Regulated ADAM17-Dependent EGF Family Ligand Release by Substrate-Selecting Signaling Pathways

Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways

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Citation Dang, M., N. Armbruster, M. A. Miller, E. Cermeno, M. Hartmann, G. W. Bell, D. E. Root, D. A. Lauffenburger, H. F. Lodish, and A. Herrlich. “Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.” Proceedings of the National Academy of Sciences 110, no. 24 (June 11, 2013): 9776-9781. © 2013 National Academy of Sciences.

As Published http://dx.doi.org/10.1073/pnas.1307478110

Publisher National Academy of Sciences (U.S.)

Version Final published version

Citable link http://hdl.handle.net/1721.1/84949

Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways

Michelle Danga,b,c,1, Nicole Armbrusterc,1, Miles A. Millerd, Efrain Cermenoa,d, Monika Hartmanne, George W. Bella, David E. Rootf, Douglas A. Lauffenburgerd, Harvey F. Lodisha,b,d,2, and Andreas Herrlicha,c,2 aWhitehead Institute for Biomedical Research, Cambridge, MA 02142; bDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; cRenal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; dDepartment of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; eLeibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany; and fBroad Institute, Cambridge, MA 02142

Contributed by Harvey F. Lodish, April 28, 2013 (sent for review December 6, 2012)

Ectodomain cleavage of cell-surface proteins by A disintegrin and on the ADAM17 ectodomain (14, 15) or its C terminus (16, 17). metalloproteinases (ADAMs) is highly regulated, and its dysregula- However, modulation of activity of the relatively few available tion has been linked to many diseases. ADAM10 and ADAM17 ADAMs does not suffice to explain substrate-specific regulation cleave most disease-relevant substrates. Broad-spectrum metallo- of cleavage (18, 19), and none of the referenced studies has protease inhibitors have failed clinically, and targeting the cleavage addressed how specificity of cleavage is achieved. Transgenic of a specific substrate has remained impossible. It is therefore nec- overexpression of ADAM17 in mice does not lead to overactivity essary to identify signaling intermediates that determine substrate of ADAM17 or increased ADAM17 substrate release, emphasiz- specificity of cleavage. We show here that phorbol ester or angio- ing the importance of posttranslational control of cleavage (20). tensin II-induced proteolytic release of EGF family members may Most reports on induced shedding (reviewed in refs. 5, 21) have not require a significant increase in ADAM17 protease activity. only used monitoring of substrate cleavage as a surrogate measure Rather, inducers activate a signaling pathway using PKC-α and of protease activity. However, only few studies unequivocally the PKC-regulated protein phosphatase 1 inhibitor 14D that is re- document induced changes of protease activity, and those were quired for ADAM17 cleavage of TGF-α, heparin-binding EGF, and small. A tight-binding ADAM17 inhibitor interacts with the cat- amphiregulin. A second pathway involving PKC-δ is required for alytic site of ADAM17 only after 12-O-tetradecanoylphorbol-13- neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation acetate (TPA; i.e., phorbol ester) stimulation (12), suggesting of serine 286 in the NRG cytosolic domain is essential for induced regulation of the catalytic site. Another convincing example of NRG cleavage. Thus, signaling-mediated substrate selection is regulated enzyme activity has been based on observed effects of clearly distinct from regulation of enzyme activity, an important oxidation on several putative disulphide bonds in the ADAM17 mechanism that offers itself for application in disease. ectodomain that result in a structural change. This involves the interaction with an extracellular redox regulator, protein disulfide epidermal growth factor receptor | transactivation isomerase (PDI). PDI down-regulation enhanced TPA-induced shedding of heparin-binding (HB) EGF, addition of exogenous PDI decreased it, and PDI addition to recombinant ADAM17 he ectodomains of many cell surface proteins are shed from the fl Tsurface (i.e., “ectodomain shedding”) by metalloproteases. reduced basal cleavage of a uorescence resonance energy trans- Ectodomain shedding generates many diverse bioactive cytokines fer (FRET) peptide. These changes correlated with altered to- and growth factors, and governs important cellular processes in the pology of antibody epitopes outside of, but not within, the developing and adult organism, including the control of growth, catalytic domain (14). However, induced HB-EGF cleavage could adhesion, and motility of cells (reviewed in refs. 1–3). EGF re- have also resulted from enhanced interaction of the substrate with ceptor activation generates signals for cell proliferation, migra- ADAM17 via the altered topology outside of the catalytic domain tion, differentiation, or survival. The 12 EGF family members are without requiring changes in protease activity. Neither study de- synthesized as cell surface transmembrane precursors. The active termined protease activity independent of substrate cleavage, still growth factors are released by A disintegrin and metalloprotein- leaving us with uncertainty whether induced substrate cleavage ases (ADAMs) and activate specific heterodimeric EGF receptors truly requires enhanced protease activity. By using stopped-flow on the cell surface connected to diverse intracellular signaling X-ray spectroscopy and other techniques, Solomon et al. showed pathways (4, 5). Increased shedding of EGF ligands has been linked that ADAM17 activity is primed by enzyme conformational changes to different clinical pathologic processes (6–10); hence, therapeutic induced by the substrate before proteolysis (22). Novel exosite control of ligand release would be beneficial. Of the 12 functional inhibitors of ADAM17 activity that bind ADAM17 outside of the ADAMs encoded in the human genome (3) only two—ADAM10 catalytic site and likely interfere with the binding of glycosylated and ADAM17—handle most of the numerous ADAM substrates, moieties of the substrate have been developed (23). Both studies in particular, the EGF ligands. However, broad-spectrum metal- further support regulation of proteolysis on the substrate level. loprotease inhibitors tested for clinical use have failed as a result of Here we identify pathway components that distinguish sub- indiscriminate blockade of substrate cleavage, leading to clinical strates of ADAM17 and parse substrate selection from regula- side effects (11). Even recently developed selective ADAM inhib- tion of protease activity. itors still affect the cleavage of many substrates (12). Modulation of the release of specific ADAM substrates has been impossible to fi date because it is unknown how cleavage speci city is regulated on Author contributions: H.F.L. and A.H. designed research; M.D., N.A., M.A.M., E.C., and A.H. the molecular level. It is therefore necessary to identify key signals performed research; M.D., N.A., M.A.M., E.C., M.H., D.E.R., D.A.L., and A.H. contributed new that determine substrate specificity of cleavage. reagents/analytic tools; M.D., N.A., M.A.M., G.W.B., D.A.L., H.F.L., and A.H. analyzed data; Ectodomain cleavage is made specific by a number of in- and H.F.L. and A.H. wrote the paper. tracellular signals; e.g., by calcium influx, by activation of G pro- The authors declare no conflict of interest. tein-coupled receptors, and the release of diacylglycerol (reviewed 1M.D. and N.A. contributed equally to this work. in refs. 3, 13). Several distinct mechanisms that modulate cleavage 2To whom correspondence may be addressed. E-mail: [email protected] or lodish@ on the level of ADAM17 have been described, including regula- wi.mit.edu. fi tion of ADAM17 expression, maturation, traf cking to the cell This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. surface (reviewed in ref. 13), and posttranslational modifications 1073/pnas.1307478110/-/DCSupplemental.

9776–9781 | PNAS | June 11, 2013 | vol. 110 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1307478110 Results A FACS sh- sh1- sh1- B PKCα and PPP1R14D - KD

shRNA Screen for Regulators of TGF-α Cleavage by ADAM17. Phorbol Co PKCα PPP1R14D > ester (i.e., TPA) stimulates most PKC isoforms (-α,-β,-γ,-δ,-e,

η θ μ sh-Co sh1-PKCα - ,- and - ), and is a commonly used cleavage stimulus in Control shedding studies. ADAM17 is the physiological effector of TPA- 75kD -- α-PKCα induced signals, whereas ADAM10 primarily responds to cal- 37kD -- α-ERK cium signals (24, 25). To identify novel genes that regulate μ TPA 1 M IP: α-PPP1R14D (#1) shedding downstream of PKC, we carried out a lentiviral shRNA (2min) gene knockdown screen targeting most human kinases and phosphatases and some of their associated components, probing their APC Fluorescence = Red > α GFP Fluorescence = Green sh-Co sh1-PPP1R14D effect on TPA-induced cleavage of TGF- , a classical ADAM17 25kD -- sh-Co sh1-PKC sh1-PPP1R14D α-PPP1R14D (#2) substrate (24). Cleavage was measured with an extensively vali- Control X-Geometric Mean (Green) 42.5 42.2 34.9 20kD -- Y-Geometric Mean (Red) 21.0 13.1 21.8 37kD37kD -- -- α-ERK dated high-throughput 96-well FACS assay (18, 19) (Fig. S1A). Red:Green Ratio 0.494 0.310 0.626 TPA X-Geometric Mean (Green) 41.5 40.6 37.7 We screened 3,500 unique lentiviral shRNAs carrying puromycin 1μM Y-Geometric Mean (Red) 5.8 9.0 17.2 resistance for selection at >3× coverage with biological duplicates (2min) Red:Green Ratio 0.140 0.222 0.456 in human Jurkat cells expressing HA–TGF-α–EGFP. Genes were Red:Green Ratio % Control Cleavage 71.7 28.2 27.0 ﬁ targeted with three to ve individual shRNAs per gene and se- C TGFα cleavage - FACS D TGFα cleavage - whole cell lysate lected with puromycin. A shRNA targeting lacZ, a protein not e

ag sh- sh1- sh1- o v

ti Co PKCα PPP1R14D present in mammalian cells (control-shRNA) was used as a con- ea α Full length TGFα-GFP

l sh1-PKC Ra c

l sh1-PPP1R14D 37kD -- α-GFP trol. After stimulation with TPA for 2 to 5 min, mean geometric o ﬂ r C-term. TGFα-GFP red and green uorescence of the cells was measured by FACS Green 37kD -- sh-Co α-ERK z ed: and normalized across samples by using -scores. Cleavage was R 0155 01550155 min

%of cont %of 0 51530 TPA (1μM) induced to approximately 50% of maximum to allow detection of TPA (1μM) min cleavage activation or inhibition in the same screen. Assuming that most tested shRNAs would not affect cleavage, we selected tar- Fig. 1. shRNA screen targeting human phosphatases and kinases identi- geted genes with two or more shRNAs that reproducibly scored at fies PKC-α and PPP1R14D as regulators of TPA-induced TGF-α cleavage. (A) least 2 z-scores (for best shRNA) and 1.5 z-scores (for other Representative FACS plots of control (sh-Co), PKC-α (sh1-PKC-α), or PPP1R14D – α– shRNAs) above or below the mean z-score of all samples. A (sh1-PPP1R14D) knockdown Jurkat cells expressing HA TGF- EGFP with or z fi without TPA (1 μM). x axis, green C-terminal fluorescence; y axis, red ectodomain positive -score identi es inhibitory shRNAs (cleavage-activating fl α genes) and a negative z-score activating shRNAs (cleavage- uorescence (anti-HA stain). Statistical analysis is shown in the table. (B)PKC- fl (90–100%) and PPP1R14D (80%) shRNA knockdown by Western blot (densi- inhibiting genes). A distribution of all shRNA red:green uores- μ α z B tometry) and (C) knockdown effect on TPA (1 M)-induced TGF- cleavage cence -scoresisshowninFig S1 . shRNAs that showed repro- measured by FACS (red:green fluorescence ratio) or (D) by anti-GFP Western blot ducible effects on TGF-α cleavage were retested after subcloning – α– β in whole-cell lysates [full-length HA TGF- EGFP (39 kDa), C-terminal cleavage into an isopropyl -D-1-thiogalactopyranoside (IPTG)-inducible product (30 kDa)]. Further details are provided in the text. For all Western blots, lentiviral expression vector. we show one representative of three to five independent experiments. For FACS experiments, we show the mean of at least four independent experiments PKC-α and Protein Phosphatase 1 Inhibitor 14D Regulate Induced performed in triplicate; data are shown as percentage of control. Cleavage of only Specific EGF Ligand ADAM17 Substrates, Including TGF-α, but Not Neuregulin. Our screen identified positive and negative regulators of TPA-induced TGF-α cleavage, including The red signal stems from surface-stained full-length HA– the cleavage activating genes PKC-α and protein phosphatase 1 TGF-α–EGFP, and the green signal stems from the C-terminal (PP1) inhibitor 14D (PPP1R14D), a PP1 inhibitor that is acti- GFP fusion. The red signal is lost after cleavage (ectodomain lost vated by PKC phosphorylation (26, 27). Fig. 1A shows repre- in supernatant before FACS stain is carried out; Fig. S1A), sentative screen FACS plots of HA–TGF-α–EGFP-expressing whereas the GFP signal migrates with the C terminus after Jurkat cells. The elliptical marker was added to highlight changes cleavage, as is also seen in the Western blot. Hence, the low red: in the relevant plot area. In control-shRNA–expressing cells, the green ratio at 15 min of the FACS plot mirrors the results from reduction of red fluorescence (ectodomain) by TPA compared the Western blot when full-length:C-terminal product ratio is with control-treated cells is dramatic, whereas green fluorescence taken into account. In PKC-α or PPP1R14D knockdown Jurkat (C terminus) is roughly maintained. In PKC-α and PPP1R14D cell Western blots, the full-length band does not diminish by knockdown cells (Fig. 1B shows Western blot confirmation), this 5 min and shows some decrease at 15 min. This is reflected in the fluorescence shift is largely absent, indicating maintained HA– accumulation of C-terminal cleavage product particularly in the TGF-α−EGFP ectodomain fluorescence on the cell surface, a re- PPP1R14D knockdown cells. Our knockdown westerns show sult of blocked cleavage. The GFP signal as measured by FACS is 90% to 100% knockdown for PKC-α and approximately 80% slightly different between the cell lines as a result of effects on knockdown for PPP1R14D in these experiments (Fig. 1B). This basal expression or basal cleavage of the reporter. This does not could explain why PPP1R14D knockdown was less effective then affect cleavage detection by red:green fluorescent ratio as it is PKC-α knockdown in blocking cleavage. Of note, the C-terminal highly linear over a wide range of reporter expression (18, 19). We cleavage product is already present in control-treated cells, also showed the effect of PKC-α or PPP1R14D knockdown in reflecting basal cleavage, also seen in the control shRNA- FACS time-course experiments (Fig. 1C) and in whole cell lysates expressing cells. We confirmed our results in HEK293T cells using anti-GFP Western blots (Fig. 1D). In Fig. 1D, the Western overexpressing the G protein-coupled angiotensin II (AngII) blot shows two bands, an upper full-length HA–TGF-α–EGFP type 1 receptor known to activate PKC (28). Broad-spectrum and a lower C-terminal cleavage product. In control shRNA- inhibition of PKC isoforms by bisindolylmaleimide 1 (BIM1) expressing Jurkat cells, the full-length band strongly diminishes (to indeed strongly inhibited TPA- and AngII-induced TGF-α cleavage approximately 10% of control) over 5 and 15 min, whereas the as measured by FACS (Fig. 2A). PKC-α or PPP1R14D down- C-terminal cleavage product accumulates over the same time frame, regulation (Fig. 2B) had the same effect as BIM1 in inhibiting suggesting strong cleavage. The full-length band appears to slightly induced TGF-α cleavage (Fig. 2C, 1 and 2). In the latter experi- recover at 15 min compared with 5 min of TPA, but the C-terminal ment, we used cell-surface anti-HA immunoprecipitation (IP) to cleavage product continues to accumulate at 15 min, further de- detect full-length HA–TGF-α−GFP because detection of the creasing the full-length:C-terminal product ratio. A similar result small cleaved cell surface fraction of TGF-α was difficult in whole- can be seen in the FACS time-course experiments in Fig. 1C that cell lysates containing a large fraction of uncleaved intracellular CELL BIOLOGY plot the red:green ratio of HA–TGF-α–EGFP-expressing cells. TGF-α. However, we have been able to observe TPA-induced

Dang et al. PNAS | June 11, 2013 | vol. 110 | no. 24 | 9777 A B FACS PKCα and PPP1R14D-KD α BIM1(10μM) BIM1(10μM) sh-Cosh1-PKC 75kD -- α-PKCα 37kD -- α-ERK CO CO IP: α-PPP1R14D (#1) ed:Green Ratio Red:Green Ratio R Red:Green Ratio %of controlcleav %of % of control cleavage % of control cleavage sh-Co sh1-PPP1R14D 25kD -- α α-PPP1R14D (#2) Fig. 2. PKC- and PPP1R14D regulate TPA- and AngII-induced TPA 1 μM (min) AngII 1μM (min) 20kD -- α μ C 37kD -- α-ERK TGF- cleavage but not the cleavage of NRG. (A) TPA (1 M) or TGFα cleavage - surface IP and whole cell lysate AngII (1 μM)-induced TGF-α cleavage with or without broad- 1 IP: α-HA-TGF α-EGFP 2 IP: α-HA-TGF α-EGFP spectrum PKC inhibitor BIM1 (10 μM) measured by FACS. (B) sh-Co sh1-PKCα sh1-PPP1R14D sh-Co sh1-PKCα sh1-PPP1R14D PKC-α (90–100%) and PPP1R14D (70%) shRNA knockdown by 37kD -- α-HA 37kD -- α-HA Western blot (densitometry). (C) Knockdown effect on TPA μ μ α 37kD -- α-ERK 37kD -- α-ERK (1 M; 1) or AngII (1 M; 2)-induced TGF- cleavage measured by 0 5 15 30 0 5 15 30 0 5 15 30 min 0 5 15 30 0 5 15 30 0 5 15 30 min cell surface IP of full-length HA–TGF-α–EGFP and by detection TPA 1μM AngII 1μM of C-terminal cleavage products (3; shown only for TPA). (D) D NRG cleavage - PKCα/PPP1R14D-KD Effect of PKC-α and PPP1R14D knockdown on TPA-induced 3 sh-Co sh1-PKCα sh1-PPP1R14D Flag–NRG–EGFP and endogenous NRG cleavage measured by sh-Co sh1-PKCα sh1-PPP1R14D 150kd-- Full length NRG-GFP 100kd-- Full length endog. C-terminal NRG Western blot. Full-length/C-terminal fragment: 50kD -- α-Tubulin α-NRG (C-term.) endogenous NRG (100 kDa/50 kDa) and Flag–NRG–EGFP (150 50kd-- C-term. NRG-GFP 30kD -- α-GFP kDa/75 kDa). (E) ADAM17 (90–100%) siRNA knockdown by (C-term. C-term. endog. NRG TGFα) Western blot (densitometry) and (F) knockdown effect on TPA 0155 30 0155 30 0 155 30 min 37kd-- α-ERK μ – – TPA 1μM (1 M)-induced Flag NRG EGFP cleavage measured by GFP 0 5 15 30 0 5 15 30 0 5 15 30 min Western blot. (G) Effect of broad-spectrum metalloprotease TPA 1μM E F G inhibition with batimastat (BB94, 10 μM) or of speciﬁc ADAM9 ADAM17- KD NRG cleavage - NRG cleavage - ADAM9/10 inhibition or ADAM10 inhibition with their cognate prodomains (pro- M] A A ADAM17 KD μ A ADAM9, 270 nM; pro-ADAM10, 250 nM) on TPA-induced NRG [10 ffer ctrl cleavage measured by GFP Western blot. For all Western blots, DMSO BB94 proADAM9 proADAM10Bu Co-siRN A17-siRN A10-siRN Co-siRNA A17-siRNA we show one representative of three to ﬁve independent 75kd-- α-ADAM17 130kd-- α-GFP α-GFP 130kd-- experiments. For FACS experiments, we show the mean of at 50kd--