Abstract
Although they are the primary determinants of substrate specificity, few E3-substrate pairs have been positively identified, and few E3’s profiled in a proteomic fashion. Praja1 is an E3 implicated in bone development and highly expressed in brain. Although it has been well studied relative to the majority of E3’s, little is known concerning the repertoire of proteins it ubiquitylates. We sought to identify high confidence substrates for Praja1 from an unbiased proteomic profile of thousands of human proteins using protein microarrays. We first profiled Praja1 activity against a panel of E2’s to identify its optimal partner in vitro. We then ubiquitylated multiple, identical protein arrays and detected putative substrates with reagents that vary in ubiquitin recognition according to the extent of chain formation. Gene ontology clustering identified putative substrates consistent with information previously known about Praja1 function, and provides clues into novel aspects of this enzyme’s function.
Similar content being viewed by others
References
Li, W., Bengtson, M. H., Ulbrich, A., Matsuda, A., Reddy, V. A., Orth, A., et al. (2008). Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle’s dynamics and signaling. PLoS One, 3, e1487.
Marblestone, J. G., Kumar, K. G., Eddins, M. J., Leach, C. A., Sterner, D. E., Sterner, M. R., et al. (2010). Novel Approach for Characterizing Ubiquitin E3 Ligase Function. Journal of Biomolecular Screening, 15, 1220–1228.
Sowa, M. E., Bennett, E. J., Gygi, S. P., & Harper, J. W. (2009). Defining the human deubiquitinating enzyme interaction landscape. Cell, 138, 389–403.
Nijman, S. M., Luna-Vargas, M. P., Velds, A., Brummelkamp, T. R., Dirac, A. M., Sixma, T. K., et al. (2005). A genomic and functional inventory of deubiquitinating enzymes. Cell, 123, 773–786.
Todi, S. V., Winborn, B. J., Scaglione, K. M., Blount, J. R., Travis, S. M., & Paulson, H. L. (2009). Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3. EMBO Journal, 28, 372–382.
Chen, Z. J., & Sun, L. J. (2009). Nonproteolytic functions of ubiquitin in cell signaling. Molecular Cell, 33, 275–286.
Haglund, K., & Dikic, I. (2005). Ubiquitylation and cell signaling. EMBO Journal, 24, 3353–3359.
Grabbe, C., & Dikic, I. (2009). Functional roles of ubiquitin-like domain (ULD) and ubiquitin-binding domain (UBD) containing proteins. Chemical Reviews, 109, 1481–1494.
Weake, V. M., & Workman, J. L. (2008). Histone ubiquitination: triggering gene activity. Molecular Cell, 29, 653–663.
Sigismund, S., Polo, S., & Di Fiore, P. P. (2004). Signaling through monoubiquitination. Current Topics in Microbiology and Immunology, 286, 149–185.
Huang, T. T., & DAndrea, A. D. (2006). Regulation of DNA repair by ubiquitylation. Nature Reviews Molecular Cell Biology, 7, 323–334.
Hochstrasser, M. (2009). Origin and function of ubiquitin-like protein conjugation. Nature, 458, 422–429.
Lee, D. H., & Goldberg, A. L. (1998). Proteasome inhibitors: valuable new tools for cell biologists. Trends in Cell Biology, 8, 397–403.
Deretic, V. (2010). Autophagy in infection. Current Opinion in Cell Biology, 22, 252–262.
Skaug, B., & Chen, Z. J. (2010). Emerging role of ISG15 in antiviral immunity. Cell, 143, 187–190.
Turnbull, E. L., Rosser, M. F., & Cyr, D. M. (2007). The role of the UPS in cystic fibrosis. BMC Biochemistry, 8(Suppl1), S11–S20.
Attaix, D., Ventadour, S., Codran, A., Béchet, D., Taillandier, D., & Combaret, L. (2005). The ubiquitin–proteasome system and skeletal muscle wasting. Essays Biochem, 41, 173–186.
Debigare, R., Cote, C. H., & Maltais, F. (2010). Ubiquitination and proteolysis in limb and respiratory muscles of patients with chronic obstructive pulmonary disease. Proceedings of the American Thoracic Society, 7, 84–90.
Rogers, N., Paine, S., Bedford, L., & Layfield, R. (2010). Review: the ubiquitin proteasome system: contributions to cell death or survival in neurodegeneration. Neuropathology and Applied Neurobiology, 36, 113–124.
Nicholson, B., Marblestone, J. G., Butt, T. R., & Mattern, M. R. (2007). Deubiquitinating enzymes as novel anticancer targets. Future Oncology, 3, 191–199.
Stork, O., Stork, S., Pape, H. C., & Obata, K. (2001). Identification of genes expressed in the amygdale during the formation of fear memory. Learning and Memory, 8, 209–219.
Mishra, L., Tully, R. E., Monga, S. P., Yu, P., Cai, T., Makalowski, W., et al. (1997). Praja1, a novel gene encoding a RING-H2 motif in mouse development. Oncogene, 15, 2361–2368.
Yoon, W. J., Cho, Y. D., Cho, K. H., Woo, K. M., Baek, J. H., Cho, J. Y., et al. (2008). The Boston-type craniosynostosis mutation MSX2 (P148H) results in enhanced susceptibility of MSX2 to ubiquitin-dependent degradation. The Journal of Biological Chemistry, 283, 32751–32752.
Yu, P., Chen, Y., Tagle, D. A., & Cai, T. (2002). PJA1, encoding a RING-H2 finger ubiquitin ligase, is a novel human X chromosome gene abundantly expressed in brain. Genomics, 79, 869–874.
Sasaki, A., Masuda, Y., Iwai, K., Ikeda, K., & Wantanabe, K. (2002). A RING finger protein Praja1 regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/Necdin family protein, Dlxin-1. The Journal of Biological Chemistry, 277, 22541–22546.
Doyle, J. M., Gao, J., Wang, J., Yang, M., & Potts, P. R. (2010). MAGE-RING protein complexes comprise a family of E3 ubiquitin ligases. Molecular Cell, 39, 963–974.
Lu, J., Lin, Y., Qian, J., Tao, S., Zhu, J., Pickart, C., et al. (2008). Functional dissection of a HECT ubiquitin E3 ligase. Molecular and Cellular Proteomics, 7(1), 35–45.
Andrews, P. S., Schneider, S., Yang, E., Michaels, M., Chen, H., Tang, J., et al. (2010). Identification of substrates of Smurf1 ubiquitin ligase activity utilizing protein microarrays. Assay and Drug Development, 8, 471–487.
Gupta, R., Kus, B., Fladd, C., Wasmuth, J., Tonikian, R., Sidhu, S., et al. (2007). Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast. Molecular Systems Biology, 3, 116.
Balut, C. M., Loch, C. M., Gao, Y., Devor, D. Role of ubiquitination and Usp8 (UBPY)-dependent deubiquitination in the endocytosis and lysosomal targeting of plasma membrane KCa3.1 (submitted).
Shi, Y., Chan, D. W., Jung, S. Y., Malovannaya, A., Wang, Y., Qin, J. (2010). A dataset of human endogenous protein ubiquitination sites. Molecular and Cellular Proteomics. doi:10.1074/mcp.M110.002089.
Meierhofer, D., Wang, X., Huang, L., & Kaiser, P. (2008). Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. Journal of Proteome Research, 7, 4566–4576.
Xu, P., Duong, D., Seyfried, N., Cheng, D., Xie, Y., Robert, J., et al. (2009). Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell, 137, 133–145.
Beissbarth, T., & Speed, T. P. (2004). GOstat: Find statistically overrepresented gene ontologies within a group of genes. Bioinformatics, 20, 1464–1465.
Benjamini, V., & Hochberg, V. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society B, 57, 289–300.
Loch, C. M., Ramirez, A. B., Liu, Y., Sather, C. L., Delrow, J. J., Scholler, N., et al. (2007). Use of high density antibody arrays to validate and discover cancer serum biomarkers. Molecular Oncology, 1, 313–320.
Wada, K., & Kamitani, T. (2006). UnpEL/USP4 is ubiquitinated by Ro52 and deubiquitinated by itself. Biochemical and Biophysical Research Communications, 342, 253–258.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Loch, C.M., Eddins, M.J. & Strickler, J.E. Protein Microarrays for the Identification of Praja1 E3 Ubiquitin Ligase Substrates. Cell Biochem Biophys 60, 127–135 (2011). https://doi.org/10.1007/s12013-011-9180-x
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12013-011-9180-x