Structure and Functions of the 20S and 26S Proteasomes. proteasome regulation and demonstrate the biological significance of proteasome phosphorylation in regulating cell proliferation and tumorigenesis. Introduction The 26S proteasome is an essential protein complex responsible for degrading the majority of cellular proteins in eukaryotes1. An impaired proteasome system often underlies neurodegenerative diseases and the aging process2, 3. On the other hand, the quick growth of malignancy cells is usually often dependent on elevated proteasome activity, and proteasome inhibitors such as Bortezomib (Velcade?) have proven to be effective against multiple myeloma and certain solid cancers4, 5. Further understanding of proteasome regulation is usually of enormous biological and clinical importance. The mature 26S proteasome consists of at least 33 unique subunits. Fourteen of them (1-7 and 1-7) form the RN-1 2HCl 20S core particle (CP), a barrel-shaped structure that encloses three types of peptidase activities (trypsin-like, caspase-like and chymotrypsin-like). The remaining 19 subunits (Rpt1-6, Rpn1-3, 5-13 and 15) constitute the 19S regulatory particle (RP) that caps the CP on one or both ends. Protein substrates destined for proteasomal degradation are captured and processed by the 19S RP before they are threaded into the 20S CP for proteolysis. During this process, the ATPase subunits (Rpt1-6) play key functions in substrate engagement, unfolding, translocation and CP gate opening6-8. Given its biological importance and biochemical complexity, the 26S proteasome is usually regulated at several levels by multiple mechanisms, ranging from transcriptional control to post-translational modifications (e.g. phosphorylation) of proteasome subunits9-14. Notably, the human 26S proteasome contains over 300 phosphorylation sites, over 99% of which have not been analyzed (http://www.phosphosite.org). It remains poorly comprehended how these RN-1 2HCl regulations are achieved biochemically and how they influence the vast biological processes that require proteasome function. Cell cycle regulation is one of the best appreciated functions of the 26S proteasome15, 16. Impaired degradation of important proteins caused by proteasome inhibitors or protein aggregation impedes cell proliferation, which underpins the pathogenesis and treatment of certain diseases4, 5, 17, 18. Recent phospho-proteomic studies have revealed a number of proteasome phosphorylation events at different cell cycle stages19-22, raising an important and intriguing question whether and how the proteasome itself is usually regulated during cell cycle to accommodate this process where protein degradation must be finely regulated. Here we show that this 26S proteasome is usually dynamically phosphorylated at RN-1 2HCl Thr25 of the 19S subunit Rpt3 in a cell cycle-regulated manner. Cells deficient of Rpt3-T25 phosphorylation exhibit reduced proliferation and decreased proteasome activity. We identify dual-specificity tyrosine-regulated kinase 2 (DYRK2) as the major kinase that phosphorylates Rpt3-T25. Loss of this single phosphorylation significantly inhibits tumor growth in vivo. Our study for the first time demonstrates the biological importance of proteasome phosphorylation in cell cycle and tumorigenesis, and suggests a possible approach of proteasome-oriented therapy by targeting proteasome kinases. RESULTS Cell cycle-dependent Rpt3-Thr25 phosphorylation Rpt3-T25 phosphorylation has been documented in several proteomic studies19, 23, 24, although its function and regulation remained unknown. To characterize this event, we generated a phospho-T25-specific antibody (Fig. 1a). T25 phosphorylation of endogenous Rpt3 was found both in vivo (Fig. 1b) and in 26S proteasomes isolated from multiple cell lines (Fig. 1c and Supplementary Fig. 1a), establishing Rpt3-T25 as a bona fide proteasome phosphorylation site. Several lines of evidence show that Rpt3-T25 phosphorylation undergoes reversible and dynamic regulation. First, the phosphorylation Rabbit Polyclonal to OR1D4/5 was increased by treating cells with Calyculin A, a potent inhibitor of the PP1 and PP2A phosphatases (Fig. 1d). Second, Rpt3-T25 phosphorylation appeared to be associated with actively proliferating cells, as it was downregulated following serum starvation (Fig. 1e) or contact inhibition (Fig. 1f), both of which arrest cells in the G0/G1 phase of cell cycle. Interestingly, Rpt3-T25 phosphorylation was first reported to be present in nocodazole-synchronized mitotic cells but not in cells at late G1 phase19. Indeed, we consistently found higher levels of Rpt3-T25 phosphorylation at G2/M phase than at the G1/S boundary in.