A Second Mutant Allele of Furin in the Processing-incompetent Cell Line, LoVo

doi:10.1074/jbc.270.44.26565

A Second Mutant Allele of Furin in the Processing-incompetent Cell Line, LoVo

EVIDENCE FOR INVOLVEMENT OF THE HOMO B DOMAIN IN AUTOCATALYTIC ACTIVATION (*)

From the (1) Institute of Applied Biochemistry
(2) Institute of Basic Medical Sciences
(3) Tsukuba Advanced Research Alliance Center, and
(4) Institute of Biological Sciences and Gene Experiment Center, University of Tsukuba, Tsukuba, Ibaraki 305, Japan the
(5) Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, and the
(6) Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven, B-3000 Leuven, Belgium

^§§ To whom correspondence should be addressed:
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
Tel.: 81-298-53-6356; Fax: 81-298-53-6006; kazunaka{at}sakura.cc.tsukuba.ac.jp.

↵^§ Present address: Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan.

Abstract

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.

Footnotes

↵^¶ The first two authors contributed equally to this work.
↵** Recipient of a fellowship from the Japanese Society for the Promotion of Science for Japanese Junior Scientists.
↵* This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, the Special Research Project on Circulation Biosystem in the University of Tsukuba, the Saneyoshi Scholarship Foundation, the Nissan Science Foundation, the Ciba-Geigy Foundation (Japan) for the Promotion of Science, and Sankyo Co., Ltd. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

RT-PCR

reverse transcriptase-polymerase chain reaction.
- Received July 6, 1995.
- Revision received August 25, 1995.