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.