Proprotein Convertase PC7 Enhances the Activation of the EGF Receptor Pathway through Processing of the EGF Precursor

Estelle Rousselet,Suzanne Benjannet,Edwidge Marcinkiewicz,Marie-Claude Asselin,Claude Lazure,Nabil G Seidah

doi:10.1074/jbc.m110.189936

Estelle Rousselet, Suzanne Benjannet + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.189936

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Abstract

Although the processing profile of the membrane-bound epidermal growth factor precursor (pro-EGF) is tissue-specific, it has not been investigated at the cellular level nor have the cognate proteinases been defined. Among the proprotein convertases (PCs), only the membrane-bound PC7, the most ancient and conserved basic amino acid-specific PC family member, induces the processing of pro-EGF into an ∼115-kDa transmembrane form (EGF-115) at an unusual VHPR(290)↓A motif. Because site-directed mutagenesis revealed that Arg(290) is not critical, the generation of EGF-115 by PC7 is likely indirect. This was confirmed by testing a wide range of protease inhibitors, which revealed that the production of EGF-115 is most probably achieved via the activation by PC7 of a latent serine and/or cysteine protease(s). EGF-115 is more abundant at the cell surface than pro-EGF and is associated with a stronger EGF receptor (EGFR) activation, as evidenced by higher levels of phosphorylated ERK1/2. This suggests that the generation of EGF-115 represents a regulatory mechanism of juxtacrine EGFR activation. Thus, PC7 is distinct from the other PCs in its ability to enhance the activation of the cell surface EGFR.

Highlights

A wide variety of cancers express EGF receptor (EGFR) [5], clinical trials of EGFR inhibitors showed moderate beneficial effects [6]
PC7 Processes Pro-EGF into an ϳ115-kDa Membrane-bound Form—We first tested the possible implication of proprotein convertases (PCs) in the processing of the cell surface pro-EGF, which may mimic the undefined kidney protease reported to be implicated in proEGF maturation [12]
We co-expressed pro-EGF in HEK293 or Neuro2A cells with PC1/3, Furin, PC5/6A, PACE4, or PC7, the only basic aa-specific PCs that can function in the trans-Golgi network and/or cell surface [16]

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Reagents—All PCs constructions V5-tagged or not (mouse PC1, human Furin, mouse PC5/6A, human PACE4, full-length human PC7, full-length and soluble PC7 (PC7 and sPC7), rat PC7-KDEL, and rat PC7-GPI) were cloned into pIRES-2-GFP vector, as described previously [23, 31, 42]. The cells were washed and incubated in serum-free medium for 3 h before cell lysis The incubation of this co-culture with mouse recombinant EGF (20 ng/ml) was used as a positive control for the phosphorylation of ERK1/2. Biosynthesis was performed 48 h posttransfection, and the cells were washed and pulse- labeled in Cys/Met-free RPMI 1640 medium containing 0.2% BSA for 2 h with 250 ␮Ci/ml of [35S]Cys/Met for all experiments except for sulfation of pro-EGF (2 h with 500 ␮Ci/ml of Na235SO4). Cells were washed with buffer A (1ϫ PBS with 7.1 ml of 35% BSA and 2 ml of 250 mg/liter glucose) and stained with a rat anti-mouse pro-EGF primary antibody (1:250, R&D Systems) for 40 min at room temperature and with anti-rat IgG conjugated with Alexa 647 (1:750, Molecular Probes) for 20 min at room temperature. A difference between experimental groups was considered statistically significant whenever the p value was Ͻ0.05

RESULTS

Roche Applied Science

DISCUSSION