A second mutant allele of furin in the processing-incompetent cell line, LoVo. Evidence for involvement of the homo B domain in autocatalytic activation.
Furin is a Golgi membrane-associated endoprotease that is involved in cleavage of various precursor proteins predominantly at Arg-X-Lys/Arg-Arg sites. Furin itself is synthesized as an inactive precursor, which is activated through intramolecular autocatalytic cleavage at an Arg-X-Lys-Arg site. We previously found that human colon carcinoma LoVo cells have a frameshift mutation within the homo B domain of furin and thereby lack processing activity toward Arg-X-Lys/Arg-Arg sites. In this study, however, we identified a second furin mutation in this cell line. The mutation, a replacement of a conserved Trp residue within the homo B domain with Arg, results in lack of processing activity of the mutant furin. The combination of both mutations can account for the recessive nature of the processing incompetence of LoVo cells. Immunofluorescence analysis with three distinct anti-furin monoclonal antibodies revealed that neither furin mutant underwent the autocatalytic activation or left the endoplasmic reticulum for the Golgi. These data indicate that the homo B domain as well as the catalytic domain is required for autocatalytic activation of furin.
Takahashi
S
S
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Nakagawa
T
T
Kasai
K
K
Banno
T
T
Duguay
S J
SJ
Van de Ven
W J
WJ
Murakami
K
K
Nakayama
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D045683:Furin; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013381:Subtilisins; D045683:Furin