Regulated ADAM17-dependent EGF family ligandrelease by substrate-selecting signaling pathwaysMichelle 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 Instituteof 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 andmetalloproteinases (ADAMs) is highly regulated, and its dysregula-tion has been linked to many diseases. ADAM10 and ADAM17cleave most disease-relevant substrates. Broad-spectrum metallo-protease inhibitors have failed clinically, and targeting the cleavageof a specific substrate has remained impossible. It is therefore nec-essary to identify signaling intermediates that determine substratespecificity of cleavage. We show here that phorbol ester or angio-tensin II-induced proteolytic release of EGF family members maynot require a significant increase in ADAM17 protease activity.Rather, inducers activate a signaling pathway using PKC-α andthe PKC-regulated protein phosphatase 1 inhibitor 14D that is re-quired for ADAM17 cleavage of TGF-α, heparin-binding EGF, andamphiregulin. A second pathway involving PKC-δ is required forneuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylationof serine 286 in the NRG cytosolic domain is essential for inducedNRG cleavage. Thus, signaling-mediated substrate selection isclearly distinct from regulation of enzyme activity, an importantmechanism that offers itself for application in disease.

epidermal growth factor receptor | transactivation

The ectodomains of many cell surface proteins are shed from thesurface (i.e., “ectodomain shedding”) by metalloproteases.

Ectodomain shedding generates many diverse bioactive cytokinesand growth factors, and governs important cellular processes in thedeveloping and adult organism, including the control of growth,adhesion, and motility of cells (reviewed in refs. 1–3). EGF re-ceptor activation generates signals for cell proliferation, migra-tion, differentiation, or survival. The 12 EGF family members aresynthesized as cell surface transmembrane precursors. The activegrowth factors are released by A disintegrin and metalloprotein-ases (ADAMs) and activate specific heterodimeric EGF receptorson the cell surface connected to diverse intracellular signalingpathways (4, 5). Increased shedding of EGF ligands has been linkedto different clinical pathologic processes (6–10); hence, therapeuticcontrol of ligand release would be beneficial. Of the 12 functionalADAMs encoded in the human genome (3) only two—ADAM10and ADAM17—handle most of the numerous ADAM substrates,in particular, the EGF ligands. However, broad-spectrum metal-loprotease inhibitors tested for clinical use have failed as a result ofindiscriminate blockade of substrate cleavage, leading to clinicalside effects (11). Even recently developed selective ADAM inhib-itors still affect the cleavage of many substrates (12). Modulation ofthe release of specific ADAM substrates has been impossible todate because it is unknown how cleavage specificity is regulated onthe molecular level. It is therefore necessary to identify key signalsthat determine substrate specificity of cleavage.Ectodomain cleavage is made specific by a number of in-

tracellular signals; e.g., by calcium influx, by activation of G pro-tein-coupled receptors, and the release of diacylglycerol (reviewedin refs. 3, 13). Several distinct mechanisms that modulate cleavageon the level of ADAM17 have been described, including regula-tion of ADAM17 expression, maturation, trafficking to the cellsurface (reviewed in ref. 13), and posttranslational modifications

on the ADAM17 ectodomain (14, 15) or its C terminus (16, 17).However, modulation of activity of the relatively few availableADAMs does not suffice to explain substrate-specific regulationof cleavage (18, 19), and none of the referenced studies hasaddressed how specificity of cleavage is achieved. Transgenicoverexpression of ADAM17 in mice does not lead to overactivityof ADAM17 or increased ADAM17 substrate release, emphasiz-ing the importance of posttranslational control of cleavage (20).Most reports on induced shedding (reviewed in refs. 5, 21) haveonly used monitoring of substrate cleavage as a surrogate measureof protease activity. However, only few studies unequivocallydocument induced changes of protease activity, and those weresmall. A tight-binding ADAM17 inhibitor interacts with the cat-alytic site of ADAM17 only after 12-O-tetradecanoylphorbol-13-acetate (TPA; i.e., phorbol ester) stimulation (12), suggestingregulation of the catalytic site. Another convincing example ofregulated enzyme activity has been based on observed effects ofoxidation on several putative disulphide bonds in the ADAM17ectodomain that result in a structural change. This involves theinteraction with an extracellular redox regulator, protein disulfideisomerase (PDI). PDI down-regulation enhanced TPA-inducedshedding of heparin-binding (HB) EGF, addition of exogenousPDI decreased it, and PDI addition to recombinant ADAM17reduced basal cleavage of a fluorescence resonance energy trans-fer (FRET) peptide. These changes correlated with altered to-pology of antibody epitopes outside of, but not within, thecatalytic domain (14). However, induced HB-EGF cleavage couldhave also resulted from enhanced interaction of the substrate withADAM17 via the altered topology outside of the catalytic domainwithout requiring changes in protease activity. Neither study de-termined protease activity independent of substrate cleavage, stillleaving us with uncertainty whether induced substrate cleavagetruly requires enhanced protease activity. By using stopped-flowX-ray spectroscopy and other techniques, Solomon et al. showedthat ADAM17 activity is primed by enzyme conformational changesinduced by the substrate before proteolysis (22). Novel exositeinhibitors of ADAM17 activity that bind ADAM17 outside of thecatalytic site and likely interfere with the binding of glycosylatedmoieties of the substrate have been developed (23). Both studiesfurther support regulation of proteolysis on the substrate level.Here we identify pathway components that distinguish sub-

strates of ADAM17 and parse substrate selection from regula-tion of protease activity.

Author contributions: H.F.L. and A.H. designed research; M.D., N.A., M.A.M., E.C., and A.H.performed research; M.D., N.A., M.A.M., E.C., M.H., D.E.R., D.A.L., and A.H. contributed newreagents/analytic tools; M.D., N.A., M.A.M., G.W.B., D.A.L., H.F.L., and A.H. analyzed data;and H.F.L. and A.H. wrote the paper.

The authors declare no conflict of interest.1M.D. and N.A. contributed equally to this work.2To whom correspondence may be addressed. E-mail: aherrlich@partners.org or lodish@wi.mit.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1307478110/-/DCSupplemental.

9776–9781 | PNAS | June 11, 2013 | vol. 110 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1307478110

ResultsshRNA Screen for Regulators of TGF-α Cleavage by ADAM17. Phorbolester (i.e., TPA) stimulates most PKC isoforms (-α, -β, -γ, -δ, -e,-η, -θ and -μ), and is a commonly used cleavage stimulus inshedding studies. ADAM17 is the physiological effector of TPA-induced signals, whereas ADAM10 primarily responds to cal-cium signals (24, 25). To identify novel genes that regulateshedding downstream of PKC, we carried out a lentiviral shRNAgene knockdown screen targeting most human kinases and phos-phatases and some of their associated components, probing theireffect on TPA-induced cleavage of TGF-α, a classical ADAM17substrate (24). Cleavage was measured with an extensively vali-dated high-throughput 96-well FACS assay (18, 19) (Fig. S1A).We screened 3,500 unique lentiviral shRNAs carrying puromycinresistance for selection at >3× coverage with biological duplicatesin human Jurkat cells expressing HA–TGF-α–EGFP. Genes weretargeted with three to five individual shRNAs per gene and se-lected with puromycin. A shRNA targeting lacZ, a protein notpresent in mammalian cells (control-shRNA) was used as a con-trol. After stimulation with TPA for 2 to 5 min, mean geometricred and green fluorescence of the cells was measured by FACSand normalized across samples by using z-scores. Cleavage wasinduced to approximately 50% of maximum to allow detection ofcleavage activation or inhibition in the same screen. Assuming thatmost tested shRNAs would not affect cleavage, we selected tar-geted genes with two or more shRNAs that reproducibly scored atleast 2 z-scores (for best shRNA) and 1.5 z-scores (for othershRNAs) above or below the mean z-score of all samples. Apositive z-score identifies inhibitory shRNAs (cleavage-activatinggenes) and a negative z-score activating shRNAs (cleavage-inhibiting genes). A distribution of all shRNA red:green fluores-cence z-scores is shown in Fig S1B. shRNAs that showed repro-ducible effects on TGF-α cleavage were retested after subcloninginto an isopropyl β-D-1-thiogalactopyranoside (IPTG)-induciblelentiviral expression vector.

PKC-α and Protein Phosphatase 1 Inhibitor 14D Regulate InducedCleavage of only Specific EGF Ligand ADAM17 Substrates, IncludingTGF-α, but Not Neuregulin. Our screen identified positive andnegative regulators of TPA-induced TGF-α cleavage, includingthe cleavage activating genes PKC-α and protein phosphatase 1(PP1) inhibitor 14D (PPP1R14D), a PP1 inhibitor that is acti-vated by PKC phosphorylation (26, 27). Fig. 1A shows repre-sentative screen FACS plots of HA–TGF-α–EGFP-expressingJurkat cells. The elliptical marker was added to highlight changesin the relevant plot area. In control-shRNA–expressing cells, thereduction of red fluorescence (ectodomain) by TPA comparedwith control-treated cells is dramatic, whereas green fluorescence(C terminus) is roughly maintained. In PKC-α and PPP1R14Dknockdown cells (Fig. 1B shows Western blot confirmation), thisfluorescence shift is largely absent, indicating maintained HA–TGF-α−EGFP ectodomain fluorescence on the cell surface, a re-sult of blocked cleavage. The GFP signal as measured by FACS isslightly different between the cell lines as a result of effects onbasal expression or basal cleavage of the reporter. This does notaffect cleavage detection by red:green fluorescent ratio as it ishighly linear over a wide range of reporter expression (18, 19). Wealso showed the effect of PKC-α or PPP1R14D knockdown inFACS time-course experiments (Fig. 1C) and in whole cell lysatesusing anti-GFP Western blots (Fig. 1D). In Fig. 1D, the Westernblot shows two bands, an upper full-length HA–TGF-α–EGFPand a lower C-terminal cleavage product. In control shRNA-expressing Jurkat cells, the full-length band strongly diminishes (toapproximately 10% of control) over 5 and 15 min, whereas theC-terminal cleavage product accumulates over the same time frame,suggesting strong cleavage. The full-length band appears to slightlyrecover at 15 min compared with 5 min of TPA, but the C-terminalcleavage product continues to accumulate at 15 min, further de-creasing the full-length:C-terminal product ratio. A similar resultcan be seen in the FACS time-course experiments in Fig. 1C thatplot the red:green ratio of HA–TGF-α–EGFP-expressing cells.

The red signal stems from surface-stained full-length HA–TGF-α–EGFP, and the green signal stems from the C-terminalGFP fusion. The red signal is lost after cleavage (ectodomain lostin supernatant before FACS stain is carried out; Fig. S1A),whereas the GFP signal migrates with the C terminus aftercleavage, as is also seen in the Western blot. Hence, the low red:green ratio at 15 min of the FACS plot mirrors the results fromthe Western blot when full-length:C-terminal product ratio istaken into account. In PKC-α or PPP1R14D knockdown Jurkatcell Western blots, the full-length band does not diminish by5 min and shows some decrease at 15 min. This is reflected in theaccumulation of C-terminal cleavage product particularly in thePPP1R14D knockdown cells. Our knockdown westerns show90% to 100% knockdown for PKC-α and approximately 80%knockdown for PPP1R14D in these experiments (Fig. 1B). Thiscould explain why PPP1R14D knockdown was less effective thenPKC-α knockdown in blocking cleavage. Of note, the C-terminalcleavage product is already present in control-treated cells,reflecting basal cleavage, also seen in the control shRNA-expressing cells. We confirmed our results in HEK293T cellsoverexpressing the G protein-coupled angiotensin II (AngII)type 1 receptor known to activate PKC (28). Broad-spectruminhibition of PKC isoforms by bisindolylmaleimide 1 (BIM1)indeed strongly inhibited TPA- and AngII-induced TGF-α cleavageas measured by FACS (Fig. 2A). PKC-α or PPP1R14D down-regulation (Fig. 2B) had the same effect as BIM1 in inhibitinginduced TGF-α cleavage (Fig. 2C, 1 and 2). In the latter experi-ment, we used cell-surface anti-HA immunoprecipitation (IP) todetect full-length HA–TGF-α−GFP because detection of thesmall cleaved cell surface fraction of TGF-α was difficult in whole-cell lysates containing a large fraction of uncleaved intracellularTGF-α. However, we have been able to observe TPA-induced

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Fig. 1. shRNA screen targeting human phosphatases and kinases identi-fies PKC-α and PPP1R14D as regulators of TPA-induced TGF-α cleavage. (A)Representative FACS plots of control (sh-Co), PKC-α (sh1-PKC-α), or PPP1R14D(sh1-PPP1R14D) knockdown Jurkat cells expressing HA–TGF-α–EGFP with orwithout TPA (1 μM). x axis, greenC-terminalfluorescence; y axis, red ectodomainfluorescence (anti-HA stain). Statistical analysis is shown in the table. (B) PKC-α(90–100%) and PPP1R14D (80%) shRNA knockdown by Western blot (densi-tometry) and (C) knockdown effect on TPA (1 μM)-induced TGF-α cleavagemeasured by FACS (red:greenfluorescence ratio) or (D) by anti-GFPWestern blotin whole-cell lysates [full-length HA–TGF-α–EGFP (39 kDa), C-terminal cleavageproduct (30 kDa)]. Further details are provided in the text. For all Western blots,we showone representative of three tofive independent experiments. For FACSexperiments, we show the mean of at least four independent experimentsperformed in triplicate; data are shown as percentage of control.

Dang et al. PNAS | June 11, 2013 | vol. 110 | no. 24 | 9777

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accumulation of C-terminal cleavage products in control shRNA-expressing cells that is significantly blocked in PKC-α orPPP1R14D knockdown cells (Fig. 2C, 3). Importantly, knock-down of either gene did not affect TPA-induced cleavage ofneuregulin (NRG) in HEK cells (Fig. 2D), also an ADAM17substrate (Fig. 2 E–G). We therefore hypothesized that PKC-αand PPP1R14D may act in the regulation of only a subset ofADAM17 substrates. We confirmed this by using specific ELISAsto detect different endogenous protein ectodomains cleaved byADAM17 (Fig. S1 C and D) in the same sample supernatant ofcells that express various cleaved metalloprotease substrates, in-cluding EGF ligands. In MDA-MB-231 breast cancer cells thatdepend on EGF ligand cleavage for proliferation and expressTGF-α, PKC-α, and PPP1R14D, PKC-α and PPP1R14D areindeed required for TPA-induced ADAM17 cleavage of TGF-α,HB-EGF, and amphiregulin (AR), but not for the basal cleavageof TNF receptor 1 (Fig. S2). In 12Z cells (which do not expressTGF-α), both genes were required for the cleavage of HB-EGF(Fig. S3). Cleavage of the ADAM10 substrate c-Met was unaffectedin both cell lines (Figs. S1–S3), suggesting that regulation byeither gene is specific for ADAM17 substrates. We were un-able to detect NRG cleavage with several different ELISAs inMDA-MB-231 cells.

PKC-δ–Dependent C-Terminal Serine Phosphorylation RegulatesInduced NRG Cleavage. The broad-spectrum PKC inhibitor BIM1blocks TPA- and AngII-induced NRG cleavage in HEK293Tcells (Fig. 3A, 1 and 2). In contrast, and consistent with the lackof effect of PKC-α down-regulation (Fig. 2D), Gö6976, an inhib-itor of only PKC-α and -β, did not block NRG cleavage (Fig. 3A,3). We confirmed these results for BIM1 and Gö6976 detectingNRG cleavage by FACS (Fig. 3 A, 4 and 5). We therefore askedwhich other TPA-regulated PKC isoforms might be responsible.PKC-δ down-regulation strongly blocked TPA-induced NRGcleavage (Fig. 3B, 1 and 2), whereas the knockdown of otherPKC isoforms had no effect. By using a phospho-specific PKCconsensus site antibody, we previously showed that TPA induces

a serine phosphorylation on the C terminus of NRG beforecleavage, which is sensitive to BIM1 inhibition (18). Serine 286(S286) is contained in the only sequence in the NRG C terminusmatching the consensus PKC phosphorylation site. Indeed, mu-tation of S286 to alanine blocked TPA-induced NRG cleavage asmeasured by FACS (Fig. 3C). We confirmed these findings byimmunofluorescence and Western blot. The TPA- and AngII-induced S286 phosphorylation was prevented by BIM1, but notby the PKC-α,β inhibitor Gö6976 (Fig. 3D, 1 and 2), consistentwith their respective effects on NRG cleavage (Fig. 3A, 1–5). Incontrast, PKC-δ knockdown abolished AngII- or TPA-inducedS286 phosphorylation (Fig. 3D, 3), suggesting that PKC-δ activityestablishes S286 phosphorylation.

TPA and AngII Induce Inhibition of PP1. Phosphorylation of serine58 in PPP1R14D by PKC activates the PP1 inhibitor by inducinga conformational change (26, 27), allowing binding of the in-hibitor to a specific PP1 complex. In IP experiments, PP1-α butnot PP1-β or PP1-γ was coprecipitated strongly with Flag-PPP1R14D from TPA-treated Jurkat cells and TPA- or AngII-treated HEK293T cells compared with controls (Fig. 4 A and B),suggesting that PP1α is the downstream effector of PPP1R14D.We confirmed the inhibitory effect of activated PPP1R14D ina biochemical PP1-α assay in vitro, by using a FRET peptidesubstrate that only reveals its fluorescence when dephosphory-lated. Only an exogenously applied PP1 inhibitor (inhibitor-2) orendogenous PPP1R14D immunoprecipitated from TPA- orAngII-treated cells significantly inhibited PP1-α activity; how-ever, PPP1R14D precipitated from control cells did not (Fig.4C). In contrast to knockdown of PPP1R14D, a regulator of onlyspecific PP1-α–containing complexes, PP1-α knockdown itselfwas not well tolerated in Jurkat or HEK293T cells, leading tosignificantly decreased cell proliferation and increased celldeath. PP1-α carries out many cellular dephosphorylation reac-tions, affecting multiple cellular functions (29).Taken together, our findings strongly suggest (i) that regula-

tion of substrate cleavage by modulating protease activity may

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Fig. 2. PKC-α and PPP1R14D regulate TPA- and AngII-inducedTGF-α cleavage but not the cleavage of NRG. (A) TPA (1 μM) orAngII (1 μM)-induced TGF-α cleavage with or without broad-spectrum PKC inhibitor BIM1 (10 μM) measured by FACS. (B)PKC-α (90–100%) and PPP1R14D (70%) shRNA knockdown byWestern blot (densitometry). (C ) Knockdown effect on TPA(1 μM; 1) or AngII (1 μM; 2)-induced TGF-α cleavage measured bycell surface IP of full-length HA–TGF-α–EGFP and by detectionof C-terminal cleavage products (3; shown only for TPA). (D)Effect of PKC-α and PPP1R14D knockdown on TPA-inducedFlag–NRG–EGFP and endogenous NRG cleavage measured byC-terminal NRG Western blot. Full-length/C-terminal fragment:endogenous NRG (100 kDa/50 kDa) and Flag–NRG–EGFP (150kDa/75 kDa). (E) ADAM17 (90–100%) siRNA knockdown byWestern blot (densitometry) and (F) knockdown effect on TPA(1 μM)-induced Flag–NRG–EGFP cleavage measured by GFPWestern blot. (G) Effect of broad-spectrum metalloproteaseinhibition with batimastat (BB94, 10 μM) or of specific ADAM9or ADAM10 inhibition with their cognate prodomains (pro-ADAM9, 270 nM; pro-ADAM10, 250 nM) on TPA-induced NRGcleavage measured by GFP Western blot. For all Western blots,we show one representative of three to five independentexperiments. For FACS experiments, we show the mean of atleast four independent experiments performed in triplicate.Data are shown as percentage of control.

9778 | www.pnas.org/cgi/doi/10.1073/pnas.1307478110 Dang et al.

not suffice to explain the observed specificity of cleavage, (ii) thatthe identified regulators may act in intracellular signalingpathways that address the C terminus of substrates to regulatecleavage, and (iii) that ADAM17 substrate cleavage is regu-lated and made specific by distinct PKC isoforms, PPP1R14Dand PP1-α.

PKC-α and PPP1R14D Regulate ADAM17 Cleavage Without SignificantlyAffecting Protease Activity. Given our finding that ADAM17 sub-strates are specifically selected, does this require, in addition,regulation at the level of protease activity? We measured basaland TPA-induced protease activity by using proteolytic activity

matrix analysis (PrAMA). PrAMA allows measurement of par-ticular protease activities independently of measuring specificphysiological substrate cleavage (30). This technique uses a panelof FRET-based peptide substrates that emit fluorescence uponcleavage. Peptide substrates can be incubated directly with livecells, with generated fluorescence measured in a plate reader.Specific protease-associated cleavage profiles were developed andvalidated with purified proteases in vitro and in ADAM-KO cellsin vivo. We used five different FRET substrates that vary in theirspecificity for ADAMs and matrix metalloproteinases to infer theactivity of ADAM17. Knockdown of ADAM17 indeed stronglyreduced PrAMA-inferred ADAM17 protease activity in MDA-MB-231 cells (Fig. 5A). Knockdown of ADAM17 (approximately80%) for both shRNAs used was confirmed by Western blot (Fig.S1D). However, baseline live-cell ADAM17 protease activity wasonly mildly reduced by 0% to 20% in PKC-α or PPP1R14DMDA-MB-231 knockdown cells (significant in only one of twoshRNAs tested per gene) compared with control (Fig. 5B). Evenmore surprisingly, 30 min TPA stimulation, although able to in-duce substantial substrate cleavage in control cells (Figs. 1–3 andFigs. S2–S4), did not increase protease activity under any condi-tion, but rather decreased it to a small degree (Fig. 5B). Weobtained similar results by using immunoprecipitated cell-surfaceADAM17 and one relevant FRET substrate for cleavage detec-tion. TPA or anisomycin, an inducer of cell stress, did not increaseactivity of cell surface ADAM17 at 5, 15, or 30 min, but a smalldecrease in protease activity was detected at 30 min in TPA-stimulated samples (Fig. 5C). Basal cell surface ADAM17 ac-tivity showed only small differences in control and knockdowncells, similar to live-cell PrAMA (Fig. S4). Overall levels of ma-ture ADAM17 were unchanged by PKC-α or PPP1R14D knock-down in MDA-MB-231 cells, with only minor changes in the ratioof pro- to mature form (Fig. 5D), arguing against transcriptionaldown-regulation or reduced ADAM17 maturation as explana-tions for the small reductions in protease activity. Cell surfacelevels of ADAM17, another factor that could influence cellsurface protease activity, were equal in control and PKC-α knock-down cells and were moderately reduced with PPP1R14D down-regulation (Fig. 5E). As basal protease activity in PPP1R14Dknockdown cells is not significantly affected (Fig. 5B), this

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TPA 1 μM (min) AngII μM (min)Fig. 3. PKC-δ and a PKC-δ–dependent serine phosphorylationof the NRG C terminus regulate its cleavage. (A) TPA (1 μM) orAngII (1 μM)-induced NRG cleavage with or without the broad-spectrum PKC inhibitor BIM1 (10 μM) or specific PKC-α,β in-hibitor Gö6976 (10 μM) measured by C-terminal NRG Westernblot, Flag-NRG-EGFP (150 kd/75 kd; 1–3), or FACS (4 and 5). (B)PKC-δ (60%) knockdown by Western blot (densitometry; 1) andknockdown effect on TPA-induced NRG cleavage (2). (C) Mu-tation of serine 286 to alanine in the NRG C terminus resultsin inhibition of TPA (1 μM)-induced cleavage (dotted line,NRGS286A; dashed line, NRG WT). (D) Effects of BIM1, Gö6976,or PKC-δ siRNA knockdown on S286 phosphorylation measuredby Western blot with a PKC-site phospho-specific antibody:TPA (1 μM; 1 and 3) or AngII (1 μM; 2 and 3). For all Westernblots, we show one representative of three to five indepen-dent experiments. For the FACS experiments, we show themean of at least four independent experiments performed intriplicate; data are shown as percentage of control.

A PPP1R14D IP - PP1 coIP

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Fig. 4. TPA and AngII induce PP1 inhibition. (A) PP1-α but not PP1-β or PP1-γ(detected by specific antibodies) coimmunoprecipitate with Flag–PPP1R14Din TPA (1 μM)-treated Jurkat cells (PPP1R14D IP input is detected byPPP1R14D antibody) or (B) HEK293T cells (shown for PP1-α only). (C) PP1-αphosphatase assay: endogenous PPP1R14D was immunoprecipitated fromTPA (1 μM) or AngII (1 μM)-treated cells and incubated with recombinantPP1-α. Phosphatase activity was measured with a FRET peptide that fluo-resces only when dephosphorylated by PP1. Inhibitor-2 was used as a positivecontrol for PP1 inhibition. For all Western blots, we show one representativeof three to five independent experiments. For the PP1 activity assay, we showthe mean of at least four independent experiments performed in triplicate.

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suggests that surface levels of active ADAM17 are in saturation.Nonetheless, PPP1R14D may regulate ADAM17 surface levels.In summary, our results indicate that the predominant effects

of PKC-α and PPP1R14D on substrate cleavage are not medi-ated by changes in ADAM17 expression, maturation, or cellsurface expression, nor are they caused by significant regulationof ADAM17 protease activity. The implication is that PKC-α andPPP1R14D (and PP1-α) directly or indirectly act on the substrateor on a third interacting protein required for cleavage regulation.

DiscussionOur results support at least two principal conclusions, asnoted below.

Induced Shedding May Not Require Significant Changes in ADAM17Protease Activity. In the present study, live-cell PrAMA or studieswith immunoprecipitated cell surface ADAM17 showed a re-duction of only 0% to 20% of basal protease activity with PKC-αor PPP1R14D down-regulation compared with control cells andsmall decreases in protease activity in response to 30-min TPAstimulation. Decreases of ADAM17 surface levels in response toTPA stimulation, either by endocytosis or possibly cleavage, havebeen reported before (14, 31). More importantly, even withoutincreases in protease activity, strong TPA-induced substratecleavage (1.5- to fivefold vs. control) was observed. PKC-α orPPP1R14D down-regulation blocked this proteolysis withoutinducing significant changes in protease activity, at least not asmeasured with currently available techniques that allow deter-mination of protease activity independent of measuring physio-logical substrate cleavage. This indicates that these signalingcomponents regulate substrate cleavage independently of a ma-jor effect on protease activity. In live-cell PrAMA experimentsusing 30-min TPA stimulations that we published previously,TPA also enhanced ADAM17 protease activity only slightly (30),

yet substrate cleavage was increased much more significantly(18). In contrast to our studies, the application of cell stress in-cluding by anisomycin for 60 min (16) increased protease activityby 20% to 25% (measured with immunoprecipitated ADAM17and one FRET-peptide substrate). Concurrently, cell stress pro-duced a small to moderate increase of ADAM17 surface levelsnot observed in our study that likely account for the observedsmall increases in protease activity after 60 min of stimulation.However, despite only small changes in protease activity in thisstudy, cell stress-induced TGF-α cleavage was increased five to10 fold vs. control cells. These findings further support the no-tion that substrate cleavage is regulated independently of a majorchange in protease activity.

Different Signaling Modules Select Distinct Substrates for ADAM17Cleavage. PKC-α and PPP1R14D act only in ADAM17 cleavageof TGF-α, HB-EGF, and AR, and possibly act in a signalingcascade by inhibiting PP1-α. PKC-δ is part of a different signalingmodule that regulates ADAM17 cleavage of NRG. PKC-α andPKC-δ are both activated by TPA, yet the signaling modules theydrive are different. This would lend a satisfying explanation tothe conundrum of how specific substrate cleavage in response tothe same cleavage stimulus, TPA, could be achieved by only onemetalloprotease, ADAM17. Additionally, different stimulus-specific responses in substrate cleavage (e.g., osmotic stress, Gprotein-coupled receptor activation) could be explained bystimulus-dependent engagement of different signaling modulesthat act on distinct subsets of substrates and/or act in differentways on the same substrate, e.g., by posttranslational modifica-tion. Cleavage of substrates depends, of course, on the level ofmature enzyme on the cell surface. This level is regulated byADAM gene expression, transport, and maturation, as well asby internalization. Signal transduction pathways affect all theseprocesses (e.g., ref. 32). The rapid choice of substrates forcleavage must, however, be defined by their posttranslationalmodifications. Such selection likely involves modifications of theC terminus of substrates, as shown here for NRG, or, alterna-tively, of the C terminus of yet unidentified regulatory proteinsthat interact with the substrates. In contrast to many substrates,the C terminus of ADAM17 is not required for induced or basalcleavage (25), although conflicting results on the regulatory roleof a C-terminal threonine 735 phosphorylation have been re-ported (16, 17, 32). To date, a number of C-terminal regulatoryevents on substrates that regulate shedding and could be influ-enced by intracellular signaling have been described. Phosphor-ylated serine residues and linkage to the actin cytoskeleton viaezrin/radixin/moesin proteins are important for PKC-dependentshedding of L-selectin (33). Calmodulin is constitutively boundto the cytoplasmic tails of L-selectin and angiotensin-convertingenzyme, and its dissociation induced by calmodulin kinase in-hibitors or TPA activates their shedding (34, 35). Shedding ofCD44, EGF, betacellulin, N-cadherin, and IL-6R are inducibleby calmodulin kinase inhibitors and calcium ionophores (36–38).Regulation of substrate cell surface levels also affects cleavage ofcertain substrates. As examples, the APP homologue APLP1binds to APP through a conserved motif in the cytoplasmic do-main and increases the shedding of APP by reduction of its en-docytosis (39). Monoubiquitination of AR’s C terminus inducesits immediate endocytosis and thereby rapidly blocks its ecto-domain cleavage by ADAM17 (40). However, not all substratesrequire their C terminus for shedding and not all C-terminalphosphorylation events induced by shedding stimuli regulatecleavage [e.g., IL-6 receptor (41), TNF-α receptor II (42), HB-EGF (43)]. Because ADAM17 and ADAM10 also do not requiretheir C-termini for stimulus-induced cleavage (25), these findingssuggest the existence of other “third” regulatory proteins thatreceive signaling input, and/or that C termini of some substratescontain negative regulatory signals for cleavage that are missingafter C-terminal deletion. As TPA induces many sheddingevents, it is not surprising that PKC isozymes have been impli-cated. Only few studies have identified specific PKC isoforms

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Fig. 5. PKC-α and PPP1R14D regulate ADAM17 cleavage without signif-icantly affecting protease activity. Determination of ADAM17 protease ac-tivity by PrAMA in (A) ADAM17 or (B) PKC-α and PPP1R14D shRNAknockdown vs. control cells. (C) Cell-surface ADAM17 protease activity wasdetected by ADAM17 surface IP and assayed with one relevant FRET sub-strate. (D) Effect of PKC-α or PPP1R14D knockdown on pro-ADAM17 (100kDa) and mature ADAM17 (75 kDa) expression levels measured by C-termi-nal ADAM17 Western blot and (E) by detecting cell surface levels ofADAM17 by FACS (ADAM17 ectodomain antibody). Cells were treated asindicated. For all Western blots, we show one representative of three to fiveindependent experiments. ADAM17/tubulin ratios were determined bydensitometry of three independent Western blots. For the protease activityassays, we show the mean of at least three independent experiments per-formed in triplicate.

9780 | www.pnas.org/cgi/doi/10.1073/pnas.1307478110 Dang et al.

involved in cleavage regulation, but none clearly distinguishedwhether protease or substrates were the target of regulation [e.g.,PKC-e/TNF-α (44); PKC-δ/HB-EGF (45); PKC-δ and PKC-e/IL-6R (46)]. In concordance with our study, PKC-α siRNA knock-down blocks TPA-induced HB-EGF cleavage (47), and PKC-δand phosphorylated C-terminal serine residues regulate cleavageof chicken NRG in neuronal cells (48).In summary, we show that the regulatory proteins PKC-α,

PPP1R14D, and PKC-δ specifically affect the cleavage of onlyselect ADAM17 substrates without significantly affecting pro-tease activity. This observation offers itself as a new avenue fortherapeutic intervention independent of the protease and pos-sibly specific for a particular disease-causing substrate.

Materials and MethodsSI Materials and Methods describes materials, retrovirus/lentivirus pro-duction and infection, IP, Western blotting and ELISAs, PP1 biochemical

assay, immunoprecipitated cell-surface ADAM17 protease assay, FACS, andshRNA FACS screen.

PrAMA. For live-cell PrAMA, IPTG-induced MDA-MB-231 human breast cancercells were seeded in a 384-well clear-bottom black-walled plate. The next day,samples were stimulated with internally quenched FRET substrates (5 μM)alone or with TPA (1 μM) for 30 min. Substrates were also added to no-cell(negative control) and trypsin (positive control). Fluorescence readings wereobtained every 10 min for 2 h at 37 °C.

ACKNOWLEDGMENTS. We thank the Massachusetts Institute of TechnologySwanson Biotechnology Center for help with image analysis; Issei Komurofor providing HEK293T- angiotensin II type 1 receptor cells; Glenn Paradisand Patti Wisniewski (FACS Facility), Kathleen Ottina (BL2plus Facility), andJen Grenier (Broad Institute) for their help; Prat Thiru and Bingbing Yuan forhelp with biostatistical analysis; and Peter Herrlich for critical reading. Thiswork was supported by National Institute of Diabetes and Digestive andKidney Diseases Grants R00DK077731 and R01-CA96504.

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