Network Analysis of UBE3A/E6AP-Associated Proteins Provides Connections to Several Distinct Cellular Processes - PubMed (original) (raw)
. 2018 Mar 30;430(7):1024-1050.
doi: 10.1016/j.jmb.2018.01.021. Epub 2018 Feb 6.
Katja Luck 2, Simone Kühnle 1, Alice Desbuleux 3, Patricia Szajner 1, Jeffrey T Galligan 1, Diana Rodriguez 1, Leon Zheng 1, Kathleen Boyland 1, Flavian Leclere 1, Quan Zhong 2, David E Hill 2, Marc Vidal 4, Peter M Howley 5
Affiliations
- PMID: 29426014
- PMCID: PMC5866790
- DOI: 10.1016/j.jmb.2018.01.021
Network Analysis of UBE3A/E6AP-Associated Proteins Provides Connections to Several Distinct Cellular Processes
Gustavo Martínez-Noël et al. J Mol Biol. 2018.
Abstract
Perturbations in activity and dosage of the UBE3A ubiquitin-ligase have been linked to Angelman syndrome and autism spectrum disorders. UBE3A was initially identified as the cellular protein hijacked by the human papillomavirus E6 protein to mediate the ubiquitylation of p53, a function critical to the oncogenic potential of these viruses. Although a number of substrates have been identified, the normal cellular functions and pathways affected by UBE3A are largely unknown. Previously, we showed that UBE3A associates with HERC2, NEURL4, and MAPK6/ERK3 in a high-molecular-weight complex of unknown function that we refer to as the HUN complex (HERC2, UBE3A, and NEURL4). In this study, the combination of two complementary proteomic approaches with a rigorous network analysis revealed cellular functions and pathways in which UBE3A and the HUN complex are involved. In addition to finding new UBE3A-associated proteins, such as MCM6, SUGT1, EIF3C, and ASPP2, network analysis revealed that UBE3A-associated proteins are connected to several fundamental cellular processes including translation, DNA replication, intracellular trafficking, and centrosome regulation. Our analysis suggests that UBE3A could be involved in the control and/or integration of these cellular processes, in some cases as a component of the HUN complex, and also provides evidence for crosstalk between the HUN complex and CAMKII interaction networks. This study contributes to a deeper understanding of the cellular functions of UBE3A and its potential role in pathways that may be affected in Angelman syndrome, UBE3A-associated autism spectrum disorders, and human papillomavirus-associated cancers.
Keywords: Angelman syndrome; autism; cervical cancer; human papillomavirus; proteomics.
Copyright © 2018 Elsevier Ltd. All rights reserved.
Figures
Figure 1. A UBE3A-centered protein interaction network
This network displays interactors of UBE3A found by AP-MS or Y2H as well as interactors of its associated proteins. All links shown in the network are supported by at least two different sources (see Methods). Highlighted are a few key protein complexes of which all apart from the ribosome are significantly enriched in the systematically built network of UBE3A using only QBCHL as source of interactions (see Figure S2).
Figure 2. Confirmation of interaction of UBE3A with several HCIPs
V5-tagged HCIPs were immunoprecipitated from T-REx 293 cells using anti-V5 magnetic beads. Protein extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blot using antibodies against HA-tag, V5-tag and Actin. HA-SRC was used as negative control and V5-PSMD4 as positive control. HA-SRC signal was present in each pulldown, including that done with the empty V5-tag vector and therefore is considered as background for this experiment. The long black line indicates the place where part of the blots was removed because it was irrelevant for the figure. HA-UBE3A: UBE3A isoform 1 C820A, catalytically inactive. EV: empty vector.
Figure 3. UBE3A partially colocalizes with HERC2, EIF3C, and MCM6 in SH-SY5Y cells
UBE3A colocalizes with HERC2 and EIF3C in the cytosol and with MCM6 predominantly in the nuclei.
Figure 4. UBE3A co-fractionates with HERC2 and NEURL4 in high molecular weight fractions in a size-exclusion experiment with SH-SY5Y cells protein extract
8 mgs. of protein extract from SH-SY5Y cells were loaded on a Superose 6 column for size-exclusion chromatography. After fractionation, 20 µl of the collected fractions (500 µl) were analyzed by SDS-PAGE and Western blot using antibodies against the UBE3A, HERC2, NEURL4, PSMD4 and PSMA5. 20 µg of protein extract were loaded as input. The position where the peaks of protein standards eluted (669, 443, 200, 150, 66 and 29 kDa) and the void volume (V) are indicated over the fractions number.
Figure 5. The HUN complex interactome
Preys of HERC2, NEURL4, MAPK6, ECI2, and ECH1 were connected if there was evidence for association or interaction from QBCHL, reciprocal IP from this study or [Al-Hakim et al] and if any pair of preys had at least one bait in common. Key protein complexes are highlighted. Oval shapes are equivalent to circles, they were used with the sole purpose of facilitating the reading of the gene symbols in the corresponding nodes.
Figure 6. UBE3A promotes the degradation of ASPP2 by the proteasome
A.UBE3A coimmunoprecipitates ASPP2. HEK 293T cells were transfected with the indicated vectors. 48 hours after transfection the HA-tagged proteins were immunoprecipitated with anti-HA agarose beads. Protein extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blot using antibodies against HA-tag and Actin. HBH-ASPP2 carrying a biotinylation signal in the HBH tag was detected using streptavidin-HRP. HA-UBE3A: UBE3A isoform 1 C820A, catalytically inactive. B. Coexpression of UBE3A reduces ASPP2 protein levels. HEK 293T cells were transfected with the corresponding vectors. 48 hours post transfection the cells were harvested and the protein extracts were analyzed by SDS-PAGE and Western blot. Proteins were detected using anti HA, V5 and actin antibodies, and streptavidin HRP. Of note, UBE3A runs as a double band while its catalytically inactive form runs as a single band. - indicates that the cells were transfected with the corresponding empty vector. WT: wild type, CA: catalytically inactive form of UBE3A.
Figure 7. Interconnectivity of the interactomes of CAMKII and the HUN complex
The network built between CAMK2D and HUN complex preys using interaction data from QBCHL, reciprocal IP and Y2H data from this study and pulldown data from [Al-Hakim et al.], shows strong connectivity and suggests an involvement of both, the CAMKII and HUN complex, in common biological processes. Interestingly, no distinct protein complexes were identified in this network. Oval shapes are equivalent to circles, they were used with the sole purpose of facilitating the reading of the gene symbols in the corresponding nodes.
Figure 8. Confirmation of interaction of CAMK2D and CAMK2A with several HCIPs
V5-tagged HCIPs were immunoprecipitated from T-REx 293 cells using anti-V5 magnetic beads. Protein extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blot using antibodies against HA-tag, V5-tag and Actin. HA-SRC was used as negative control and V5-CAMK2B as positive control. The long black line indicates the place where part of the blots was removed because it was irrelevant for the figure. EV: empty vector.
References
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- Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell. 1993;75:495–505. -PubMed
- Yamamoto Y, Huibregtse JM, Howley PM. The human E6-AP gene (UBE3A) encodes three potential protein isoforms generated by differential splicing. Genomics. 1997;41:263–6. -PubMed
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