Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation. Multiple receptors tyrosine kinases can trigger Rpt2–Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2–Y439 in vitro and negatively regulates 26S proteasome activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase nonreceptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2–Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anticancer therapies.
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We thank Drs. Xin-Hua Feng, Hai Song, Bin Zhao (Life Sciences Institute, Zhejiang University, LSI-ZJU), Zhong-Yin Zhang (Purdue University), Shigeo Murata (The University of Tokyo), and Susan Lindquist (Massachusetts Institute of Technology) for crucial constructs, cell lines, and reagents. We are grateful for the critical and encouraging comments from Drs. Kun-Liang Guan, Anne-Claude Gingras, Benjamin Neel, Tony Hunter, and Tony Tiganis. We appreciate the technical assistance from Xiaorui Jiang, Fei Zhang, and the Core Facilities of LSI-ZJU. XG was supported by Natural Science Foundation of China (31671391, 31870762), Zhejiang Natural Science Foundation (LR18C050001), Fundamental Research Funds for the Central Universities (2016QN81011), and the startup funding from Zhejiang University. BY was funded by Natural Science Foundation of China (91953103). ZW was supported by Natural Science Foundation of China (31671039) and National Key Research and Development Plan of the Ministry of Science and Technology of China (2016YF0501000). LH was funded by NIH (R01GM074830).
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Chen, L., Zhang, Y., Shu, X. et al. Proteasome regulation by reversible tyrosine phosphorylation at the membrane. Oncogene 40, 1942–1956 (2021). https://doi.org/10.1038/s41388-021-01674-z