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.epidermal growth factor receptor | transactivation T he ectodomains of many cell surface proteins are shed from the surface (i.e., "ectodomain shedding") by metalloproteases. Ectodomain shedding generates many diverse bioactive cytokines and growth factors, and governs important cellular processes in the developing and adult organism, including the control of growth, adhesion, and motility of cells (reviewed in refs. 1-3). EGF receptor activation generates signals for cell proliferation, migration, differentiation, or survival. The 12 EGF family members are synthesized as cell surface transmembrane precursors. The active growth factors are released by A disintegrin and metalloproteinases (ADAMs) and activate specific heterodimeric EGF receptors on the cell surface connected to diverse intracellular signaling pathways (4, 5). Increased shedding of EGF ligands has been linked to different clinical pathologic processes (6-10); hence, therapeutic control of ligand release would be beneficial. Of the 12 functional ADAMs encoded in the human genome (3) only two-ADAM10 and ADAM17-handle most of the numerous ADAM substrates, in particular, the EGF ligands. However, broad-spectrum metalloprotease inhibitors tested for clinical use have failed as a result of indiscriminate blockade of substrate cleavage, leading to clinical side effects (11). Even recently developed selective ADAM inhibitors still affect the cleavage of many substrates (12). Modulation of the release of specific ADAM substrates has been impossible to date because it is unknown how cleavage specificity is regulated on the molecular level. It is therefore necessary to identify key signals that determine substrate specificity of cleavage.Ectodomain cleavage is made specific by a number of intracellular signals; e.g., by calcium influx, by activation of G protein-coup...

ADAM-10 and -17 regulate endometriotic cell migration via concerted ligand and receptor shedding feedback on kinase signaling

“…In contrast, ADAM-17 knockdown did not show an effect on basal cell motility (Fig. 4F), possibly due in part to previously reported adhesion-related protein functions (23)(24)(25). Notably, however, ADAM-17 activity (as inferred using PrAMA) did not significantly correlate with cell motility in a positive manner in the CSR dataset, and ADAM-17 IP+activity results significantly anticorrelated with features of cell migration.…”

α-Cyperone Inhibits PMA-Induced EPCR Shedding through PKC Pathway

“…To clarify the molecular mechanisms underlying the α-cyperone effects on EPCR shedding induced by 2 µM PMA, we first tested the protein, mRNA level and activity of TACE in HUVECs. TACE has been reported to mediate a number of protein cleavage events, [9][10][11][12][13] including EPCR 8) and its function mainly depends on its activity and protein expression, which we found could be inhibited by α-cyperone. It is well known that TACE is regulated by MAPK signaling which has been reported that ERK and p38 could activate TACE, 33,34) so we measured the effect of α-cyperone on the phosphorylation of MAPK induced by PMA.…”

“…α-Cyperone Inhibits PKC Activation Induced by PMA A series of experiments showed that TACE is also regulated by the PKC pathway [9][10][11][12][13] ; furthermore, PMA is also a potent PKC activator. All of these reports indicate that the activation of the PKC pathway is involved in EPCR shedding, but which PKC isoform is playing the pivotal role in this process is still unclear.…”

“…8) Furthermore, numerous in vitro experiments show that TACE is regulated by activated protein kinase C (PKC). [9][10][11][12][13] PKC is widely distributed in various tissues, organs and cells. In quiescent cells, PKC exists mainly in the cytoplasm.…”

Distinct Intracellular Domain Substrate Modifications Selectively Regulate Ectodomain Cleavage of NRG1 or CD44

“…This indicates that ectodomain cleavage requires tight regulation. How ectodomain cleavage is regulated and made substrate specific is largely unknown to date.The metalloproteases ADAM10 and ADAM17 are involved in the cleavage of most substrates that undergo regulated cleavage induced by intracellular signaling pathways, which are, in turn, activated by G protein-coupled receptors (GPCRs) or RTKs (2) involving the activation of protein kinase C (PKC) isoforms (5,28,29). An obvious way to regulate cleavage is modulation of the availability and activity of the enzymes.…”