title: A unique cathepsin-like protease isolated from CV-1 cells is involved in rapid degradation of retinoblastoma susceptibility gene product, RB, and transcription factor SP1.
abstract: The regulation of transcription factors by kinase or phosphatase has been well-described. However, little is known about the inactivation of transcription factors or the nuclear regulators by proteolytic degradation. In this report, we purified a specific protease, SPase, from nuclear extracts of the green monkey kidney cell line, CV-1. Studies of biochemical characteristics and substrate specificity indicated that SPase is a cathepsin B-like cysteinyl protease. However, the two tryptic peptide sequences derived from the purified SPase are either identical or highly homologous to those of human cathepsin L, and furthermore, SPase shares immunoreactivity with both anti-human cathepsin L and anti-mouse cathepsin L antibody. The SPase was shown to be localized in both cytoplasm and nucleus when subcellular compartments of CV-1 cells were fractionated. Transcription factor, SP1, and retinoblastoma susceptible gene product, RB, are substrates of SPase while other nuclear factors such as c-Jun and c-Fos are not. These results implied that SPase plays an integral role in regulating a set of proteins in the nuclei. In vivo treatment of CV-1 cells with cysteinyl protease inhibitor, E-64d, protected RB from degradation. SPase failed to degrade underphosphorylated RB present in TPA induced terminally differentiated HL-60 or U937 cells. Phosphorylation of RB may cause conformational changes, thus facilitating proteolytic digestion. These observations suggest that an alternative pathway inactivates the function of RB in controlling cell growth. Therefore, a possible role of SPase may be to affect the stability of important regulators involved in controlling cellular proliferation and differentiation.
authors.affiliation: Department of Surgery, School of Medicine, University of California, Los Angeles 90095-1782, USA.
authors.forename: T
authors.initials: T
authors.lastname: Nishinaka
authors.forename: Y H
authors.initials: YH
authors.lastname: Fu
authors.forename: L I
authors.initials: LI
authors.lastname: Chen
authors.forename: K
authors.initials: K
authors.lastname: Yokoyama
authors.forename: R
authors.initials: R
authors.lastname: Chiu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D056668:Cathepsin L; D002403:Cathepsins / Q000276:immunology; D002455:Cell Division; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D010450:Endopeptidases; D018922:HL-60 Cells / Q000187:drug effects; D000882:Haplorhini; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D016160:Retinoblastoma Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
chemical_list: D002457:Cell Extracts; D015853:Cysteine Proteinase Inhibitors; D009687:Nuclear Proteins; D016160:Retinoblastoma Protein; D016329:Sp1 Transcription Factor; D002403:Cathepsins; D010450:Endopeptidases; C105586:CV-1 SPase; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse