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. 2012 Mar;11(3):M111.014407.
doi: 10.1074/mcp.M111.014407. Epub 2011 Oct 29.

Characterization of MRFAP1 Turnover and Interactions Downstream of the NEDD8 Pathway

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Free PMC article

Characterization of MRFAP1 Turnover and Interactions Downstream of the NEDD8 Pathway

Mark Larance et al. Mol Cell Proteomics. .
Free PMC article

Abstract

The NEDD8-Cullin E3 ligase pathway plays an important role in protein homeostasis, in particular the degradation of cell cycle regulators and transcriptional control networks. To characterize NEDD8-cullin target proteins, we performed a quantitative proteomic analysis of cells treated with MLN4924, a small molecule inhibitor of the NEDD8 conjugation pathway. MRFAP1 and its interaction partner, MORF4L1, were among the most up-regulated proteins after NEDD8 inhibition in multiple human cell lines. We show that MRFAP1 has a fast turnover rate in the absence of MLN4924 and is degraded via the ubiquitin-proteasome system. The increased abundance of MRFAP1 after MLN4924 treatment results from a decreased rate of degradation. Characterization of the binding partners of both MRFAP1 and MORF4L1 revealed a complex protein-protein interaction network. MRFAP1 bound to a number of E3 ubiquitin ligases, including CUL4B, but not to components of the NuA4 complex, including MRGBP, which bound to MORF4L1. These data indicate that MRFAP1 may regulate the ability of MORF4L1 to interact with chromatin-modifying enzymes by binding to MORF4L1 in a mutually exclusive manner with MRGBP. Analysis of MRFAP1 expression in human tissues by immunostaining with a MRFAP1-specific antibody revealed that it was detectable in only a small number of tissues, in particular testis and brain. Strikingly, analysis of the seminiferous tubules of the testis showed the highest nuclear staining in the spermatogonia and much weaker staining in the spermatocytes and spermatids. MRGBP was inversely correlated with MRFAP1 expression in these cell types, consistent with an exchange of MORF4L1 interaction partners as cells progress through meiosis in the testis. These data highlight an important new arm of the NEDD8-cullin pathway.

Figures

Fig. 1.
Fig. 1.
MRFAP1 and MORF4L1 are up-regulated in response to NEDD8 inhibition. a, SILAC analysis of U2OS cells following NEDD8 inhibition. The abundance of ∼2,500 proteins is indicated on the y axis using a log2 scale. The abundance of each protein indicated by the position of the dot on the y axis was determined by summing up all individual light and heavy peptide intensities detected for each protein. The relative fold decrease or increase is shown using the log2 ratio (heavy/light) on the x axis, with 1 μm MLN4924 treatment for 18 h (heavy) over DMSO only (light) (n = 2). b, immunoblotting of subcellular fractionation from U2OS cells treated with 1 μm MLN4924 treatment for 18 h or DMSO only (n = 3). c, immunoblotting of total cell lysates from the indicated cell types treated with 1 μm MLN4924 treatment for 18 h or DMSO only (n = 2). d, immunofluorescence microscopy of endogenous MRFAP1 in U2OS cells treated with 1 μm MLN4924 treatment for 18 h or DMSO only. The white arrowheads indicate change in nuclear size after MLN4924 treatment (n = 3). The scale bar indicates 10 μm.
Fig. 2.
Fig. 2.
MRFAP1 is stabilized by NEDD8 inhibition. a, immunoblotting of total cell lysates from U2OS cells treated with 1 μm MLN4924 for 18 h or DMSO only. A time course of cycloheximide treatment followed to examine protein degradation (n = 3). b, immunoblotting of total cell lysates from either LAP1-MRFAP1 wild type, LAP1-MRFAP1 7KR, or LAP1-MRFAP1-P2A-MORF4L1-mCherry after a time course of cycloheximide treatment to examine protein degradation (n = 2). c, immunoblotting of total cell lysates from U2OS cells treated with 10 μm MG132 or DMSO only. A time course of cycloheximide treatment was used to examine protein degradation. MG132 and cycloheximide were added at the same time for each time point (n = 3).
Fig. 3.
Fig. 3.
MRFAP1 interacts with ubiquitin E3 ligases. a, SILAC GFP-IP analysis of U2OS cells stably expressing inducible LAP1-MRFAP1 wild type. GFP immunoprecipitates from uninduced (light), doxycycline-induced (medium), and doxycycline-induced cells with simultaneous MLN4924 treatment (heavy) were mixed and analyzed together by LC-MS/MS. The log2 ratio (medium/light) is indicated on the x axis. The log2 ratio (heavy/light) is indicated on the y axis. High ratios indicate high abundance of interaction compared with control. Proteins of interest are indicated (n = 2). b, immunoblotting of GFP-immunoprecipitated LAP1-MRFAP1. The cells were treated with 1 μm MLN4924 for 18 h or DMSO only (n = 3).
Fig. 4.
Fig. 4.
MRFAP1 is part of a complex protein-protein interaction network. a, SILAC GFP-IP analysis of U2OS cells stably expressing inducible LAP1-MORF4L1. b, SILAC GFP-IP analysis of U2OS cells stably expressing inducible LAP1-CUL4B. GFP immunoprecipitates from uninduced (light), doxycycline-induced (medium), and doxycycline induced cells with simultaneous MLN4924 treatment (heavy) were mixed and analyzed together by LC-MS/MS. The log2 ratio (medium/light) is indicated on the x axis. The log2 ratio (heavy/light) is indicated on the y axis. High ratios indicate high abundance of interaction compared with control. Proteins of interest are indicated (n = 1).
Fig. 5.
Fig. 5.
Human normal tissue expression pattern of MRFAP1. a, immunohistochemistry imaging of endogenous MRFAP1 in selected tissues. The brown color indicates staining (n = 3). The red arrows indicate nuclear staining in spermatogonia of testis and ciliated epithelial cells of the fallopian tube. b, immunohistochemistry imaging of endogenous protein as indicated in testis tissue. The brown color indicates staining (n = 3). c, immunofluorescence microscopy of endogenous MRFAP1 in murine testis tissue (n = 3). The red arrows indicate nuclear staining in spermatogonia of testis. The scale bar indicates 10 μm.
Fig. 6.
Fig. 6.
Model for the regulation of MRFAP1 by the NEDD8 pathway. The NuA4 histone acetyltransferase complex is recruited to chromatin via the binding of MORF4L1 to tri/di-methylated lysine 36 on histone H3. Cells treated with MLN4924 have a defect in NEDD8 conjugation; therefore Cul4b would be less active in MRFAP1 degradation. An increase in free MRFAP1 protein displaces MRGBP from MORF4L1 and creates a stable MORF4L1-MRFAP1 complex.

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