The WW-HECT protein Smurf2 interacts with the Docking Protein NEDD9/HEF1 for Aurora A activation
- Finola E Moore†1,
- Evan C Osmundson†1,
- Jennifer Koblinski2, 3,
- Elena Pugacheva4,
- Erica A Golemis5,
- Dipankar Ray1, 6Email author and
- Hiroaki Kiyokawa1, 3Email author
© Moore et al; licensee BioMed Central Ltd. 2010
Received: 15 June 2010
Accepted: 8 September 2010
Published: 8 September 2010
The multi-functional adaptor protein NEDD9/HEF1/Cas-L regulates cell motility, invasion and cell cycle progression, and plays key roles in cancer progression and metastasis. NEDD9 is localized to the centrosome and is required for activation of Aurora A kinase in mitosis. Here we demonstrate that the HECT-WW protein Smurf2 physically associates with NEDD9 and is required for the stability of NEDD9 protein. Smurf2 depletion results in a marked decrease in NEDD9 protein levels, by facilitating polyubiquitination and proteasomal degradation of NEDD9. Conversely, forced overexpression of Smurf2 results in upregulation of endogenous NEDD9 protein, confirming the role for Smurf2 in NEDD9 stability. Cells with Smurf2 depletion fail to activate Aurora A at the G2/M boundary, leading to a marked delay in mitotic entry. These observations suggest that the stable complex of Smurf2 and NEDD9 is required for timely entry into mitosis via Aurora A activation.
Smurf2 (Smad ubiquitination regulatory factor 2) is a HECT-E3 ligase that negatively regulates TGF-β signaling . Smurf2 targets TGF-β type I receptor, Smad1, Smad2, Smad7, and the transcriptional repressor SnoN for degradation by the proteasome [1–4]. In addition to its role in TGF-β signaling, Smurf2 functions in diverse biological pathways, including those controlling the cell cycle and cell polarity/cytoskeletal remodeling [5–9]. Previous work from our laboratory demonstrated that Smurf2 protein levels vary during the cell cycle, peaking during mitosis . The localization of Smurf2 also undergoes dynamic regulation. Smurf2 is at the centrosome from G1 through prophase, then localizes to the spindle midzone during anaphase, and the midbody during cytokinesis . To date, the best-defined role of Smurf2 in mitosis involves its binding to and stabilization of Mad2, which is required for the spindle assembly checkpoint .
Smurf2 contains WW domains, which mediate interactions with proteins that have PPxY motifs , while Mad2 does not possess any PPxY motif, suggesting other mitosis-relevant partners might exist for Smurf2. For further insight into the cell cycle-regulatory role of Smurf2, we used a candidate-based approach to select for potential Smurf2 interactors, examining those proteins that both contain a PPxY-motif and exhibit a similar subcellular localization pattern. NEDD9 (neural precursor cell expressed, developmentally down-regulated 9, also called HEF1, human enhancer of filamentation 1 and Cas-L Crk-associated substrate related, lymphocyte-type) is a scaffold protein that contains a PPxY motif . NEDD9 displays similar protein expression and localization pattern as Smurf2, rising in G2 and decreasing after mitosis, localizing to the centrosome, midzone, and midbody . The localization of NEDD9 to the centrosome is required for proper mitotic entry . The cell cycle-regulatory function of NEDD9 is mediated, at least partly, by its role for the activation of Aurora A kinase. Centrosomal Aurora A activity is a critical step for mitotic entry from the G2 phase, required for the initial activation of Cyclin B-CDK1 at the centrosome . Among the elements recruited to the centrosome at the G2/M boundary are the activators of Aurora A, such as Ajuba, TPX2 and NEDD9. Thus, NEDD9 plays a significant role in triggering coordinated activation of the mitotic kinase cascade from Aurora A to Cyclin B-CDK1 and perhaps other mitotic kinases required for proper progression of mitosis .
To date, the upstream mechanisms that control the level of NEDD9 protein during mitotic progression have been poorly understood. Here we demonstrate that Smurf2 regulates NEDD9 levels by preventing its proteasomal degradation and this control is rate-limiting for Aurora A activation and mitotic entry. Our data indicate a novel regulatory pathway critical for timely mitotic entry.
Smurf2 and NEDD9 interact
Depletion of Smurf2 destabilizes NEDD9
Regulation of NEDD9 by Smurf2 is mediated by the proteasome
Overexpression of Smurf2 stabilizes NEDD9 in a ligase-independent manner
Smurf2 depletion results in delayed mitotic entry with impaired Aurora A activation
In the present study we have demonstrated the novel regulation of the multi-functional scaffold protein NEDD9 by the WW-HECT protein Smurf2. Physical interaction with Smurf2 leads to stabilization of NEDD9 protein via suppression of polyubiquitination and subsequent proteasomal degradation. Interestingly, stabilization does not appear to depend on the E3 ligase activity of Smurf2. Depletion of Smurf2, as well as NEDD9 depletion, results in impaired activation of Aurora A at the G2/M boundary. These results support the notion that Smurf2 is a critical regulator of entry into mitosis, extending our recent study on the role for Smurf2 in Mad2 regulation and the Spindle Assembly Checkpoint.
Aurora A activation during late G2 is a critical step for commitment to mitosis, and prerequisite for proper activation of Cyclin B-CDK1 and other mitotic kinases . Centrosomal Aurora A activity governs the timing of mitotic entry, triggering nuclear envelop breakdown at prophase . Recent studies demonstrated the requirement for NEDD9 in Aurora A activation and suggested that this scaffold protein is a critical component of mitosis regulation [12, 14]. NEDD9 expression is regulated in a cell cycle-dependent manner and peaks in G2 and M, when it accumulates at the centrosome together with Aurora A. NEDD9 together with other Aurora A activators such as TPX2 and Ajuba stimulates autophosphorylation of Aurora A at Thr288, which is required for full activation of the kinase. Aurora A then phosphorylates NEDD9 at Ser296, leading to dissociation of the complex and allowing Aurora A to interact with other substrates. Our finding that Smurf2 promotes Aurora A activation does not exclude possible effects of Smurf2 on other Aurora A regulators such as TPX2 and Ajuba. The mitotic function of NEDD9 could be related to its key role in focal adhesion-dependent migration [reviewed in [19, 20]]. NEDD9 associates with focal adhesion kinase (FAK) and a Src family kinase. Subsequent Src-mediated phosphorylation of NEDD9 creates active SH2 sites, which bind to the adaptor protein Crk. Crk association subsequently recruits DOCK180 and C3G, eliciting signals to the GTPases Rac and Rap, respectively. A number of recent studies suggested the presence of crosstalk between the focal adhesion attachment signaling and the centrosome-based mitosis signaling [21–23]. Multiple components of integrin-mediated migratory signaling including NEDD9 and Pak have been shown to associate with and activate Aurora A at the centrosome. Another centrosomal protein GIT1, which is required for Pak localization to the centrosome, binds to the focal adhesion protein Paxillin. Furthermore, the mitotic LATS1 kinase in complex with the focal adhesion protein Zyxin localizes to microtubules proximal to the centrosome and regulates mitotic initiation . It is noteworthy that LATS1 also possesses a PPxY motif for potential association with the WW domains of Smurf2, although its significance remains to be determined.
Smurf2 also plays multiple roles in cell migration and mitotic regulation [5, 25]. Among the substrates for Smurf2-mediated polyubiquitination are TGF-β type 1 receptor, the GTPase Rap1B, and its closely related homolog, Smurf1 [1, 8, 26]. Smurf1 polyubiquitinates RhoA, talin head domain and hPEM2 [27, 28]. These proteins are all involved in the control of cell migration. Moreover, a recent study demonstrated that Smurf2 and Smurf1 are critical regulators of planar cell polarity. Mice deficient for Smurf1 and Smurf2 display defects in planar cell polarity that leads to perturbed stereocilia alignment in neurosensory cells of the cochlea and failed closure of the neural tube . Our recent work provided evidence that Smurf2 is also a regulator of mitosis . Smurf2 expression fluctuates during the cell cycle, with a peak around the G2/M boundary. Smurf2 localizes to the centrosome from interphase until late mitosis, when it moves to the mitotic midbody together with the chromosome passenger complex. Smurf2-depleted cells exhibit multiple defects associated with impaired spindle assembly checkpoint such as premature activation of the anaphase promoting complex (APC) in prometaphase, misaligned and lagging chromosomes during the metaphase to anaphase transition, and failed cytokinesis. These defects are attributable partly to a marked decrease in the spindle checkpoint protein Mad2, as a consequence of accelerated proteasomal degradation. The present study demonstrates that Smurf2 depletion also downregulates NEDD9, which results in impaired Aurora A activation and delayed mitotic entry. The integrin signaling including NEDD9, which governs the basal cell adhesion to the extracellular matrix, determines the orientation of the cell division plane together with the cadherin-mediated planar adhesion signaling. Thus, the crosstalk involving Smurf2, NEDD9 and Aurora A may function as effectors of attachment-sensing mitotic checkpoint. Also, Smurf2 and NEDD9 may collaborate in RhoA activation critical for not only migration but also cytokinesis [26, 29]. Taken together, these data imply that in proliferating cell types Smurf2 controls various protein complexes that are critical for different phases of mitosis, i.e., the NEDD9-Aurora A centrosomal complex in G2 and prophase, the Mad2 spindle checkpoint complex in prometaphase, and the RhoA complex in cytokinesis. Since Smurf2 is known to play diverse roles in the biology of non-proliferative differentiated cells, it will be important to determine whether the mitosis-promoting function of Smurf2 is one of cell type-specific events or a more conserved mechanism of proliferation.
The mechanism with which Smurf2 controls NEDD9 stability remains to be elucidated. The stability of NEDD9 protein is regulated by phosphorylation and subsequent polyubiquitination . In response to TGF-β signals, NEDD9 undergoes polyubiquitination facilitated by physical interaction with Smad3 [17, 31]. Additionally, another member of the WW-HECT family, AIP4 (atrophin 1 interacting protein 4)/Itch, can also target NEDD9 for degradation in a TGF-β-dependent manner . Further, APC/CCdh1 targets NEDD9 for degradation at the end of mitosis . We found that phosphorylated and hyperphosphorylated NEDD9 are stabilized by Smurf2. Though Smurf2 is known as a negative regulator of TGF-β signaling, the NEDD9-stabilizing action of Smurf2 seems unlikely to depend on altered TGF-β signaling. HeLa cells are not typically responsive to TGF-β signals . Further, we found that depletion of Smad3, Smurf1, or AIP4/Itch failed to rescue NEDD9 levels in cells with Smurf2 depletion (data not shown). We believe that the Smurf2 regulation of NEDD9 in mitotic entry occurs through a different mechanism from Smurf2 regulation of Mad2 in the Spindle Assembly Checkpoint. It is likely that Smurf2 interacts with Mad2 and NEDD9 at distinct subcellular locations during mitosis. At the kinetochore and its proximity, Smurf2 may target an intermediary E3 ligase for degradation to stabilize Mad2. In contrast, Smurf2 at the centrosome binds and stabilizes NEDD9 apparently in a ligase-independent fashion. Currently several hypotheses are being evaluated regarding NEDD9 stabilization by Smurf2. Our observation that the catalytically inactive mutant of Smurf2 could also stabilize NEDD9 levels excludes the possibility that Smurf2 targets an intermediary ligase for NEDD9 degradation. Consistent with the ligase-independent function of Smurf2 is a previous report that overexpression of wild-type or ligase-dead Smurf2 induces senescence . Further, AIP4/Itch stabilizes Smad7/TGFβRI complex independently of its ligase activity . Smurf2 also interacts with Smad7, and does not immediately induce its degradation . Interestingly, NEDD9 has been shown to interact with Smad7 . These data also exclude a model in which NEDD9 is stabilized by monoubiquitination. Smurf2 may sequester NEDD9 away from locations in the cell where it could encounter its E3 ligase. Alternatively, Smurf2 may instead mask regulatory epitopes for ubiquitination. Smurf2 may serve as an adaptor for an unidentified regulator that counteracts with another E3 ligase promoting NEDD9 degradation. The ongoing studies are expected to identify the E3 ligase that targets NEDD9 for degradation in response to Smurf2 depletion, and to reveal missing components of the Smurf2-dependent mitosis-regulatory pathway.
Both Smurf2 and NEDD9 are overexpressed in multiple types of cancers. Smurf2 upregulation has been associated with poor prognosis in cancers including esophageal squamous cell carcinoma and renal cell carcinoma [37, 38]. Smurf2 has also been found to be upregulated in breast cancer tissue and cell lines as well as ovarian and prostate cancer cell lines . Jin and colleagues found that depletion of Smurf2 by siRNA inhibited migration and invasion, overexpression of Smurf2 led to enhanced migration and invasion . Together, these data suggest that Smurf2 promotes tumor cell migration and invasion. Increased levels of NEDD9 have been found in lung adenocarcinoma , glioblastoma , and melanoma . NEDD9 was identified as one of a few critical genes that mediate metastasis in melanoma  and breast cancer . Mice null for Nedd9 are resistant to MMTV-polyoma T-induced tumorigenesis , recapitulating the significant role for NEDD9 in tumor development. It will be important to determine whether Smurf2 and NEDD9 levels correlate with each other in human cancers. Future studies using human cancer specimens should provide insight into the putative oncogenic interaction of these two proteins in the regulation of cell cycle progression and genomic instability of cancer cells.
The present work demonstrates that Smurf2 positively regulates NEDD9, which is required for Aurora A activation and proper mitotic entry. These data suggest that Smurf2 plays diverse roles in mitotic regulation.
Cell lines and reagents
HeLa human cervical carcinoma cells (ATCC) were cultures under standard conditions of complete medium containing DMEM, 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml Penicillin/Streptomycin. CN34 breast cancer cells were cultured as described in . Antibodies used in this study are NEDD9/HEF1/Cas-L (2G9), Ubiquitin (P4D1), normal rabbit IgG, normal mouse IgG from Santa Cruz Biotechnology (Santa Cruz, California); Smurf2 from Upstate/Millipore (Lake Placid, NY); FLAG M2 and β-Actin (Clone AC-15) from Sigma Aldrich; and Myc from Invitrogen.
Plasmids and siRNA reagents
siRNA was ordered from Dharmacon/Thermo Fisher Scientific for Smurf2 (#1), NEDD9 (smartpool) and control (#3). The sequence for siSm2 was 5'-GAUGAGAACACUCCAAUUAUU-3'. NEDD9 and its mutants were sub-cloned into Myc vector from Sigma Aldrich. FLAG-Smurf2WT and FLAG-Smurf2(C716A) in pCs2+ vector were kindly provided by Gerald Thomsen at Stony Brook University. Smurf2 was sub-cloned into mCherry vector from Clontech. Smurf2si-resistant was created by site-directed mutagenesis (Quickchange from Stratagene) of FLAG-Smurf2 at 4 nucleotides within the region targeted by siSmurf2#1: T631C, G634A, T640G, A643G. For protein and RNA extractions, cells were reverse transfected with 50 nM siRNA using RNAiMax from Invitrogen, then harvested 48 h later. When DNA was transfected, 1 μg of each plasmid per 6D dish was transfected with Lipofectamine2000 from Invitrogen.
For immunoblotting or immunoprecipitation, cells were lysted by sonication in lysis buffer as described previously . Unless otherwise noted, 30 μg total protein lysate was loaded onto gel. Co-immunoprecipitation was performed in HeLa cells that were either asynchronous synchronized at mitosis by 2 mM thymidine 18 h, release for 9 h, 400 ng/μl nocodazole for 14 h. 400 μg total protein was incubated with 1.5 μg of antibody overnight at 4°C. Protein A (for rabbit Smurf2 IPs) or G (for mouse NEDD9 IPs, both from Zymogen) beads were added for 1 h 4°C. Immunoprecipitated materials were loaded onto 2 different gels and probed by Western blot accordingly. For immunoprecipitation with NEDD9 antibody, lysates were pre-cleared with protein G beads for 30 minutes 4°C before incubation with NEDD9 antibody. For NEDD9 immunoprecipitation for ubiquitination, cells were treated for 4 h with 2 μM MG132 44 h post-transfection. Entire immunoprecipitates were loaded onto one gel, gel was transferred onto PVDF membrane as usual, then prepared for Ubiquitin blotting by treatment with 6 M guanidium chloride, 20 mM Tris pH 7.5, 1 mM PMSF (fresh), 5 mM β-mercaptoethanol (fresh) for 30 minutes, 4°C.
RNA was extracted with Agilent kit, 2 μg RNA was used to synthesize cDNA with the Invitrogen Superscript II kit, PCR was performed with 2 μl of cDNA, 27 cycles, Tm or 50°C.
HeLa cells were grown on coverslips and fixed in ice cold methanol for 20 minutes to overnight. Centrosome staining was followed as described previously .
Neural precursor cell expressed, developmentally down-regulated 9, also called HEF1: Human enhancer of filamentation 1, and Cas-L: Crk-associated substrate related, lymphocyte-type
Transforming growth factor
Target protein for Xenopus kinesin-like protein 2
Cyclin dependent kinase 1
LATS2: Large tumor suppressor
green fluorescent protein
short interfering RNA
We thank Qingshen Gao, Kenji Fukasawa, Thomas McGarry, Navdeep Chandel for their critical suggestions, Joan Massague for CN34 cells, and Limin Sun, Brian Zwecker, Thomas O'Grady and Gina Kirsammer for their technical expertise. The work was supported in part by grants provided to H. K. from the National Institutes of Health (CA112282, CA100204, and HD38085), the Northwestern Memorial Foundation, the Searle Leadership Fund, the Zell Fund, the H Foundation, the Lynn Sage Cancer Research Foundation, and the Phi Beta Psi Sorority. The work of EG is supported by grant from the National Institutes of Health (CA63366).
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