism behind cancer development. For example, p27KIP1 protein levels are reduced in >60% of all cancers, despite no known genetic mutations and no reduction in protein synthesis rates (35). Thus, an increase in p27 proteolysis almost certainly underlies the decrease in cellular p27 protein levels. In fact, mechanisms of p27 degradation are continually being elucidated and involve signaling pathways that are known to be activated in cancer. This type of regulation of protein expression led to the hypothesis that the overexpression of RNF11 was an oncogenic

FIGURE 4. RNF11 binds 14-3-3 in an AKT-dependent manner. A. and deleterious occurrence. Cells were transfected with wild-type GST-RNF11 vectors and subjected The effects of the TGF-h signaling pathway are regulated by to GST-pulldowns. RNF11-bound proteins were probed with 14-3-3, GST, numerous cellular proteins (36-38). Binding of TGF-h to the and phospho-AKT substrate antibodies. Some cells were treated with TGF-h or LY2940002. B. Whole cell extracts from cells in (A) were probed receptors at the cell surface leads to the phosphorylation/ with antibodies against phosphorylated (activated) AKT. activation of the receptor-activated Smads 2 and 3 (R-Smad).

FIGURE 5. 14-3-3/RNF11 binding needs the phosphate group. A. Whole cell extracts from MCF-7 cells transfected with dominant-negative or constitutively active AKT and probed with AKT, FLAG, and phospho-specific GSK3h antibodies. B. FLAG-RNF11 was immunoprecipitated from lysates in (A) and bound proteins probed with 14-3-3 antibodies. C. MCF-7 cells were transfected with wild-type and T135E RNF11 expression vectors in the presence or absence of LY2940002 and treated as in (B).

Activated R-Smads bind to Smad4 and the heterodimer RNF11 protein levels and consequently alter RNF11 function. translocates to the nucleus where it regulates the transcription If RNF11 can act to restore TGF-h signaling by competitively of TGF-h-responsive genes. Smad7 can bind to the TGF-h inhibiting/altering Smurf2 interaction and function, why receptor which prevents phosphorylation of the R-Smads. In doesn’t RNF11 overexpression prevent or oppose malignant addition, when complexed with the E3 ligase Smurf2, Smad7 transformation? In some cell types and disease scenarios (41), acts to degrade the TGF-h receptor (11). These two actions the constitutive expression of Smad7 may down-regulate suppress the TGF-h signaling pathway and are part of the steady state RNF11 levels and disrupt protein function, normal feedback loop that regulates the activity of the TGF-h thereby rendering RNF11 incapable of restoring TGF-h pathway. However, inappropriate activation of these inhibitory responsiveness. Also, it may be that RNF11 has cell type– mechanisms can render a cell insensitive to TGF-h, effectively specific functions and depends on the partner proteins to shutting down this pathway. Because one of the main which it is bound. RNF11 complexes may inhibit/degrade a ramifications of activation of the TGF-h pathway is cell tumor suppressor in one cell type, making it oncogenic, cycle arrest (37, 39, 40), hyperactivation of the Smad7/Smurf2 whereas in another cell, RNF11 may degrade/inhibit an complex may lead to inappropriate initiation of the cell cycle. oncogenic factor, giving RNF11 a tumor suppressor function. In fact, TGF-h insensitivity is a common characteristic of This potential for differential RNF11 function may be many breast cancers. Thus, overexpression of RNF11, a determined by its amino acid sequence (Fig. 1). We identified protein that binds Smurf2, could alter Smurf2 activity. RNF11 multiple potential RNF11 regulatory domains, the most contains a PY motif that is identical to that found in the TGF- attractive of which being an AKT phosphorylation site h inhibitory Smad7 and seems to actually inhibit Smuf2 (T135) contained within a 14-3-3 binding domain. Phosphor- activity and restore TGF-h sensitivity when Smurf2 is ylation by AKT has been shown to promote binding to 14-3-3 overexpressed (5). However, unlike Smad7, RNF11 cannot (18, 19), and this study was undertaken to establish whether bind Smurf2 when its PY domain is altered (5, 11). This this is the case for RNF11. In addition, constitutive activation implies that Smad7 could retain association with Smurf2 in a of AKT confers TGF-h resistance to epithelial cells (42). manner that is implicitly different than RNF11/Smurf2 Inhibition of AKT activity with LY2940002 restores TGF-h association, thereby allowing for the formation of a tripartite sensitivity and represents the rationale behind our choosing complex. Indeed, we observe that RNF11, Smurf2 and Smad7 WM35 and WM239 cells as model systems to evaluate interact at the molecular level and influence steady state RNF11 regulation by AKT. RNF11 is indeed phosphorylated protein levels (Fig. 6C). Interestingly, in an assay looking at by AKT in vitro (Fig. 2A) and in vivo at T135 (Fig. 3) by the activity of the tripartite complex on TGF-h signaling, we AKT. This phosphorylation at T135 enhances RNF11 binding observe that the tripartite complex impacts negatively on to 14-3-3 and redistributes RNF11 from being predominantly TGF-h signaling. However, when a PY mutant Smad7 is cytoplasmic to mostly nuclear. Thus, 14-3-3 binding to substituted in the tripartite complex, there is slight, but RNF11, mediated by phosphorylation at T135 by AKT, is significant increase in TGF-h responsiveness (P < 0.001; data likely an important regulatory mechanism for RNF11 function. not shown). We were likely unable to achieve a complete This may help explain why RNF11 is elevated in so many restoration of function with the coexpression of the Smad7 PY breast cancers. AKT has also been shown to be elevated in mutant with RNF11 and Smurf2, due to the fact that although breast cancer, which, likely not coincidentally, is a cancer that the Smad7 PY mutant is still capable of binding to Smurf2, it often becomes insensitive to TGF-h. TGF-h insensitivity can binds at a lowered efficiency (11). However, these observa- be brought about by activated AKT in mammary epithelial tions indicate that Smad7/Smurf2 complexes can affect cells (17, 42), and TGF-h responsiveness can be restored

restore the integrity of the TGF-h signaling pathway and prevent tumor development. This hypothesis is further supported by our observations that the RNF11 (T135E) mutant, which is not phosphorylated by AKT and does not bind 14-3-3, is in fact better at enhancing TGF-h signaling than is wild-type RNF11. AKT activation not only promotes RNF11/14-3-3 interaction, but leads to a nuclear predominance of RNF11. This may affect RNF11 interaction with Smurf2, resulting in a loss of the restorative effects of RNF11 on TGF-h signaling. Thus, elevation of RNF11 may be an initial response by a cell to restore proper TGF-h signaling in response to the onset of malignant transformation. In the event of other cell signaling alterations, such as activated AKT, RNF11 is unable to restore homeostasis and the cell continues to overexpress RNF11 in a failing attempt to cope with ongoing transformation. So, instead of being the protein underlying oncogenic change, RNF11 overexpression may actually serve as an early marker of cell transformation. Detection of RNF11 overexpression may allow for earlier identification of tumor development and improve the efficiency of cancer treatment. Of course, the determination of other ramifications of RNF11 overexpression needs to be firmly established for treatment strategies to be FIGURE 6. Mechanism of RNF11 protein degradation. A. MCF-7 cells improved. were transfected with wild-type FLAG-RNF11 either with or without transfected constitutively active AKT. Cells were separated into nuclear Another interesting observation is that when RNF11 is and cytoplasmic components by digitonin permeabilization and probed for transfected into cells with constitutively active AKT, a lower FLAG-RNF11. B. HEK293 and WM239 cells were transfected with either level of RNF11 expression is observed compared with that in wild-type or T135E mutant FLAG-RNF11 in the presence or absence of the proteasome inhibitor MG132. C. HEK293 cells were cotransfected with cells where AKT is normally active (Fig. 4A). This can be Smurf2 and various amounts of GST-RNF11 in the absence or presence prevented by treatment with the proteasome inhibitor MG132, of different amounts of HA-Smad7 expression vectors. suggesting that activated AKT leads to ubiquitin-mediated degradation of RNF11. In addition, T135E RNF11 does not subsequently by pharmacologically inhibiting AKT activity. exhibit this lowered expression in AKT-active cells. Because We propose that one of the ramifications of the phosphory- T135E RNF11 does not bind 14-3-3, it is likely that AKT- lation of RNF11 by AKT is that RNF11 is sequestered away dependent binding of RNF11 to 14-3-3 leads to the degradation from Smurf2 by 14-3-3. This sequestration may explain why of RNF11. Because constitutively active AKT changes the RNF11 is elevated in breast cancer and likely unable to distribution of RNF11 from a predominantly cytoplasmic to

FIGURE 7. Disruption of 14-3-3 binding and AKT phosphorylation at T135 enhances RNF11 function. TGF-h signaling assays were conducted using a TGF-h-responsive (3TPLux) reporter system. HepG2 cells were transiently transfected with 3TPLux reporter constructs alone or in combination with expression vectors for Smurf2, wild-type RNF11, and mutant RNF11 (T135E), which does not bind to 14-3-3 and is not phosphorylated by AKT. Cells were cultured in the presence or absence of 10 ng/mL TGFh-1 and reporter activity was assayed using a dual luciferase activity kit. Statistical differ- ences between experimental conditions was determined using unpaired t tests; *, P = 0.007.

mostly nuclear pattern of expression, AKT phosphorylation Detection system (Promega, Madison, WI) as described by the may selectively induce degradation of cytoplasmic RNF11. manufacturer. All luciferase values were normalized to a Alternatively, 14-3-3 may act to actively transport RNF11 from cotransfected Renilla Luciferase activity as an internal control. the cytoplasm to the nucleus following the phosphorylation of All transfections were conducted using LipofectAMINE 2000 RNF11 at threonine 135 by AKT. (Invitrogen, Burlington, Ontario, Canada). We noted that RNF11 was not entirely degraded when AKT was activated. This suggested a non–AKT-dependent mecha- In vitro Phosphorylation Assays nism of RNF11 degradation. When RNF11 was cotransfected Bacterial recombinant vectors encoding HIS-tagged RNF11 withbothSmad7andSmurf2,noRNF11proteinwas were expressed and isolated using a ProBond HIS-tag isolation detectable, whereas cotransfection with Smurf2 alone had kit (Invitrogen). RNF11 protein purification was verified by virtually no effect on RNF11 degradation. Interestingly, Coomassie staining of SDS-PAGE gels and MS/MS-mass cotransfection of RNF11 with Smad7 and Smurf2 also resulted spectrometry. Ten nanograms of RNF11 and BCA2, a novel in elimination of Smurf2 protein. Thus, it seems that RNF11 ring finger protein which also contains an AKT phosphoryla- and Smurf2 are degraded together in the presence of Smad7. tion site within a 14-3-3 binding domain, were incubated with This suggests a novel mechanism for Smurf2 degradation. It recombinant active AKT (Cell Signaling Technologies) and has been shown that Smad7 can bind to Smurf2 even if the 32Pg-ATP for 30 minutes at 37jC. Reactions were also Smad7 PY domain is mutated. Thus, it may be that RNF11, conducted using recombinant GSK3h as a positive control. whose PY domain is identical to that of Smad7, forms a tripartite complex with Smurf2 and Smad7, resulting in the Two-dimensional Phosphopeptide Mapping degradation of RNF11 and Smurf2. This represents a unique WM239 cells, which have constitutively active AKT, were self-sacrificing mechanism of Smurf2 degradation mediated by transiently transfected with wild-type FLAG-RNF11 expressing RNF11. cDNAs. Cells were starved in phosphate-free media containing We are beginning to unravel the complexity of RNF11 5% dialyzed phosphate-depleted fetal bovine serum for 4 hours. function. We do know that RNF11 binds a wide range of Cells were then incubated with 32P-orthophosphate (1 mCi/ cellular targets that encompass a vast array of important life plate) for 3 hours. Cells were then lysed in 0.5% NP40 buffer functions (6). RNF11 binds to and seems to inhibit the activity containing protease and phosphatase inhibitors. FLAG-RNF11 of Smurf2, possibly by initiating Smurf2 degradation, thereby proteins were immunoprecipitated from lysates using FLAG alleviating the suppressive effects of Smurf2 on cell signaling. antibody-conjugated affinity gel (Sigma). Immunoprecipitated RNF11 binds to 14-3-3 in an AKT-dependent fashion, and this proteins were separated on 12% SDS-PAGE gels and radio- binding may act to sequester RNF11 away from its various labeled proteins visualized by autoradiography. FLAG-RNF11 cellular activities. More research is necessary to fully proteins were excised and subjected to trypsin digestion. The understand RNF11 function, but it is becoming more apparent resultant RNF11 fragments were applied to a thin-layer that this protein is an integral part of cellular regulation. chromatography plate and separated in the first dimension Moreover, RNF11 is likely to be regulated by other cell using a Hunter apparatus (Amersham Pharmacia, Montreal, signaling pathways. Quebec, Canada). Peptide fragments were separated in the second dimension using thin-layer chromatography. Phosphor- ylated fragments were visualized using autoradiography. Materials and Methods Subsequent experiments were conducted using WM35 cells, Cell Lines and Luciferase Assays which possess normal AKT activity, and WM239 cells treated In order to determine whether AKT phosphorylation affects with the phosphoinositide-3-kinase inhibitor LY2940002 RNF11, FLAG-RNF11 vectors were transiently transfected into (25 Amol/L, Calbiochem, San Diego, CA). In addition, WM35 and WM239 human melanoma cell lines. WM239 cells RNF11 vectors which harbored a threonine 135 to glutamate harbor a PTEN deletion and a resultant elevation in AKT kinase (T135E) mutation were generated using a site-directed activity compared with the parental WM35 line. In addition, mutagenesis kit (Invitrogen). The effect of this T135E mutation wild-type and mutant RNF11 expressing vectors were tran- on RNF11 phosphorylation was also evaluated. siently transfected into MCF-7 mammary epithelial cells in the absence or presence of cDNAs encoding constitutively active or dominant-negative AKT. Acknowledgments Standard dual luciferase assays were conducted as follows. We thank Jeff Wrana and Liliana Attisano for the generous gift of Smad and Smurf vectors, and Joe Testa for AKT plasmids. 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Michael K. Connor, Peter B. Azmi, Venkateswaran Subramaniam, et al.

Mol Cancer Res 2005;3:453-461.

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