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, 1833 (1), 21-32

The Novel Interaction Between Microspherule Protein Msp58 and Ubiquitin E3 Ligase EDD Regulates Cell Cycle Progression

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The Novel Interaction Between Microspherule Protein Msp58 and Ubiquitin E3 Ligase EDD Regulates Cell Cycle Progression

Mario Benavides et al. Biochim Biophys Acta.

Abstract

Microspherule protein Msp58 (or MCRS1) plays a role in numerous cellular processes including transcriptional regulation and cell proliferation. It is not well understood either how Msp58 mediates its myriad functions or how it is itself regulated. Here, by immunoprecipitation, we identify EDD (E3 identified by differential display) as a novel Msp58-interacting protein. EDD, also called UBR5, is a HECT-domain (homologous to E6-AP carboxy-terminus) containing ubiquitin ligase that plays a role in cell proliferation, differentiation and DNA damage response. Both in vitro and in vivo binding assays show that Msp58 directly interacts with EDD. Microscopy studies reveal that these two proteins co-localize in the nucleus. We have also found that depletion of EDD leads to an increase of Msp58 protein level and extends the half-life of Msp58, demonstrating that EDD negatively regulates Msp58's protein stability. Furthermore, we show that Msp58 is upregulated in multiple different cell lines upon the treatment with proteasome inhibitor MG132 and exogenously expressed Msp58 is ubiquitinated, suggesting that Msp58 is degraded by the ubiquitin-proteasome pathway. Finally, knockdown of either Msp58 or EDD in human lung fibroblast WI-38 cells affects the levels of cyclins B, D and E, as well as cell cycle progression. Together, these results suggest a role for the Msp58/EDD interaction in controlling cell cycle progression. Given that both Msp58 and EDD are often aberrantly expressed in various human cancers, our findings open a new direction to elucidate Msp58 and EDD's roles in cell proliferation and tumorigenesis.

Figures

Figure 1
Figure 1
Identification of a novel nuclear protein complex containing Msp58 and EDD. A. Nuclear extract from HeLa S3 cells or HeLa S3 cells stably expressing FLAG-Msp58 were immunoprecipitated with anti-FLAG M2 agarose. The eluates were examined by silver staining. EDD was identified by mass-spectrometry (LC-MS/MS). B. The eluates were immunoblotted with anti-FLAG and anti-EDD antibodies. Lanes 1 and 4: 5% of input, lanes 2 and 5: eluate from IP of the nuclear extract of the FLAG-Msp58 HeLa S3 stable line, and lanes 3 and 6: eluate from IP of the nuclear extract of HeLa S3 cells (control). C. Msp58 was co-purified with FLAG-EDD. Total lysates from HEK293T cells transiently expressing FLAG-EDD and GFP-Msp58 were immunoprecipitated with anti-FLAG M2 agarose. Cells co-transfected with FLAG-EDD and GFP were used as control. Inputs (lanes 1, 2, 5 and 6) and 3X-FLAG peptide eluates (lanes 3, 4, 7, 8) were analyzed by immunoblotting with specified antibodies.
Figure 2
Figure 2
Msp58 interacts with EDD via the EDDFR4 (1976-2474) region. A. Schematic diagram of EDD and its derivative fragments that were used for in vitro binding assays. B. The EDD fragments that contain the FR4 (1976-2474) region specifically interact with Msp58. Recombinant GST or GST-Msp58 proteins immobilized on the glutathione beads were incubated with 35S-Met labeled EDD fragments (FR12, FR35, FR45 and FR4). The eluates were resolved by SDS-PAGE, followed by autoradiography. 15% of input was loaded to show the expression levels of proteins. C. Coomassie blue staining of the lysates of uninduced (U, lanes 1 and 3) and induced (I, lanes 2 and 4) bacteria, co-expressing His6-Msp58 with EDD-FR5-S or EDD-FR4-S. Asterisk points to a degradation product of EDD-FR4-S. D. Immunoblotting of samples, as described in C, with anti-His6 tag or anti-S tag antibodies. E. Msp58 directly binds EDD-FR4, but not EDD-FR5. Lysates of induced bacteria, as described in C, were purified with cobalt beads. The input (I, lanes 1 and 3) and the eluates (E, lanes 2 and 4) were immunoblotted with anti-S tag antibody.
Figure 3
Figure 3
Two independent regions of Msp58 can bind EDD. A. Schematic diagram of Msp58 with predicted domains and its derivative fragments used to test their binding with EDD. B. The lysates of uninduced (U, lanes 1, 3 and 5) and induced (I, lanes 2,4 and 6) bacteria, co-expressing EDDFR4-S with His6-Msp58 (or His6- Msp581-296 or His6-Msp58297-462) were resolved by SDS-PAGE, followed by immunoblotting with anti-His6 tag and anti-S tag antibodies. C. Msp58297-462 contains an EDD interacting domain. S-protein agarose was incubated with the lysate from bacteria co-expressing EDDFR4-S with His6-Msp58 (or Msp58 fragments). The unbound (U, lanes 1, 3 and 5) and bound (B, lanes 2,4 and 6) proteins were examined by immunoblotting with anti-His6 tag antibody. Asterisks point to degradation fragments. D. The lysates of uninduced (U, lanes 1 and 3) and induced (I, lanes 2 and 4) bacteria, co-expressing EDDFR4-S with His6-Msp581-342 (lane 1, 2) or Msp58343-462 (lane 3, 4), were examined as described in B. E. Both His6-Msp581-342 (lane 4) and Msp58343-462 (lane 6) are able to bind EDD. The experiments were performed as described in C. His6-Msp581-296 (lanes 1 and 2) served as a negative control. Asterisks point to degradation fragments.
Figure 4
Figure 4
EDD co-localizes with Msp58 in the nucleoplasm of mammalian cells. A. Immnunostaining of HeLa cells with mouse monoclonal antibody mAb414 and rabbit polyclonal anti-Msp58 (top) or anti-EDD (bottom) antibodies. B. WI-38 (top) and HeLa (bottom) cells were immunostained with rabbit polyclonal anti-Msp58 and mouse polyclonal anti-EDD antibodies. C. HeLa cells transfected with GFP-Msp58 expression construct were immunostained with the rabbit polyclonal antibodies against EDD. Bars: 10 µm.
Figure 5
Figure 5
EDD negatively regulates the protein level and stability of Msp58. A. Depletion of EDD resulted in higher Msp58 protein levels in WI-38, HeLa and HEK 293T cells. Seventy-two hours after transfection with either EDD (lanes 2, 4 and 6) or control (lanes 1, 3 and 5) siRNAs, the cells were harvested and analyzed by immunoblotting with the specified antibodies. B. EDD knockdown increased Msp58 protein stability. HeLa cells were transfected with EDD or control siRNAs, and seventy-two hours after transfection cells were treated with CHX. At the indicated time points, cells were harvested and analyzed by immunoblotting. C. A semi-log graph showing the half-lives of Msp58 as shown in B. The trendlines are derived from the raw data regression.
Figure 6
Figure 6
Msp58 is regulated by the ubiquitin-proteasome pathway. A. Msp58 protein level is controlled by the proteasome. Before harvest, WI-38, HeLa and HEK 293T cells were treated with cycloheximide (CHX; lanes 2, 3, 5, 6, 8 and 9), with (lanes 3, 6 and 9) or without (lanes 2, 5 and 8) MG132, for 6 hours. The lysates were analyzed by immunoblotting with specified antibodies. B. GFP-Msp58 was ubiquitinated in vivo and knockdown of EDD had no obvious effect on the level of ubiquitinated GFP-Msp58. HEK 293T cells were co-transfected with HA-ubiquitin and GFP-Msp58 expression constructs, with or without EDD siRNAs. Cells co-transfected with HA-Ubiquitin and GFP were used as control. Lysates were immunoprecipitated with anti-HA affinity matrix and the inputs (lanes 1–3 and 7–9) and eluates (lanes 4–6 and 10–12) were examined by immunoblotting with specified antibodies.
Figure 7
Figure 7
Msp58 and EDD regulate cell cycle progression. A. Representative FACS histograms of WI-38 cells transfected with specified siRNAs. B. Analysis of the G2/M phase of the cell cycle. Bars show relative populations of WI-38 cells transfected with specified siRNAs as compared with the cells transfected with GFP siRNAs. Data presented as means±SD (n=5). C. Representative immunoblots showing the levels of cyclins (B, D1, D3, and E) in different siRNAs-transfected samples. The protein bands were semi-quantified using ImageJ software, and the numbers on top of blots represent the relative levels of cyclins normalized to control siRNA-transfected sample.

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