A high-resolution systems-wide screen for substrates of the SCFTrCP ubiquitin E3 ligase

<p><em>presented in: HUPO World Congress: The proteome quest to understand biology and disease in Madrid, Spain, 2013</em></p> <p> </p> <p>Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases, the largest family of enzymes in mammals. Therefore, reliable assays aimed at the identification of substrates of ubiquitin ligases are crucial, not only to unravel the molecular mechanisms by which the UPS controls protein degradation, but also for drug discovery purposes since many established UPS substrates are oncoproteins or tumor suppressors. Here, we develop a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for identifying in a systems-wide manner bone fide substrates of SCFβTrCP, a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are trademarked by a multi-subunit architecture typically comprising the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. SCFβTrCP binds, via its WD40 repeats, the DpSGXX(X)pS di-phosphorylated motif in its substrates. Our combined workflow recovers 27 previously reported SCFβTrCP substrates, of which 22 are confidently verified by two independent statistical protocols, confirming the reliability of this approach. Besides known substrates, we identify 221 proteins that, besides harboring the DpSGXX(X)pS motif, also interact specifically with the WD40 repeats. From this list, we highlight several putative novel SCFβTrCP substrates with their putative degron motifs as well as phosphorylation and ubiquitylation sites. Thus, we demonstrate that the integration of structural information, AP-MS and degron motif mining constitutes a generic, specific and effective screen for the identification of substrates of ubiquitin ligases.</p

(A) Nuclear and nonnuclear fractions were prepared as described in Materials and methods

After separation, each fraction was brought to 1% Triton X-114 and phase separation was initiated by heating the samples to 37°C. The aqueous and detergent phases of each fraction were analyzed for Rac1, RhoGDI, and lamin B by immunoblotting. Immunoblots were quantified with [I]protein A and phosphorimaging, and the percentage of total protein in each fraction in the detergent phase was calculated (mean ± SEM; = 3). (B) Triton X-114 partition as shown in A was performed on the nuclear fractions of COS-1 cells treated overnight with or without 10 μM simvistatin. Aq, aqueous; Det, detergent phases. (C) Selected images of GFP-Rac1 in COS-1 and ECV cells showing prominent decoration of the nuclear envelope (arrowhead). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

PAE (A) or NIH 3T3 (B) cells stably expressing GFP-Rac1 at levels below endogenous were examined by time-lapse confocal microscopy over one division cycle

Arrowheads indicate representative parent and daughter cells. Note that nuclear Rac1 is high immediately preceding mitosis and that GFP-Rac1 is excluded from the nuclei of the daughter cells immediately after cell division. See Videos 1 and 2 (available at ). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

(A) PAE cells stably expressing GFP-Rac1 at levels below endogenous were synchronized in G1/S by serum starvation followed by hydroxyurea and then released

The percentage of cells with nuclear Rac1 was determined hourly and plotted as mean ± SEM ( = 4). (B) Aliquots of the cells analyzed in A were scraped from plates at the indicated times and analyzed for stage of the cell cycle by propidium iodide and cytofluorimetry. (C) Unsynchronized COS-1 cells were transfected with GFP-Rac1 and, after 16 h, fixed and stained for cyclin A. Although the cell expressing GFP-Rac1 in the nucleus (arrows) stained for cyclin A, a marker of G2/M, those excluding the protein from the nucleus (arrowheads) did not. This correlation held for 93% of transfected cells examined (>100). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

COS-1 cells were separated into nuclear and nonnuclear fractions as described in Materials and methods

Equal cell equivalents of each fraction were analyzed by SDS-PAGE and immunoblots (inset) for the indicated proteins. Immunoprecipitated proteins were detected and quantified with [I]protein A and phosphorimaging, and the percentage of total protein in the nuclear fraction was calculated (mean ± SEM; = 3).<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

(A) Confocal images of asynchronous T98G cells expressing GFP-Rac1 with the percent of the transfected population represented by each pattern indicated

T98G (B) or IMR-90 (C) cells were synchronized by serum deprivation for 72 h and then induced to cycle by refeeding with 10% FBS. Aliquots of cells were harvested at the times indicated and assayed for Rac1 and the indicated control proteins by immunoblotting. Bar, 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

(A) PAE cells stably expressing GFP-Rac1 at levels below endogenous Rac1 (Fig

S1, available at ) were scored for the percentage of cells showing strong nuclear fluorescence before and 16 h after the addition of increasing amounts of the indicated compounds. Representative cells at the indicated dose are shown with the percentage of cells showing each phenotype indicated (left), and cumulative dose-response data are shown on the right (mean ± SEM; = 3). Bars, 10 μm. (B) Endogenous Rac1, Ras, and RCC1 were measured in the nuclear fractions as described in before and after the addition of 50 μM apigenin for 24 h (mean ± SEM; = 4).<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

Phenotypic assays on wild type and <i>asb11^cul</i> embryos.

<p>(<b>A</b>), Morphological analysis of wild type and mutant embryos at 48 and 72hpf. (<b>B</b>), (<i>left</i>) Anterior view of wild type and mutant embryos at 10hpf after whole mount <i>in situ</i> hybridization, WISH, using probe against <i>d-asb11</i>. (<i>right</i>) Graph shows the quantification of the respective expressions using qPCR. (<b>C</b>), (<i>left</i>) Endogenous d-Asb11 in wild type (WT), heterozygous (asb11+/−) and mutant (<i>asb11^cul</i>) embryos at 12 hpf was detected by immunoblotting using anti-d-Asb11 antibody. (<i>right</i>) Graph quantifies 3 individual experiments, with 30 embryos/genotype/experiment.</p

Cullin box is essential for DeltaA degradation and for maintaining a cell proliferating state <i>in vivo</i>.

<p>(<b>A</b>) Zebrafish embryos were injected with Myc-tagged <i>deltaA</i> (MT-DeltaA) and <i>d-asb11</i> (Asb11) or <i>asb11^cul</i> (Asb11^cul) mRNA at one-cell stage. (<i>lower panel)</i> Lysates of 12 hpf embryos were analyzed by immunoblotting for the presence of DeltaA. (higher panel) Graph quantifies 2 individual experiments, each with 30 injected embryos/group. (<b>B</b>), Fluorescent whole-mount antibody labeling of wild type (WT) and <i>asb11^cul</i> embryos at 24 hpf for the mitotic marker anti-phosphohistone-3 (PH 3) antibody (<i>green</i>) and the neuronal marker Hu(C). Graph shows the number of positive cells per area (5 somites from beginning of yolk extension) of 5 embryos for each genotype.</p

<i>her4::gfp</i> transactivation and premature differentiation of neural cells in <i>asb11^cul</i>.

<p>(<b>A</b>), the <i>her4::gfp</i> reporter was co-injected with <i>myc-tag</i> (MT) mRNA as a control, myc-tagged <i>d-asb11</i> full length (MT-Asb11) or myc-tagged <i>asb11^cul</i> (MT-Asb11^cul) mRNA in zebrafish embryos. Injected embryos were treated with (+) (n = 25) or without (−) (n = 25) DAPT, from 1.5 hpf. At 14 hpf, embryos were analyzed for <i>her4</i> transactivation based on the intensity of the GFP signal. Positive embryos were counted and percentages of embryos presenting weak (blue), medium (green) or strong (red) signal were given. (<b>B</b>), Wild type (<i>left panel</i>) and mutant (<i>middle panel</i>) embryos at 12 hpf were analyzed for WISH using probe against <i>ngn1</i>. (<i>right</i>) Graph quantifies expression of <i>ngn1</i> using qPCR. (<b>C</b>) Wild type (<i>left panel</i>) and mutant (<i>right panel</i>) polster of embryos at 16 hpf were analyzed for WISH using probe against <i>islet1</i>.</p