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Mutations in the deubiquitinase gene USP8 cause Cushing’s disease Martin Reincke1,13, Silviu Sbiera1,2,13, Akira Hayakawa3,13, Marily Theodoropoulou4,13, Andrea Osswald1, Felix Beuschlein1, Thomas Meitinger5–7, Emi Mizuno-Yamasaki3, Kohei Kawaguchi3, Yasushi Saeki8, Keiji Tanaka8, Thomas Wieland5, Elisabeth Graf5, Wolfgang Saeger9, Cristina L Ronchi10, Bruno Allolio2,11, Michael Buchfelder12,13, Tim M Strom5,6,13, Martin Fassnacht1,2,10,13 & Masayuki Komada3,13 Cushing’s disease is caused by corticotroph adenomas of the pituitary. To explore the molecular mechanisms of endocrine autonomy in these tumors, we performed exome sequencing of 10 corticotroph adenomas. We found somatic mutations in the USP8 deubiquitinase gene in 4 of 10 adenomas. The mutations clustered in the 14-3-3 protein binding motif and enhanced the proteolytic cleavage and catalytic activity of USP8. Cleavage of USP8 led to increased deubiqutination of the EGF receptor, impairing its downregulation and sustaining EGF signaling. USP8 mutants enhanced promoter activity of the gene encoding proopiomelanocortin. In summary, our data show that dominant mutations in USP8 cause Cushing’s disease via activation of EGF receptor signaling. Cushing’s disease (CD) is caused by pituitary corticotroph adenomas hypersecreting adrenocorticotropin (ACTH). Chronic elevation of ACTH is followed by excessive adrenal glucocorticoid secretion, inducing the characteristic phenotype of weight gain, central obesity, skin changes, myopathy, disturbed mood and impaired reproductive function. If left untreated, patients die from infections and cardiovascular consequences of glucocorticoid excess. CD was first described in 1932 by the neurosurgeon Harvey Cushing1, who pioneered surgical removal of the underlying pituitary adenomas. To this day, firstline therapy is transsphenoidal surgery, which results in remission in 65–90% of patients. The pathophysiology of corticotroph adenoma has remained obscure since its first description. CD is occasionally observed as a manifestation of genetic tumor syndromes, such as multiple endocrine neoplasia type 1, familial isolated pituitary adenoma, McCune-Albright syndrome and Carney complex2,3. However, an extensive search for somatic mutations in CD adenomas by direct sequencing of candidate genes and by microarray studies has been largely unsuccessful4. Another avenue of investigation is suggested by the observation that epidermal growth factor receptor (EGFR) expression has been described in hormonally active pituitary adenomas5. Its expression is especially strong in CD and correlates with ACTH expression on a cellular level5. More recently, Fukuoka et al. demonstrated that the EGFR-mediated pathway is essential for the synthesis of proopiomelanocortin (Pomc), the precursor of ACTH. In a

mouse corticotroph tumor cell line, transfection of EGFR enhances ACTH secretion via MAP kinase-dependent pathways6. Accordingly, in mice, blocking EGFR activity with gefitinib, an EGFR tyrosine kinase inhibitor, inhibited ACTH hypersecretion and corticotroph tumor cell proliferation, and enhanced apoptosis. Recently, we and other groups identified recurrent activating mutations in the catalytic subunit of protein kinase A as the underlying cause of sporadic cortisol-producing adenomas of the adrenal cortex7–10 using exome sequencing. This outcome prompted us to look for mutation in CD. RESULTS USP8 mutations in corticotroph adenomas To identify the underlying genetic basis driving tumor growth and ACTH secretion in corticotroph adenomas, we subjected available tumor/germline DNA sample pairs from 10 patients with CD to exome sequencing (Supplementary Table 1). This approach revealed a small number of protein-altering mutations (median 7, range 3–23 per tumor) that were present only in the tumor tissue (Supplementary Table 2). Remarkably, within this small set of genetic hits, 4 of 10 tumors had somatic missense mutations in the gene encoding ubiquitin-specific protease 8 (USP8): three tumors had single point mutations and one tumor the double mutation p.[Leu713Arg;Tyr717Cys] (Fig. 1a and Supplementary Fig. 1). The affected amino acids are highly conserved across a variety of vertebrate species (Fig. 1b). All mutations were

1Medizinische

Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, Munich, Germany. 2Department of Medicine I, Endocrine and Diabetes Unit, University Hospital, University of Würzburg, Würzburg, Germany. 3Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan. 4Department of Endocrinology, Max Planck Institute of Psychiatry, Munich, Germany. 5Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany. 6Institute of Human Genetics, Technische Universität München, Munich, Germany. 7DZHK (German Centre for Cardiovascular Research) partner site, Munich Heart Alliance, Munich, Germany. 8Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. 9Institut für Neuropathologie der Universität Hamburg, Hamburg, Germany. 10Comprehensive Cancer Center Mainfranken, University of Würzburg, Würzburg, Germany. 11Comprehensive Heart Failure Center, University of Würzburg, Würzburg, Germany. 12Neurochirurgische Klinik, Klinikum der Universität Erlangen, Erlangen, Germany. 13These authors contributed equally to this work. Correspondence should be addressed to M.F. ([email protected]) or M.R. ([email protected]). Received 16 July; accepted 17 November; published online 8 December 2014; doi:10.1038/ng.3166

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© 2014 Nature America, Inc. All rights reserved.

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Figure 1  Identification of somatic USP8 mutations in corticotroph adenomas. (a) Hotspot somatic mutations are found between amino acid 713 and 720. (b) Homology alignment of the USP8 hotspot mutation region: the boxed human USP8 region is highly conserved between species. (c) Localization of the identified mutations and alignment with the human USP8 functional regions: MIT domain (MIT); rhodanese-like domain (Rhod); SH3-binding motif (SBM); 14-3-3 binding motif (14-3-3) and deubiquitinase catalytic domain (DUB).

located between amino acid 713 and 720, close to the protein’s catalytic domain (Fig. 1c). On the basis of these initial results, we sequenced the entire coding sequence of USP8 in 43 additional adenomas (including 7 corticotroph adenomas, 2 Nelson tumors, 14 somatotroph adenomas, 10 prolactinomas and 10 nonfunctional adenomas; Supplementary Table 3), thereby identifying 2 additional mutations in 7 corticotroph adenomas (total: 6/17, 35% of tumors), but no mutations in the other pituitary adenomas, including the 2 Nelson tumors. Whole-exome and targeted sequencing indicated that both the wild-type and mutant alleles were present in tumor tissue, consistent with a heterozygous state of the USP8 mutation. In our series patients harboring corticotroph tumors with USP8 mutations were all female (6 of 6 versus 5 of 11 in wild-type tumors, Fisher’s exact test, P ≤ 0.05) and seemed to have smaller tumors (maximum diameter by MRI 8 ± 6 versus 13 ± 8 mm, Mann-Whitney U-test, two-sided, P = n.s.), with lower plasma ACTH levels (50 ± 46 versus 90 ± 48 pg/ml, Mann-Whitney U-test, two-sided, P = n.s.) and lower serum cortisol after suppression with 1 mg dexamethasone (7.8 ± 9.7 versus 21.1 ± 12.5 µg/dl, Mann-Whitney U-test, two-sided, P ≤ 0.05 compared to USP8–wild-type adenomas). USP8, also known as UBPY, is a deubiquitinase (DUB) that belongs to the ubiquitin-specific protease (USP) family11. Ubiquitination is a reversible post-translational protein modification that regulates the fate and function of various proteins in eukaryotic cells12,13. Specifically, ubiquitination of cell surface proteins targets them for degradation in the lysosome14–16. Conjugated ubiquitin (Ub) molecules can be removed from target proteins by DUBs17,18. Among the ~90 DUBs encoded by the human genome, USP8 has been implicated in the lysosomal trafficking of ligand-activated EGFR19–24. We previously demonstrated that USP8, by removing Ub from cell surface receptors including activated EGFR, impedes receptor downregulation19,25. We also demonstrated that 14-3-3 proteins bind to the consensus 14-3-3 binding motif RSYSSP in mouse USP8 at amino acid positions 677–682 and that a mutation in the binding motif at Ser680 (corresponding to human Ser718) inhibits 14-3-3 binding and increases DUB activity toward its cellular substrate EGFR26. The high incidence of USP8 mutations in corticotroph adenomas clustered around the 14-3-3 binding motif (Fig. 1) prompted us to investigate its expression in the pituitary gland and the mechanistic consequences of the identified mutations. 

In the normal human pituitary, immunohistochemistry revealed mainly cytoplasmic USP8 staining in 7–15% of the endocrine cells that co-localized with most anterior pituitary hormones (Fig. 2a,b). The USP8-mutated adenomas displayed high USP8 immunoreactivity exclusively in the nuclei (Fig. 2e,f), and the intensity of staining was significantly higher than in normal pituitary glands and USP8 wildtype adenomas (Supplementary Fig. 2). In contrast, non-mutated corticotroph adenomas showed heterogeneity for USP8 expression levels and subcellular distribution: five adenomas were characterized by a predominantly cytoplasmic expression pattern (Fig. 2d), whereas four showed a mainly nuclear staining pattern (Fig. 2c and Supplementary Fig. 2). It is thereby tempting to speculate that in a subgroup of corticotroph adenomas without USP8 mutations, other mechanisms might contribute to increased USP8 activation. USP8 mutants lose 14-3-3 protein binding Because all USP8 mutations identified in corticotroph adenomas were positioned within or adjacent to the 14-3-3 binding motif, we examined 14-3-3 protein binding in relation to USP8 mutant status. 14-3-3ε protein fused to glutathione S-­transferase (GST) did not pull down Flag-tagged USP8-p.Ser718Pro, -p.Ser718Cys, -p.Pro720Arg or -p.Ser718del, whereas it bound weakly to USP8-p.[Leu713Arg;Tyr717Cys] (Fig. 3a). As determined by co-­immunoprecipitation, USP8-p.Ser718Pro, -p.Ser718Cys, -p.Pro720Arg and -p.Ser718del also lost 14-3-3 binding, whereas USP8-p.[Leu713Arg;Tyr717Cys] retained weak binding (Fig. 3b). A mouse USP8 mutant lacking 14-3-3 binding has been shown to have higher DUB activity26. We therefore examined whether this is also the case for USP8 mutants identified in the human corticotroph adenomas. We expressed Flag-tagged USP8 proteins in COS-7 cells and immunoprecipitated them with anti-Flag antibody, then eluted USP8 proteins from the antibody with a Flag peptide. We verified the purity and amount of each protein by Coomassie staining (Fig. 3c, 130 kDa). Because EGFR, the substrate of USP8, undergoes Lys63linked polyubiquitination for lysosomal trafficking27, we incubated the purified USP8 proteins with Lys63-linked Ub oligomers (Fig. 3d). DAPI

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Figure 2  USP8 protein expression in corticotroph adenomas with USP8 mutations as compared to normal tissue. (a) Normal testis. (b) Normal pituitary. (c) Sample from patient 2: corticotroph adenoma with wild-type USP8. (d) Sample from patient 4: corticotroph adenoma with wild-type USP8. (e) Sample from patient 7: corticotroph adenoma with USP8-p.Ser718Pro mutation. (f) Sample from patient 9: corticotroph adenoma with USP8-p.[Leu713Arg; Tyr717Cys] mutation. Magnification, 1,600×; inset, 4,800× (before typesetting). Scale bars, 15 µm.

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USP8 mutants undergo proteolytic cleavage During the course of the experiments summarized in Figure 3, we found that USP8 mutants with higher DUB activity (p.Ser718Cys, p.Pro720Arg and p.Ser718del) exhibit a minor ~90-kDa fragment that was barely detectable in wild-type USP8 (Fig. 3e, closed arrowhead). There was a clear positive correlation between the abundance of the 90-kDa fragment and the DUB activity in the mutants, raising the possibility that USP8 is catalytically activated by proteolytic cleavage. Coomassie staining of USP8 preparations stored for 6 weeks at 4 °C showed that, as compared to preparations immediately after immuno­ purification (Fig. 3c), the stored samples contained larger amounts of the 90-kDa fragment, along with another fragment 40 kDa in size (Fig. 4a). As anti-Flag immunoblotting detected only the 90-kDa fragment (Fig. 3e and data not shown), and the Flag epitope was tagged to the N terminus of USP8, we concluded that the 90-kDa and 40-kDa fragments correspond to the N- and C-terminal regions, respectively. The cleavage efficiency was much higher for USP8-p.Ser718del and -p.Pro720Arg, and somewhat higher for USP8-p.Ser718Pro and -p.Ser718Cys, than for wild-type USP8. In contrast, cleaved fragments of USP8-p.[Leu713Arg;Tyr717Cys] were not detectable. Immunoblotting of the lysates of cells transfected with Flag-tagged USP8 mutants (except for USP8- p.[Leu713Arg;Tyr717Cys]) revealed the 90-kDa fragment (Fig. 4b, top). Furthermore, endogenous 90-kDa Nature Genetics  ADVANCE ONLINE PUBLICATION

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Incubation with USP8-p.Ser718del resulted in almost undetectable levels of Ub oligomers, suggesting that USP8-p.Ser718del has the highest DUB activity. Comparison of the band intensity for Ub oligomers showed that USP8-p.Pro720Arg has the second-highest activity and USP8-p.Ser718Pro and USP8-p.Ser718Cys are slightly more active than wild-type USP8, while USP8-p.[Leu713Arg;Tyr717Cys] is as active as wild-type USP8. We next examined the DUB activity of the USP8 mutants toward ubiquitinated EGFR immunoprecipitated from EGF-stimulated HeLa cells (Fig. 3e). Roughly consistently with their activity toward Ub oligomers, USP8-p.Ser718del, -Pro720Arg and -p.Ser718Pro deubiquitinated EGFR more efficiently than wildtype USP8, while USP8-p.Ser718Cys and -p.[Leu713Arg;Tyr717Cys] had activity equivalent to that of wild type. As expected, catalytically inactive mutant USP8CA, in which Cys786 in the catalytic core is replaced by alanine, exhibited no activity (Fig. 3d,e).

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Figure 3  14-3-3 binding and DUB activity of corticotroph adenoma USP8 mutants. (a) Top, anti-Flag immunoblots of GST-14-3-3ε fusion protein immobilized on glutathione beads and incubated with lysates of COS-7 cells expressing the wild-type and mutant Flag-tagged USP8 proteins. Bound USP8 proteins were detected by anti-Flag immunoblotting. Middle, amounts of USP8 proteins in the lysates as assessed by anti-Flag immunoblotting. Bottom, amount of GST fusion protein used as determined by Coomassie staining. (b) Top and second from top, anti-14-3-3 (top) and anti-Flag (second) immunoblots of anti-Flag immunoprecipitates of lysates of COS-7 cells expressing the Flag-tagged USP8 proteins. Third and fourth from top, anti-14-3-3 (third) and anti-Flag (fourth) immunoblots of lysates of COS-7 cells expressing the Flag-tagged USP8 proteins. (c) Coomassie staining of the Flag-tagged USP8 proteins expressed in COS-7 cells, immunoprecipitated from their lysates with anti-Flag antibody and eluted from the antibody with a Flag peptide. (d) Anti-Ub immunoblots of Lys63-linked Ub oligomers (dimer-heptamer) incubated with the USP8 proteins prepared in c for 1 h at 37 °C. (e) Top, anti-Ub immunoblots of EGFR immunoprecipitated from EGF-treated cells and incubated with the USP8 proteins prepared in c for 1 h at 37 °C. Middle and bottom, amounts of EGFR (middle) and USP8 proteins (bottom) in the reactions as assessed by anti-EGFR and anti-Flag immunoblotting, respectively. Open and closed arrowheads indicate full-length and cleaved USP8 forms, respectively.

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fragment was detected by immunoblotting of untransfected HeLa and COS-7 cell lysates with anti-USP8 antibody (Fig. 4b, bottom), suggesting that this cleavage also occurs in vivo. When USP8-p.Pro720Arg and -p.Ser718del were C-terminally tagged with the Flag epitope, immunoblotting with anti-Flag antibody detected the 40-kDa but not the 90-kDa fragment in the transfected cell lysates (Fig. 4c). To determine the cleavage site in USP8, we immunopurified USP8-p.Pro720Arg and -p.Ser718del at larger scales, excised the bands for the 90-kDa and 40-kDa fragments—as well as 130-kDa full-length forms—from a Coomassie-stained gel (Fig. 4d, left), digested them with trypsin, and identified their tryptic peptides by liquid ­ chromatography–tandem mass spectrometry (LC-MS/MS) (Fig. 4d, right; see Supplementary Table 4 for complete list of the identified peptides from each band of USP8-p.Ser718del). For both USP8-p.Pro720Arg and -p.Ser718del, tryptic peptides from the 90-kDa fragment covered the region from the N terminus to Lys714, with the C-terminal fragment being DREPSKLK714. The 40-kDa fragment covered the region from Arg715 to the C terminus, with the N-terminal fragments being R715SYSSR and R715SYSPDITQAIQEEEK for USP8p.Pro720Arg and -p.Ser718del, respectively. These results thereby identified the cleavage site between Lys714 and Arg715 immediately upstream of the 14-3-3 binding motif R715SYSSP. Another DUB, USP1, undergoes autocleavage downstream from the Ub C terminus–like diglycine motif by its own catalytic domain28. This is not the case for USP8 because the cleavage occurred downstream from a lysine residue (Fig. 4d) and catalytically inactive USP8CA also underwent the cleavage (Fig. 4b). We speculate that the cleavage of immunopurified USP8 mutants during storage (Fig. 4a) was mediated by a co-purified USP8-associated protease, whose accessibility to wild-type USP8 was impeded by 14-3-3 binding. 

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USP8 mutants inhibit EGFR downregulation To study the effect of the USP8 mutants and C40 forms on receptor downregulation, we examined EGFR endocytosis in HeLa cells ectopically expressing USP8 constructs. In the absence of EGF, EGFR

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Cleaved USP8 has elevated DUB activity 192 Because the cleaved C-terminal 40-kDa frag112 ment (hereafter referred to as USP8-C40) 85 60 contains the complete catalytic domain, 47 we examined its DUB activity. Flag-tagged 35 Coomassie USP8-C40 and its catalytically inactive mutant USP8-C40CA were immunopurified from transfected COS-7 cells using anti-Flag antibody (Fig. 4e). The molar concentrations of wild-type USP8 and USP8-C40 were adjusted by comparing their band intensities upon anti-Flag immunoblotting (Supplementary Fig. 3a). When incubated with Lys63 Ub oligomers, USP8-C40 cleaved them much more efficiently than did full-length USP8, whereas USP8-C40CA was inactive, suggesting that the proteolytic cleavage leads to elevated DUB activity of USP8 (Fig. 4f). In addition, USP8-C40 also deubiquitinated EGFR more efficiently than did the full-length USP8 (Fig. 4g). We further constructed USP8C40 mutants harboring the corticotroph adenoma–associated USP8 mutations (except for USP8-p.[Leu713Arg;Tyr717Cys], which does not undergo cleavage) and examined their DUB activity. None of the USP8-C40 mutants were more active than wild-type USP8-C40, suggesting that susceptibility to the cleavage, but not the amino acid replacements or deletion themselves, is responsible for the elevated DUB activity of USP8 mutants (Supplementary Fig. 3b). Probing the USP8-C40 preparations with anti-14-3-3 antibody showed that not only the mutant C40 forms but also wild-type C40 lost 14-3-3 binding (Supplementary Fig. 3c).



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Figure 4  Corticotroph adenoma USP8 mutants undergo proteolytic activation. (a) Coomassie staining to detect immunopurified USP8 proteins (prepared as in Fig. 3c) after storage at 4 °C for 6 weeks. Open and closed arrowheads indicate full-length and cleaved USP8, respectively. (b,c) Immunoblots with the indicated antibodies of lysates from COS-7 cells transfected with Flag-USP8 proteins (b, top), untransfected HeLa and COS-7 cells (b, bottom) and COS-7 cells transfected with C-terminally Flag-tagged USP8 proteins (c). (d) LC-MS/MS analysis of trypsin digests of cleaved 90-kDa and 40-kDa fragments of USP8-p.Pro720Arg and -p.Ser718del excised from a Coomassiestained gel (shown at left). Identified peptides are underlined in the USP8 sequences (amino acids 701–750 are represented) with peak areas of precursor ions shown. (e) Coomassie staining of full-length, C40 and C40CA forms of Flag-tagged USP8 expressed in COS-7 cells, immunoprecipitated from their lysates with anti-Flag antibody and eluted from the antibody with a Flag peptide. (f) Anti-Ub immunoblots of Lys63-linked Ub oligomers (dimer-heptamer) incubated with equal molar numbers of the USP8 proteins prepared in e for 1 h at 37 °C. (g) Top, anti-Ub immunoblots of EGFR immunoprecipitated from EGF-treated cells and incubated with equal molar numbers of the USP8 proteins prepared in e for 1 h at 37 °C. Bottom, amount of EGFR in the reactions as assessed by anti-EGFR immunoblotting.

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was localized to the plasma membrane in all the USP8 construct– expressing cells, suggesting that the USP8 mutations do not affect the steady-state level of cell surface EGFR (Supplementary Fig. 4). After 1 h of EGF stimulation, EGFR was transported to Hrs-positive early endosomes and LAMP2-positive late endosomes/lysosomes in untransfected cells (Supplementary Fig. 5). Confocal imaging of these cells showed that EGFR was normally endocytosed in cells expressing wild-type USP8 (Fig. 5a). In striking contrast, substantial amounts of EGFR remained on the plasma membrane in all the USP8 mutant–expressing cells, although USP8-p.[Leu713Arg;Tyr717Cys] was less effective than the other mutants (Fig. 5a). USP8-C40 also elevated the cell surface EGFR level, whereas catalytically inactive USP8-C40CA had no effect (Fig. 5a), suggesting that the corticotroph adenoma USP8 mutants and USP8-C40 inhibit downregulation of ligand-activated EGFR from the plasma membrane in a DUB activity– dependent manner. Quantification of the proportion of EGFRpositive area per cell in the confocal images, which is high (~90%) when EGFR is dispersed throughout the cell surface and lower (~60%) when it is internalized and concentrated in endosomes, confirmed the effects of the USP8 mutants on EGFR downregulation described above (Fig. 5c). We speculate that inhibition of EGFR downregulation by overexpression of the USP8 mutants (except for USP8p.[Leu713Arg;Tyr717Cys]) is mediated by their cleaved C40 forms. In contrast, USP8-p.[Leu713Arg;Tyr717Cys] was neither cleaved aDVANCE ONLINE PUBLICATION  Nature Genetics

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USP8 mutants sustain EGF signaling by recycling EGFR To elucidate the mechanism by which the USP8 mutants inhibit EGFR downregulation, we examined the effect of the corticotroph adenoma USP8 mutants and the cleaved USP8-C40 form on EGFR activity in Nature Genetics  ADVANCE ONLINE PUBLICATION

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* (Fig. 4) nor hyper-active (Fig. 3). However, although to a lesser extent, its ectopic * expression also elevated the cell surface L713R b EGFR level in EGF-treated cells (Fig. 5a,c). * To determine whether the p.Leu713Arg or * p.Tyr717Cys mutation is responsible for the effect, we constructed the two single mutants and examined them in the same fashion (Fig. 5b). Whereas USP8-p.Leu713Arg was mainly localized to the nucleus and had * only a very weak effect on EGFR endocy* tosis, USP8-p.Tyr717Cys was cytoplasmic * and effectively inhibited EGFR endocytosis, suggesting that the p.Tyr717Cys mutation is important in the pathogenesis of CD (Fig. 5b,c). The molecular basis for how USP8-p.[Leu713Arg;Tyr717Cys] causes cor* ticotroph adenoma remains to be elucidated. Consistent with the nuclear localization of endogenous USP8-p.Ser718Pro and USP8-p.[Leu713Arg;Tyr717Cys] in corticotroph adenomas (Fig. 2), Flag-tagged USP8-p.Ser718Pro and USP8-p.[Leu713Arg;Tyr717Cys], as well as USP8-p.Pro720Arg and -p.Ser718del at least partly, were found in the nucleus in HeLa cells (Fig. 5a and Supplementary Fig. 4). Because USP8-C40 was cytoplasmic (Fig. 5a and Supplementary Fig. 4), the nuclear localization of the USP8 mutants is probably not relevant to their effect on EGFR downregulation. We propose, however, that nuclear localization of USP8 may indicate the presence of USP8 mutations in corticotroph adenomas.

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Figure 5  Corticotroph adenoma USP8 mutants and USP8-C40 inhibit EGFR downregulation. (a,b) Immunostaining of HeLa cells transfected with the indicated Flag-tagged USP8 proteins (corticotroph adenoma–derived mutants in a and the two single mutants from the doublemutant protein in b) and treated with EGF for 1 h. The cells were triple-stained with anti-Flag antibody (magenta), anti-EGFR antibody (green) and DAPI (blue). Asterisks indicate cells not expressing Flag-USP8 proteins. Scale bars, 10 µm. (c) Proportion (%) of EGFR-positive area per cell in three cells in the confocal images from a and b, calculated by measuring the area of EGFR-positive region (green) in a cell and the area of the whole cell. The experiment was repeated 4 or more times, and results of a representative experiment are shown as mean ± s.d. Wild-type: P = 0.055 to mock (EGF−), p.Ser718Pro: P = 0.003 to mock (EGF+), p.Ser718Cys: P = 0.003 to mock (EGF+), p.Pro720Arg: P = 0.004 to mock (EGF+), p.[Leu713Arg;Tyr717Cys]: P = 0.208 to mock (EGF−), p.Ser718del: P = 0.007 to mock (EGF+), C40: P = 0.007 to mock (EGF+), C40CA: P = 0.014 to mock (EGF−), p.Leu713Arg: P = 0.001 to mock (EGF−), p.Tyr717Cys: P = 0.004 to mock (EGF+) (t-test, two-sided).

EGFR (green)

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living cells. When COS-7 cells were incubated with EGF for 5 min, robust ubiquitination of EGFR was observed (Fig. 6a, top). Although the ubiquitination level was only slightly reduced by overexpression of wild-type USP8, it was significantly reduced by overexpression of USP8-p.Ser718del or USP8-C40, suggesting that these are more active than wild-type USP8 in deubiquitinating EGFR in vivo (Fig. 6a, top). Probing the anti-EGFR immunoprecipitates with anti-Flag antibody showed that USP8-C40 constitutively binds to EGFR with higher affinity than wild-type USP8 or the p.Ser718del mutant, suggesting that EGFR is a bona fide substrate for USP8-C40 (Fig. 6a, third from top). The 90-kDa fragment was detected in cells expressing p.Ser718del but not wild-type USP8, supporting the idea of proteolytic activation of the mutant (Fig. 6a, bottom). Because the 90-kDa fragment was not co-precipitated with EGFR, USP8 probably binds to EGFR via the C40 region (Fig. 6a, third from top). 

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Figure 6  Corticotroph adenoma USP8 mutants and USP8-C40 promote EGFR recycling and sustain EGF signaling. (a) Immunoblotting with the indicated antibodies of EGFR immunoprecipitated from lysates of COS-7 cells transfected with wild-type, C40 or p.Ser718del mutant of USP8 and treated with (+) or without (−) EGF for 5 min. (b) Immunostaining of HeLa cells transfected with EGFP-Rab11A S25N fusion protein together with Flag-tagged USP8-C40 or C40CA and then treated with EGF for 1 h. The cells were stained with anti-EGFR (red) and anti-Flag (blue) antibodies. Arrowheads indicate co-localization of EGFR and Rab11A S25N on recycling endosomes. Asterisks indicate cells not expressing Flag-USP8 proteins. (c) Immunostaining of HeLa cells transfected with the indicated Flag-tagged USP8 proteins and treated with EGF for 2 h. The cells were double-stained with anti-Flag (magenta) and anti-phospho-Erk1/2 (green) antibodies. Relative cytoplasmic phospho-Erk1/2 fluorescence intensity per unit area, compared to that in surrounding untransfected cells (*1), is indicated on each cell. Scale bars, 10 µm.

Rab11A is a small GTPase that regulates recycling of cell surface proteins from early endosomes to the plasma membrane via the recycling endosome29. We transfected HeLa cells with Rab11AS25A, a GDP-fixed mutant of Rab11A that inhibits the recycling pathway, together with USP8-C40 or its inactive form C40CA, and treated them with EGF for 1 h. In mock- and C40CA-expressing cells, EGFR was normally endocytosed and did not accumulate in Rab11AS25A­positive recycling endosomes (Fig. 6b). In contrast, in cells expressing wild-type C40, we observed EGFR accumulation at the recycling endosome, suggesting that when deubiquitinated in excess by USP8C40, endocytosed EGFR takes the recycling pathway back to the plasma membrane (Fig. 6b, arrowheads). EGF stimulation leads to transient activation of the extracellular signal–regulated kinases Erk1 and Erk2 (ref. 30). The level of activated (Thr202- and Tyr204-phosphorylated) Erk1/2 was very low in the absence of EGF and, 30 min after EGF addition, increased to similar levels in untransfected HeLa cells and those ectopically expressing wild-type and the mutant USP8 proteins (Supplementary Figs. 6 and 7). After 2 h, however, the level of phosphorylated Erk1/2 was higher in cells expressing corticotroph adenoma USP8 mutants (except for USP8- p.[Leu713Arg;Tyr717Cys]) or C40 than in untransfected and wild-type USP8–expressing cells (Fig. 6c). Catalytically inactive C40CA did not produce such an effect (Fig. 6c), suggesting that inhibition of EGFR downregulation by corticotroph adenoma USP8 mutants and C40 leads to prolonged EGF signaling. USP8 mutants potentiate Pomc promoter activity In a final step, we examined the role of USP8 in ACTH synthesis in the mouse AtT-20 corticotroph adenoma cell line. Overexpression of human EGFR in AtT-20 cells, which do not express EGFR endogenously6, increased Pomc promoter activity, and this effect was enhanced by co-transfection with wild-type USP8 (P < 0.001 to EGFR, Fig. 7a). The catalytically inactive USP8 had no effect 

on EGFR-induced Pomc promoter activity. The cleaved USP8-C40 significantly increased Pomc promoter activity compared to wildtype USP8, an effect that did not occur with catalytically inactive C40CA (P < 0.001, Fig. 7a). In accordance with the results of the EGFR downregulation experiments in HeLa cells, overexpressing USP8-p.Ser718Pro, -p.Ser718Cys, -p.Pro720Arg, -p.Ser718del and -p.[Leu713Arg;Tyr717Cys] in AtT-20 cells significantly increased Pomc promoter activity compared to that seen with wild-type USP8 (P < 0.01, Fig. 7b). Dividing the double mutant USP8-p.[Leu713Arg;Tyr717Cys] into single mutant constructs revealed that the single p.Leu713Arg mutant had minimal effect, whereas p.Tyr717Cys effectively increased Pomc promoter activity (P = 0.003, Supplementary Fig. 8). In addition to its effect on the Pomc promoter, wild-type USP8 also caused increased basal and EGF-induced ACTH synthesis in EGFRoverexpressing AtT-20 cells (P < 0.01, Fig. 7c), whereas cells stably coexpressing USP8-C40 (P < 0.01, Fig. 7c) and USP8 mutants (P < 0.01, Fig. 7d) had increased ACTH secretion compared to those expressing wild-type USP8. Furthermore, both wild-type and mutant USP8s increased Pomc transcription in EGFR-overexpressing AtT-20 cells (Supplementary Fig. 9). Altogether, these data demonstrate that the USP8 mutants induce Pomc promoter activity and transcription as well as ACTH secretion. Erk1/2 plays an important role in mediating EGFR action on the Pomc promoter6. Erk1/2 kinase inhibition with PD098059 compromised, but did not completely abolish, the effects of wild-type USP8 and C40 on EGF-induced Pomc transcription and promoter activity (Supplementary Fig. 10a,b). Furthermore, PD098059 abolished the stronger stimulatory action of the USP8 mutants over wild-type USP8 on the Pomc promoter (Supplementary Fig. 10c). Overexpression of wild-type USP8, C40, and its mutants in EGFR-expressing AtT-20 cells increased phosphorylated Erk1/2 levels, but did not change Akt phosphorylation at Ser473, demonstrating that USP8 has a specific mode of action through the Erk1/2 signaling cascade (Supplementary aDVANCE ONLINE PUBLICATION  Nature Genetics

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Figure 7  USP8 mutants potentiate Pomc promoter activity and ACTH secretion in EGFR- overexpressing AtT-20 cells. (a) Pomc promoter activity in cells co-transfected with EGFR and USP8 wild-type, catalytically inactive (CA), cleaved C40 form or catalytically inactive C40 CA form. Data are luciferase/β-galactosidase ratio, means of three experiments with each transfection condition in triplicates and presented as percentage of empty vector control (pME-Flag + pRC/CMV; mock). RLA, relative luciferase activity. *P < 0.05 compared to mock (t-test and Mann-Whitney rank-sum test). Error bars, s.d. (b) The same experiments as in a in cells co-transfected with EGFR and USP8 mutants. Data are presented as percentage of wild-type USP8. *P < 0.01 compared to wild-type USP8 (t-test). Error bars, s.d. (c) ACTH secretion from stable transfectants of EGFR and wild-type USP8 or C40 treated with vehicle or 100 ng/ml EGF for 48 h. Data are ratios of ACTH (determined by radioimmunoassay; pg/ml) to cell viability values (obtained with the WST-1 colorimetric assay; OD450nm) and presented as percentage of EGFR. A representative experiment of 3 transfection experiments is shown as means of 3 measurements, with each transfection and treatment condition in triplicates. **P < 0.01 compared to each EGFR transfectant, #P < 0.05 to vehicle-treated EGFR (t-test). Error bars, s.d. (d) The same experiments as in c in stable transfectants of EGFR and USP8 mutants. Data are presented as percentage of EGFR + wild-type USP8. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to each EGFR + wild-type USP8, #P < 0.05 to vehicle-treated EGFR (t-test and Mann-Whitney rank-sum test). Error bars, s.d.

Fig. 11). We then searched for putative Erk1/2 targets that could mediate the effect of USP8. The Pomc promoter contains two nuclear receptor subfamily (Nur) binding sites (a Nur77-binding response element, NBRE, that binds Nur monomers and a Nur response element, NurRE, that binds its homo- or heterodimers) and an activating protein 1 (AP1) binding site31. Wild-type USP8 and its mutants had no significant effect on Nur transcriptional activities, but they increased AP1 transcriptional activity (P < 0.05), which is in line with the enhanced EGFR signal transduction observed in the presence of USP8 mutants (Supplementary Fig. 12). Finally, wild-type USP8 triggered basal and EGF-induced cell growth (Supplementary Fig. 13a). Although the USP8 mutants and C40 potentiated basal and EGF-induced ACTH secretion over EGFR wild-type USP8, they did not significantly promote additional cell growth in this assay (Supplementary Fig. 13). Ub Ub

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DISCUSSION The COSMIC database summarizes 85 USP8 mutations found in about 8,500 cancer genomes. Of these only three are located in the hotspot area between amino acid 713 and 720 that we describe; these were detected in a colon cancer, a urothelial carcinoma and an unclassified tumor. These data indicate that USP8 mutations are rarely found in cancers (1%) and highlight their specificity for corticotroph adenoma. In summary, our data show that one-third of corticotroph adenomas in CD harbor USP8 mutations. The mutations impair binding of 14-3-3 proteins, thus increasing proteolytic cleavage of USP8. The resulting C-terminal 40-kDa USP8 fragment has high DUB activ-

ity, increasing deubiquitination of its cellular substrate EGFR. This leads to increased recycling of endocytosed EGFR and substantial accumulation of EGFR on the plasma membrane. High EGFR levels, in turn, stimulate Pomc gene transcription and increase plasma ACTH levels. As such, mutant USP8 acts concomitantly via the EGFR on corticotroph tumorigenesis and enhanced ACTH secretion (Fig. 8). In contrast, the proliferative action of the mutant USP8 did not ­significantly exceed that of wild-type USP8, which possibly reflects the usually small size and low proliferative index of corticotroph adenomas. Upregulation and nuclear localization of mutant USP8 may also be responsible for the pathogenesis through as-yet-unidentified

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© 2014 Nature America, Inc. All rights reserved.

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Figure 8  Schematic representation showing the proposed mechanisms how USP8 mutations lead to increased ACTH secretion and tumorigenesis in corticotroph.

Nature Genetics  ADVANCE ONLINE PUBLICATION



Articles mechanisms. These results not only identify the first of the so far enigmatic driver mutations in corticotroph adenomas, but also elucidate a novel mechanism by which the EGFR pathway is constitutively activated in human tumors. However, because mutations in the 14-3-3 binding motif of USP8 are rarely found in other tumors, we cannot exclude the possibility that corticotroph-specific USP8 substrate(s) other than EGFR are responsible for the pathogenesis of CD. URLs. COSMIC database, http://cancer.sanger.ac.uk/cancergenome/ projects/cosmic/.

© 2014 Nature America, Inc. All rights reserved.

Methods Methods and any associated references are available in the online version of the paper. Accession codes. Disease-causing variants are provided in the supplementary information and will be submitted to ClinVar (www. ncbi.nlm.nih.gov/clinvar/) with accession codes SCV000192007SCV000192011. Furthermore, exome data are available on request within a scientific cooperation. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank Minoru Fukuda and Mitsunori Fukuda for the LAMP2 antibody and EGFP-Rab11AS25A expression vector, respectively. The study was supported in by the Else Kröner-Fresenius-Stiftung (grant 2012_A103 to M.R.), Bundesministerium für Bildung und Forschung (grant BMBF 01EO1004-D2 to M.F. and B.A.), the Wilhelm Sander-Stiftung (grant 2012.095.1 to B.A.), and Grants-in-aid from the Ministry of Education, Culture, Science and Technology of Japan (grant 24112003 to M.K. and 24112008 to Y.S.). W.S. is supported by funds from Novartis AG, Pfizer and NovoNordisk and Ipsen for the Hypophysenregister der Arbeitsgemeinschaft Hypophyse of the German Society of Endocrinology. M.T. is supported by a grant from the German Federal Ministry of Education and Research (01EX1021B, Spitzencluster M4, Verbund Personalisierte Medizin, Teilprojekt NeoExNET (PM1)). We thank B. Mauracher, P. Rank, J. Stalla and J.L. Monteserin-Garcia for excellent technical assistance. AUTHOR CONTRIBUTIONS M.R., M.F. and M.K. planned the study, conceived and designed the experiments, analyzed the data and wrote the paper. S.S., A.H., M.T., F.B., T.M., E.M.-Y., K.K., T.W., E.G. W.S., B.A. and T.M.S. designed and performed experiments and analyzed data. A.O., Y. S., K.T. and C.L.R. performed experiments. M.B. provided tumor tissue and clinical data. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Cushing, H. The basophil adenomas of the pituitary body and their clinical manifestations. Bull. Johns Hopkins Hosp. 50, 127–195 (1932). 2. Melmed, S. Pathogenesis of pituitary tumors. Nat. Rev. Endocrinol. 7, 257–266 (2011). 3. Stratakis, C.A. et al. The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes. Clin. Genet. 78, 457–463 (2010).



4. Dworakowska, D. & Grossman, A.B. The molecular pathogenesis of corticotroph tumours. Eur. J. Clin. Invest. 42, 665–676 (2012). 5. Theodoropoulou, M. et al. Expression of epidermal growth factor receptor in neoplastic pituitary cells: evidence for a role in corticotropinoma cells. J. Endocrinol. 183, 385–394 (2004). 6. Fukuoka, H. et al. EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas. J. Clin. Invest. 121, 4712–4721 (2011). 7. Beuschlein, F. et al. Recurrent somatic mutations underlie corticotropin-independent Cushing’s syndrome. N. Engl. J. Med. 370, 1019–1028 (2014). 8. Cao, Y. et al. Activating hotspot L205R mutation in PRKACA and adrenal Cushing’s syndrome. Science 344, 913–917 (2014). 9. Goh, G. et al. Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors. Nat. Genet. 46, 613–617 (2014). 10. Sato, Y. et al. Recurrent somatic mutations underlie corticotropin-independent Cushing’s syndrome. Science 344, 917–920 (2014). 11. Nomura, N. et al. Prediction of the coding sequences of unidentified human genes. II. The coding sequences of 40 new genes (KIAA0041-KIAA0080) deduced by analysis of cDNA clones from human cell line KG-1. DNA Res. 1, 223–229 (1994). 12. Ciechanover, A. The ubiquitin-proteasome proteolytic pathway. Cell 79, 13–21 (1994). 13. Kulathu, Y. & Komander, D. Atypical ubiquitylation—the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523 (2012). 14. Clague, M.J., Liu, H. & Urbé, S. Governance of endocytic trafficking and signaling by reversible ubiquitylation. Dev. Cell 23, 457–467 (2012). 15. Haglund, K. & Dikic, I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J. Cell Sci. 125, 265–275 (2012). 16. Tanno, H. & Komada, M. The ubiquitin code and its decoding machinery in the endocytic pathway. J. Biochem. 153, 497–504 (2013). 17. Reyes-Turcu, F.E., Ventii, K.H. & Wilkinson, K.D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu. Rev. Biochem. 78, 363–397 (2009). 18. Komander, D., Clague, M.J. & Urbé, S. Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550–563 (2009). 19. Mizuno, E. et al. Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes. Mol. Biol. Cell 16, 5163–5174 (2005). 20. Bowers, K. et al. Degradation of endocytosed epidermal growth factor and virally ubiquitinated major histocompatibility complex class I is independent of mammalian ESCRTII. J. Biol. Chem. 281, 5094–5105 (2006). 21. Mizuno, E., Kobayashi, K., Yamamoto, A., Kitamura, N. & Komada, M. A deubiquitinating enzyme UBPY regulates the level of protein ubiquitination on endosomes. Traffic 7, 1017–1031 (2006). 22. Row, P.E., Prior, I.A., McCullough, J., Clague, M.J. & Urbé, S. The ubiquitin isopeptidase UBPY regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation. J. Biol. Chem. 281, 12618–12624 (2006). 23. Alwan, H.A. & van Leeuwen, J.E. UBPY-mediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation. J. Biol. Chem. 282, 1658–1669 (2007). 24. Niendorf, S. et al. Essential role of ubiquitin-specific protease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo. Mol. Cell. Biol. 27, 5029–5039 (2007). 25. Mukai, A. et al. Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt. EMBO J. 29, 2114–2125 (2010). 26. Mizuno, E., Kitamura, N. & Komada, M. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase. Exp. Cell Res. 313, 3624–3634 (2007). 27. Huang, F., Kirkpatrick, D., Jiang, X., Gygi, S. & Sorkin, A. Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol. Cell 21, 737–748 (2006). 28. Huang, T.T. et al. Regulation of monoubiquitinated PCNA by DUB autocleavage. Nat. Cell Biol. 8, 339–347 (2006). 29. Welz, T., Wellbourne-Wood, J. & Kerkhoff, E. Orchestration of cell surface proteins by Rab11. Trends Cell Biol. 24, 407–415 (2014). 30. Roberts, P.J. & Der, C.J. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 26, 3291–3310 (2007). 31. Philips, A. et al. Novel dimeric Nur77 signaling mechanism in endocrine and lymphoid cells. Mol. Cell. Biol. 17, 5946–5951 (1997).

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ONLINE METHODS

Patient cohort. Patients with pituitary tumors were recruited through the Department of Neurosurgery, University Hospital Erlangen, and the Department of Medicine IV, University of Munich. 17 corticotroph adenomas, 2 Nelson tumors, 10 prolactinomas, 14 growth hormone–secreting adenomas and 10 nonfunctional adenomas were studied (Supplementary Table 1). In all cases, the diagnosis was confirmed histologically after surgical resection. The patients gave written informed consent, and the study was approved by the ethics committee of each individual institution. Diagnosis of ACTH-dependent Cushing’s syndrome was based on the combination of typical clinical signs and symptoms of hypercortisolism (recent weight gain, truncal obesity, moon face, buffalo hump, muscle weakness, easy bruising, striae distensae, acne, low-impact fractures, mood changes, irregular menstruation, infertility, impotency) and biochemical hallmarks of hypercortisolism, i.e. elevated urinary excretion of free cortisol, increased late-night salivary or serum cortisol, and non-suppressible serum cortisol after 1 mg dexamethasone (>1.8 µg/dl; >50 nmol/l). In addition, the patients had inadequately high or elevated plasma ACTH levels. Before surgery, the patients underwent various tests and procedures to confirm pituitary dependence, such as ACTH response to human CRH, cortisol response to high-dose dexamethasone (8 mg), MRI of the pituitary, and sinus petrosus inferior catheterization. All patients had expert histologic confirmation of corticotroph adenomas by one of us (W.S.). Nucleic acid extraction. DNA was extracted from a total of 53 adenomas with 19 paired peripheral DNA samples as described previously7. Exome and USP8 sequencing. Exomes were enriched in solution and indexed with SureSelect XT Human All Exon 50Mb kits (Version 4, Agilent Technologies). Sequencing was performed as 100-bp paired-end runs on HiSeq2000 systems (Illumina). Pools of 12 indexed libraries were sequenced on four lanes. Image analysis and base calling were performed using Illumina Real Time Analysis. Variant detection and USP8 sequencing is described in the Appendix. DNA was amplified using intron-spanning primers as provided in Supplementary Table 5. PCR was performed on 100 ng of DNA (4 µl of RT product) in a final volume of 25 µl containing 0.75 mM MgCl2 (without MgCl2 for cDNA), 400 nM of each primer, 200 µM deoxynucleotide triphosphate and 1.25 U Platinum Taq DNA Polymerase (Invitrogen). Cycling conditions were with an annealing temperature of 58 °C (56 °C for cDNA). Direct sequencing of PCR products was performed using the ABI Prism Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI Prism 3700 DNA Analyzer (Applied Biosystems). Sequencing primers were used as given in Supplementary Table 5. Functional regions of USP8 and sequence alignment. Sequence similarity analysis was performed using the ClustalW2 program (http://www.ebi.ac.uk/ Tools/msa/clustalw2/). Thereby, human USP8 (NCBI GenBank ID: 9101) was compared with sequences from other species and with those of other human USP8, respectively. Representation of human USP8 functional regions is based on information from the genome database ENSEMBL for variant USP-001 (ENSP00000379721). Sequence alignments for the different species and different mutations were performed using CLC Sequence Viewer 6 for Mac (CLC bio A/S) based on sequences retrieved from the National Center for Biotechnology Information (NCBI) genomic information repository. cDNA expression plasmids. The cDNA for human USP8 was obtained from Life Technologies and inserted into the N-terminally Flag-tagged mammalian expression vector pME-Flag. The cDNA for C-terminally Flag-tagged USP8 was amplified by PCR using full-length USP8 cDNA as a template and inserted into the mammalian expression vector pME. Introduction of point mutations into USP8 cDNA was performed using the QuikChange site-directed mutagenesis system (Stratagene). The cDNA for USP8-C40 was amplified from full-length USP8 cDNA by PCR and inserted into pME-Flag. The expression vectors for GST and 14-3-3ε fusion protein26 and EGFR (pRC/CMV-hEGFR32) were constructed as described previously. The EGFP-Rab11AS25A expression vector33 was provided by Mitsunori Fukuda (Tohoku University, Sendai, Japan). Luciferase reporter plasmids. The Pomc-luc construct has 770 bp of the rat Pomc promoter with all the necessary elements for the pituitary-specific

doi:10.1038/ng.3166

expression of Pomc upstream to the reporter gene luciferase34. The NurREluc has 3 copies of 28 bp of the Nur response element (NurRE) that binds Nur77 homodimers or Nur77/Nurr1 heterodimers inserted upstream to the minimal Pomc promoter (−34/+63), while the NBRE-luc contains 3 copies of Nur77-binding response element (NBRE) that binds Nur77 or Nurr1 monomers31. The AP1-luc has 7 repeats of the AP1-responsive sequence upstream to luciferase (Stratagene). Cell culture and DNA transfection. HeLa and COS-7 cells provided by R. Masaki (Kansai Medical University, Moriguchi, Japan) and N. Kitamura (Tokyo Institute of Technology, Yokohama, Japan), respectively, were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 100 units/ml penicillin and 0.1 mg/ml streptomycin at 37 °C and 5% CO 2. To stimulate cells with EGF, they were grown in the presence of 0.5% FCS for 24 h and subsequently incubated with human EGF (100 ng/ml; PeproTech). DNA was transfected into the cells using FuGENE6 (Promega) for 48 h or jetPRIME (Polyplus-transfection) for 24 h. Mouse AtT-20/D16vF2 corticotrophinoma cells (ATCC CCL-89) were obtained and authenticized from the American Type Culture Collection. Cells did not exceed passage 14 and were cultured in DMEM supplemented with 10% FCS, 2 nmol/l glutamine and 105 IU/l penicillin-streptomycin at 37 °C and 5% CO2. Cell culture materials were from Life Technologies, Nunc and Sigma-Aldrich. Cells were plated and, 24 h later, transfected with pME-Flag-USP8 vectors and pRC/CMV-hEGFR using SuperFect (Qiagen) according to the manufacturer’s instructions. For experiments with reporter plasmids, cells were assayed 24 h after transfection. The transfection efficacy was determined by cotransfection with the RSV-β-gal construct, and results are presented as luciferase:β-galactosidase activity ratio. In all transfection experiments, the empty vectors pME-Flag and pRC/ CMV were used as negative control, and when indicated, results are expressed as percentage of empty plasmid control. For stable transfections, AtT-20 cells were transfected with pRC/CMV-hEGFR plus pME-Flag or pME-Flag-USP8 vectors and selected using 500 µg/ml neomycin. Selected cells were serum deprived for 20 h and treated with vehicle or 100 ng/ml EGF for 48 h. The supernatant was collected for ACTH determination, and cell viability was determined using the WST-1 colorimetric assay (Roche Molecular Biochemicals)35. ACTH determination. ACTH was determined by a radioimmunoassay as previously described36. ACTH values were normalized with cell viability values assessed with the non-radioactive colorimetric WST-1 assay at 450nm (Roche Molecular Biochemicals) and are presented as (pg/ml)/OD450nm. Quantitative RT-PCR. Total RNA was extracted by the TRIzol (Invitrogen) method according to the manufacturer’s instructions. 1 µg RNA was reverse transcribed using QuantiTect reverse transcription kit (Qiagen). qPCR was performed using QuantiFast SYBR Green PCR kit (Qiagen) and reactions were run on LightCycler (Roche). Primers were against murine Pomc (5′-gaagatgcc gagattctgct-3′ and 5′-tttcagtcaggggctgttc-3′) and Tfiib (5′-tggagatttgtccac catga-3′ and 5′-gaattgccaaactcatcaaaact-3′). Pomc expression was normalized to TFIIB transcript levels. Immunoprecipitation and immunoblotting. HeLa and Cos-7 cell lysates were prepared by solubilizing cells in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM NaF, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin and 1 µg/ml pepstatin A, and collecting the supernatants after centrifugation. Immunoprecipitation and immunoblotting of the lysates were performed using standard procedures. Anti-Flag (1 µg; clone M2, SigmaAldrich) and anti-EGFR (1 µg; clone 6F1, MBL) antibodies were used for immunoprecipitation. Primary antibodies for immunoblotting were: anti-USP8 (1:200; ref. 37), 14-3-3β and other 14-3-3 isoforms (2 µg/ml; clone H-8, Santa Cruz Biotechnology), anti-Ub (1 µg/ml; clone FK2, MBL), anti-Ub (5 µg/ml; clone P4G7, Covance), anti-EGFR (0.5 µg/ml), anti-Flag (4 µg/ml), and antiα-tubulin (0.02 µg/ml; #ab15246, Abcam) antibodies. Secondary antibodies were peroxidase-­conjugated anti-mouse IgG and anti-rabbit IgG antibodies (GE Healthcare). Blots were detected using ECL Western Blotting Detection Reagents (GE Healthcare) and ImageQuant LAS 4000mini (GE Healthcare). AtT-20 cell lysates were prepared in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Roche). Samples were separated

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by PAGE and blotted using standard procedures. Primary antibodies were against phospho-Erk1/2 (Thr202/Tyr204) (1:2,000; #9101), Erk1/2 (1:4,000; #9102), phospho-Akt-Ser473 (1:500; #9271) and Akt (1:2,000; #9272, all made in rabbit, Cell Signaling Technology). An anti-rabbit horseradish peroxidase–conjugated secondary antibody was used (1:2,000; #7074, Cell Signaling). Enhanced chemiluminescent solution was purchased from Roche. Each experiment was performed in duplicates.

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GST pull-down assay. The GST and 14-3-3ε fusion protein was purified from transformed Escherichia coli cells using glutathione-Sepharose beads (GE Healthcare). The fusion protein (4 µg) was immobilized on glutathioneSepharose beads (10 µl) and incubated with cell lysates in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM NaF, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin and 1 µg/ml pepstatin A for 16 h at 4 °C. The beads were washed with the same buffer, and bound proteins were detected by immunoblotting. DUB assay. Flag-tagged USP8 proteins were expressed in COS-7 cells, immunoprecipitated from their lysates using agarose beads conjugated with anti-Flag antibody (anti-FLAG M2 affinity gel, Sigma-Aldrich), and eluted from the beads by incubation with 100 µl of PBS containing the Flag peptide (150 µg/ml, Sigma-Aldrich). Purity and concentration of eluted USP8 proteins were assessed by Coomassie staining using purified bovine serum albumin as a standard. Immunopurified USP8 proteins (~50 ng; ~20 nM) were incubated in 20–30 µl of PBS containing 5 mM MgCl2 and 2 mM dithiothreitol for 1 h at 37 °C with 0.5 µg (~700 nM) of Lys63-linked Ub oligomers (dimerheptamer, Boston Biochem) or ubiquitinated EGFR immunoprecipitated from EGF-treated HeLa cells. Reaction products were separated by SDS-PAGE and detected by immunoblotting. LC-MS/MS analysis. Protein bands were excised from a Coomassie-stained gel, diced into 1-mm3 pieces and destained with 50 mM ammonium bicarbonate (AMBC)/50% acetonitrile (ACN) and with 50 mM AMBC/30% ACN for 1 h each. After complete dehydration with 100% ACN wash, ~5 µl of 10 ng/µl trypsin (Trypsin Gold, Promega) in 50 mM AMBC was added to the gel pieces and incubated for 16 h at 37 °C. Digested peptides were extracted by 50 µl of 50% ACN/0.1% trifluoroacetic acid (TFA) for 1 h shaking. The peptides were recovered into fresh Protein Lobind tubes (Eppendorf) and an additional extraction was performed with 70% ACN/0.1% TFA for 30 min. The extracted peptides were concentrated to 10 µl by Speed-vac and were prepared in 0.1% TFA. Shotgun analysis was carried out using a nano liquid chromatograph (Easy nLC 1000, Thermo Fisher Scientific) coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific). Reversed phase chromatography was performed using the EASY-nLC 1000 with a binary buffer system consisting of 0.1% formic acid (FA) in water (solvent A) and 100% ACN/0.1% FA (solvent B) with a flow rate of 300 nl/min. The peptides were directly loaded on a reverse-phase column (75 µm inner diameter × 120 mm length, 3 µm C18 Reprosil-Pur, Nikkyo Technos) and separated using a 140 min 2-step gradient (0–40% for 120 min and 40–100% for 20 min of solvent B). The Q Exactive was operated in the data dependent MS/MS mode, using Xcalibur software, with survey scans acquired at a resolution of 70,000 at m/z 200. Up to the top 10 most abundant isotope patterns with charge 2~4 from the survey scans were selected with an isolation window of 2.0 m/z and fragmented by HCD with normalized collision energies of 28. The maximum ion injection times for the survey scan and the MS/MS scans were 60 ms, respectively, and the ion target values were set to 3e6 and 1e5, respectively. Selected sequenced ions were dynamically excluded for 10 s. To minimize carryover, we subjected 50 fmol of BSA digests (KYA tech) and 0.1% TFA between each LC-MS/MS analysis. Raw files were searched by Protein Discoverer software version 1.3 (Thermo Fisher Scientific) using SEQUEST search engine against a modified SwissProt database, in which the sequences of the USP8 mutants were added to UniProtKB/Swiss-Prot protein database (version 2012_10). The precursor and fragment mass tolerances were set to 10 ppm and 20 mmu, respectively. Methionine oxidation, protein amino-terminal acetylation, serine/threonine/ tyrosine phosphorylation and diglycine modification of lysine side chains were set as variable modifications for database searching. Peptide identification was filtered at 1% false discovery rate. To determine USP8 cleavage site, we

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compared the peak areas of identified peptides calculated by the precursor ion quantification node in Protein Discoverer software (Supplementary Table 5). Immunofluorescence detection. For immunofluorescence staining of corticotroph adenoma tissue sections, formalin-fixed and paraffin-embedded (FFPE) tissues were deparafinized twice in 100% xylol for 10 min and subsequently rehydrated in ethanol solutions of decreasing concentration for 5 min each. Antigen retrieval was performed for 13 min in a pressure cooker in citrate buffer, pH 6.5. Nonspecific binding was blocked for 1 h with 20% human AB serum in PBS. The slides were then incubated for 1 h at room temperature with a mixture of polyclonal rabbit anti-human USP8 (1:15, HPA004869, Sigma-Aldrich) and monoclonal mouse anti-human ACTH (1:50, M3501, Dako) in 20% human AB serum in PBS. As negative control served the Dako Universal mouse and rabbit negative control (Dako). Staining was developed in the dark with a mixture of 1:1000 donkey anti-rabbit–Alexa Fluor 594 and donkey anti-mouse–Alexa Fluor 488 (both from Life Technologies). Mounting and nuclei staining were performed with Vectashield-DAPI (Vector Laboratories). Visualization and acquisition were performed with a Leica microscope. Staining intensity was evaluated with a grading score of 0, 1, 2, or 3, which corresponded to negative, weak, moderate, or strong staining intensity. Testis tissue was used as positive control and normal human pituitary tissue from autopsies (n = 3) as internal control. The percentage of positive tumor cells was calculated for each specimen and scored 0 if 0% were positive, 0.1 if 1–9%, 0.5 if 10–49%, and 1 if ≥50%. A semiquantitative H-score was then calculated by multiplying the staining intensity grading score with the proportion score as previously described38. For immunofluorescence staining of HeLa cells, cells were fixed with 4% paraformaldehyde in PBS (anti-EGFR and anti-Hrs) or with 100% methanol (anti-phospho-Erk1/2 and anti-LAMP2) for 10 min on ice, permeabilized in 0.2% Triton X-100 in PBS, and blocked in 5% FCS in PBS. Cells were then incubated with rabbit anti-Flag (1 µg/ml; #F7425, Sigma-Aldrich), anti-EGFR (5 µg/ml), anti-phospho-p44/42 Erk1/2 (Thr202/Tyr204) (1:150; #clone E10, Cell Signaling), anti-Hrs39 (1:1,000), and anti-LAMP2 (1:2,000, provided by Minoru Fukuda, The Burnham Institute, La Jolla, CA, USA) antibodies. Secondary antibodies were Alexa Fluor 488–, 594–, and 633–conjugated antimouse IgG and anti-rabbit IgG antibodies (1:1,000; Invitrogen). Nuclei were stained with DAPI (1 µg/ml; Nacalai Tesque) during incubation with secondary antibodies. Fluorescence images were captured with a laser-scanning confocal microscope (LSM 780, Carl Zeiss). For quantitative measurement, the fluorescence intensity, the area of fluorescent regions in cells, and the area of whole cells in confocal images were measured using ImageJ (National Institutes of Health, Bethesda, MD, USA). When measuring the area of fluorescent regions, images were subjected to the threshold function using the same threshold for all images. Statistical analysis. If not stated otherwise, group results are expressed as mean ± s.d. Data between groups were compared using Fisher’s exact test, Student’s t-test and the Mann-Whitney U-test, as appropriate. The significance level of P < 0.05 was considered to be statistically significant.

32. Morino, C. et al. A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor. Exp. Cell Res. 297, 380–391 (2004). 33. Ishida, M., Ohbayashi, N., Maruta, Y., Ebata, Y. & Fukuda, M. Functional involvement of Rab1A in microtubule-dependent anterograde melanosome transport in melanocytes. J. Cell Sci. 125, 5177–5187 (2012). 34. Liu, B., Hammer, G.D., Rubinstein, M., Mortrud, M. & Low, M.J. Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice. Mol. Cell. Biol. 12, 3978–3990 (1992). 35. Páez-Pereda, M. et al. Retinoic acid prevents experimental Cushing syndrome. J. Clin. Invest. 108, 1123–1131 (2001). 36. Stalla, G.K. et al. Ketoconazole inhibits corticotropic cell function in vitro. Endocrinology 122, 618–623 (1988). 37. Kato, M., Miyazawa, K. & Kitamura, N. A deubiquitinating enzyme UBPY interacts with the Src homology 3 domain of Hrs-binding protein via a novel binding motif PX(V/I)(D/N)RXXKP. J. Biol. Chem. 275, 37481–37487 (2000). 38. Olaussen, K.A. et al. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N. Engl. J. Med. 355, 983–991 (2006). 39. Komada, M., Masaki, R., Yamamoto, A. & Kitamura, N. Hrs, a tyrosine kinase substrate with a conserved double zinc finger domain, is localized to the cytoplasmic surface of early endosomes. J. Biol. Chem. 272, 20538–20544 (1997).

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