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

Abstract

Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.

Keywords: epidermal growth factor receptor; transactivation.

Fig. 1.

shRNA screen targeting human phosphatases and kinases identifies 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 or without TPA (1 μM). x axis, green C-terminal fluorescence; y axis, red ectodomain fluorescence (anti-HA stain). Statistical analysis is shown in the table. (B) PKC-α (90–100%) and PPP1R14D (80%) shRNA knockdown by Western blot (densitometry) and (C) knockdown effect on TPA (1 μM)-induced TGF-α cleavage measured by FACS (red:green fluorescence ratio) or (D) by anti-GFP Western blot in whole-cell lysates [full-length HA–TGF-α–EGFP (39 kDa), C-terminal cleavage product (30 kDa)]. Further details are provided in the text. For all Western blots, we show one representative of three to five independent experiments. For FACS experiments, we show the mean of at least four independent experiments performed in triplicate; data are shown as percentage of control.

Fig. 2.

PKC-α and PPP1R14D regulate TPA- and AngII-induced TGF-α cleavage but not the cleavage of NRG. (A) TPA (1 μM) or AngII (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 by Western blot (densitometry). (C) Knockdown effect on TPA (1 μM; 1) or AngII (1 μM; 2)-induced TGF-α cleavage measured by cell surface IP of full-length HA–TGF-α–EGFP and by detection of C-terminal cleavage products (3; shown only for TPA). (D) Effect of PKC-α and PPP1R14D knockdown on TPA-induced Flag–NRG–EGFP and endogenous NRG cleavage measured by C-terminal NRG Western blot. Full-length/C-terminal fragment: endogenous NRG (100 kDa/50 kDa) and Flag–NRG–EGFP (150 kDa/75 kDa). (E) ADAM17 (90–100%) siRNA knockdown by Western blot (densitometry) and (F) knockdown effect on TPA (1 μM)-induced Flag–NRG–EGFP cleavage measured by GFP Western blot. (G) Effect of broad-spectrum metalloprotease inhibition with batimastat (BB94, 10 μM) or of specific ADAM9 or ADAM10 inhibition with their cognate prodomains (pro-ADAM9, 270 nM; pro-ADAM10, 250 nM) on TPA-induced NRG cleavage measured by GFP Western blot. For all Western blots, we show one representative of three to five independent experiments. For FACS experiments, we show the mean of at least four independent experiments performed in triplicate. Data are shown as percentage of control.

Fig. 3.

PKC-δ and a PKC-δ–dependent serine phosphorylation of the NRG C terminus regulate its cleavage. (A) TPA (1 μM) or AngII (1 μM)-induced NRG cleavage with or without the broad-spectrum PKC inhibitor BIM1 (10 μM) or specific PKC-α,β inhibitor Gö6976 (10 μM) measured by C-terminal NRG Western blot, Flag-NRG-EGFP (150 kd/75 kd; 1–3), or FACS (4 and 5). (B) PKC-δ (60%) knockdown by Western blot (densitometry; 1) and knockdown effect on TPA-induced NRG cleavage (2). (C) Mutation of serine 286 to alanine in the NRG C terminus results in 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 measured by Western blot with a PKC-site phospho-specific antibody: TPA (1 μM; 1 and 3) or AngII (1 μM; 2 and 3). For all Western blots, we show one representative of three to five independent experiments. For the FACS experiments, we show the mean of at least four independent experiments performed in triplicate; data are shown as percentage of control.

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–PPP1R14D in TPA (1 μM)-treated Jurkat cells (PPP1R14D IP input is detected by PPP1R14D antibody) or (B) HEK293T cells (shown for PP1-α only). (C) PP1-α phosphatase assay: endogenous PPP1R14D was immunoprecipitated from TPA (1 μM) or AngII (1 μM)-treated cells and incubated with recombinant PP1-α. Phosphatase activity was measured with a FRET peptide that fluoresces only when dephosphorylated by PP1. Inhibitor-2 was used as a positive control for PP1 inhibition. For all Western blots, we show one representative of three to five independent experiments. For the PP1 activity assay, we show the mean of at least four independent experiments performed in triplicate.

Fig. 5.

PKC-α and PPP1R14D regulate ADAM17 cleavage without significantly affecting protease activity. Determination of ADAM17 protease activity by PrAMA in (A) ADAM17 or (B) PKC-α and PPP1R14D shRNA knockdown vs. control cells. (C) Cell-surface ADAM17 protease activity was detected by ADAM17 surface IP and assayed with one relevant FRET substrate. (D) Effect of PKC-α or PPP1R14D knockdown on pro-ADAM17 (100 kDa) and mature ADAM17 (75 kDa) expression levels measured by C-terminal ADAM17 Western blot and (E) by detecting cell surface levels of ADAM17 by FACS (ADAM17 ectodomain antibody). Cells were treated as indicated. For all Western blots, we show one representative of three to five independent experiments. ADAM17/tubulin ratios were determined by densitometry of three independent Western blots. For the protease activity assays, we show the mean of at least three independent experiments performed in triplicate.