Core cysteine residues in the PAN domain are critical for HGF/c-MET signaling

Abstract

The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in > 28,000 proteins across 959 genera but its role in protein function is not fully understood. The PAN domain was initially characterized to be present in numerous proteins including hepatocyte growth factor (HGF). Dysregulation of HGF-mediated signaling results in numerous deadly cancers. All biological impacts of HGF in cell proliferation are triggered by binding of HGF to its cell surface receptor, cellular mesenchymal-epidermal transition (c-MET). Here, we show that four PAN domain cysteine residues are essential for HGF/c-MET signaling. Mutating these residues resulted in retardation of perinuclear localization, cellular internalization of HGF and its receptor, c-MET, and c-MET ubiquitination. Our observations indicate that the PAN domain of HGF is required for the c-MET binding and subsequent c-MET autophosphorylation and phosphorylation of its downstream targets, protein kinase B (AKT), extracellular signal-regulated kinase (ERK), and signal transducer and activator of transcription 3 (STAT3). Furthermore, transcriptional activation of HGF/c-MET signaling-related genes including matrix metalloproteinase-9 (MMP9), ETS translocation variant 1, 4, and 5 (ETV1, ETV4, ETV5), and early growth response 1 (EGR1) was impaired and cell proliferation was attenuated. These results suggest that core cysteine residues in the PAN domain are critical for HGF/c-MET interaction, c-MET mediated signal transduction, and cell survival. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway. SignificanceHGF/c-MET signaling induces multifunctional cellular responses. Dysregulation of HGF/c-MET signaling cascade can lead to tumorigenesis by transforming normal cells to tumor cells. This work defines the importance of core cysteine residues in the PAN domain of HGF in downstream activation of HGF/c-MET signaling. To understand the role of cysteines in the PAN domain, PAN mutants of HGF were used to stimulate c-MET signaling in cells and the impact was delineated by determining phosphorylation and transcription of downstream targets. Mutations in core cysteines in the HGF-PAN domain completely blocked downstream phosphorylation and perinuclear accumulation of c-MET. These results suggest an indispensable role for the cysteine-rich PAN domain in HGF/c-MET interaction and could set the stage for future therapies that selectively disrupt the MET signaling cascade with limited off-target effects in tumors overexpressing HGF/c-MET.