Copper modulates zinc metalloproteinase-dependent ectodomain shedding of key signaling and adhesion proteins and promotes the invasion of prostate cancer epithelial cells

A disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) are zinc metalloproteinases (ZMPs) that catalyse the 'ectodomain shedding' of a range of cell surface proteins including signalling and adhesion molecules. These 'sheddases' are associated with the invasion and metastasis of a range of cancers. Increased serum and tumour tissue levels of copper are also observed in several cancers although little is known about how the metal might promote disease progression at the molecular level. In the current study, we investigated whether copper might regulate the ectodomain shedding of two key cell surface proteins implicated in the invasion and metastasis of prostate cancer, the Notch ligand Jagged1 and the adhesion molecule E-cadherin, and whether the metal was able to influence the invasion of the prostate cancer epithelial cell line PC3. Physiological copper concentrations stimulated the ZMP-mediated proteolysis of Jagged1 and E-cadherin in cell culture models whilst other divalent metals had no effect. Copper-mediated Jagged1 proteolysis was also observed following the pre-treatment of cells with cycloheximide and in a cell-free membrane system, indicating a post-translational mechanism of sheddase activation. Finally, the concentrations of copper that stimulated ZMP-mediated protein shedding also enhanced PC3 invasion; an effect which could be negated using a sheddase inhibitor or copper chelators. Collectively, these data implicate copper as an important factor in promoting prostate cancer cell invasion and indicate that the selective post-translational activation of ZMP-mediated protein shedding might play a role in this process.

Ectodomain shedding of the Notch ligand Jagged1 is mediated by ADAM17, but is not a lipid-raft-associated event

Notch signalling is an evolutionarily conserved pathway involved in cell-fate specification. The initiating event in this pathway is the binding of a Notch receptor to a DSL (Delta/Serrate/Lag-2) ligand on neighbouring cells triggering the proteolytic cleavage of Notch within its extracellular juxtamembrane region; a process known as proteolytic 'shedding' and catalysed by members of the ADAM (a disintegrin and metalloproteinase) family of enzymes. Jagged1 is a Notch-binding DSL ligand which is also shed by an ADAM-like activity raising the possibility of bi-directional cell-cell Notch signalling. In the present study we have unequivocally identified the sheddase responsible for shedding Jagged1 as ADAM17, the activity of which has previously been shown to be localized within specialized microdomains of the cell membrane known as 'lipid rafts'. However, we have shown that replacing the transmembrane and cytosolic regions of Jagged1 with a GPI (glycosylphosphatidylinositol) anchor, thereby targeting the protein to lipid rafts, did not enhance its shedding. Furthermore, the Jagged1 holoprotein, its ADAM-cleaved C-terminal fragment and ADAM17 were not enriched in raft preparations devoid of contaminating non-raft proteins. We have also demonstrated that wild-type Jagged1 and a truncated polypeptide-anchored variant lacking the cytosolic domain were subject to similar constitutive and phorbol ester-regulated shedding. Collectively these data demonstrate that Jagged1 is shed by ADAM17 in a lipid-raft-independent manner, and that the cytosolic domain of the former protein is not a pre-requisite for either constitutive or regulated shedding