Unusual proteolytic activation of pro-hepatocyte growth factor by plasma kallikrein and coagulation factor XIa.
Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an alpha-chain containing four Kringle domains (K1-K4) and a serine protease domain-like beta-chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro-HGF) into the biologically active two chain form by a single cleavage at the Arg(494)-Val(495) bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC(50)) of (125)I-labeled pro-HGF during a 4-h period were 10 and 17 nm. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N-terminal sequencing they were identified as the normal cleavage site Arg(494)-Val(495) and the novel site Arg(424)-His(425) located in the K4 domain of the alpha-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg(494) --> Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg(494)-Val(495) site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved alpha-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/c-Met pathway, such as tissue repair and angiogenesis.
Peek
Mark
M
Department of Physiology, Genentech Inc., South San Francisco, California 94080, USA.
Moran
Paul
P
Mendoza
Nerissa
N
Wickramasinghe
Dineli
D
Kirchhofer
Daniel
D
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D015956:Factor XIIa / Q000037:antagonists & inhibitors; D015945:Factor XIa / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000097:blood*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014633:Valine / Q000737:chemistry
D011994:Recombinant Proteins; D006639:Histidine; D017228:Hepatocyte Growth Factor; D001120:Arginine; D019859:Proto-Oncogene Proteins c-met; D007610:Kallikreins; D015945:Factor XIa; D015956:Factor XIIa; D014633:Valine