The Potentiation Role of Hepatopoietin on Activator Protein-1 Is Dependent on Its Sulfhydryl Oxidase Activity

Abstract

Hepatopoietin (HPO) is a novel hepatotrophic growth factor that stimulates hepatocyte proliferation by two pathways. In the first, intracellular HPO specifically modulates the activator protein-1 (AP-1) pathway through JAB1 (Jun activation domain-binding protein 1), whereas in the second, extracellular HPO triggers the mitogen-activated protein kinase pathway by binding its specific receptor on the cell surface. In this report we demonstrate that HPO is a flavin-linked sulfhydryl oxidase, and the invariant CXXC (Cys-Xaa-Xaa-Cys) motif in HPO is essential for the enzyme activity of HPO but not for its dimerization nor for its binding ability with JAB1. Two intramolecular disulfides were identified in HPO by mass spectrometry, one of which is formed by the redox CXXC cysteine residues. HPO site-directed mutants (Cys/Ser) at active sites, which lost sulfhydryl oxidase activity, could not increase c-Jun phosphorylation and failed to potentiate JAB1-mediated AP-1 activation. However, the mutants still have mitogenic stimulation and mitogen-activated protein kinase activation effects on HepG2 cells. Thus, it can be concluded that the potentiation role of HPO on AP-1 is dependent on its sulfhydryl oxidase activity. Hepatopoietin (HPO)1 /augmenter of liver regeneration (ALR) is a novel human hepatotrophic growth factor. Since LaBrecque et al.(1) first reported hepatic stimulator substance in 1975, HPO has recently been the subject of intense investigation (2-10). Recombinant HPO can stimulate proliferation of hepatocytes as well as hepatoma cells in vitro, promote liver regeneration and recovery of damaged hepatocytes, and rescue acute hepatic failure in vivo (6, 7). In 1999 we identified the existence of HPO-specific receptor on the surface of these cells (8). Furthermore, we proposed that extracellular HPO stimulates proliferation of hepatocytes and enhances liver regeneration by activating the MAPK signaling pathway under the mediation of HPO receptor (9). Intriguingly, we further found that intracellular HPO can specifically modulate the AP-1 pathway through JAB1 via a MAPK-independent pathway and that HPO enhances the increased phosphorylation level of cJun through JAB1 but has no effect on the expression of transfected c-Jun or endogenous c-Jun N-terminal kinase nor on phosphorylation of c-Jun N-terminal kinase (10).Cytokines and growth factors stimulate AP-1 activity through several pathways (11), whereas the intracrine HPO regulates AP-1 transcriptional activity by an additional mechanism different from other cytokines and growth factors with mitogenic effects. It seems strange that intracellular and extracellular cytokine HPO have dissimilar actions in signal transduction. Recently, it was reported that the ERV1/HPO family belongs to sulfhydryl oxidase (SOX) participating in disulfide bond formation (12-17). The SOX proteins contain a conserved CXXC motif and a non-covalent FAD adjacent to CXXC, which are vital to their catalytic activity (18,19). Sulfhydryl oxidases generally form dimers in vivo and catalyze t...

Divergent Molecular Evolution of the Mitochondrial Sulfhydryl:Cytochrome c Oxidoreductase Erv in Opisthokonts and Parasitic Protists

“…6E). Similar spectra were recorded for mutants of the interchange residue Cys 130 in ScErv1 (16) and Cys 145 in human ALR (49,50). Thus, a CTC similar to many other flavoproteins also seems to be a conserved feature of the Erv family.…”

African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

“…In addition to this, intracellular HPO has been shown to interact with the transcriptional coactivator Jun activation domain-binding protein 1, designated JAB1, to regulate AP-1 transcriptional activity (23). Moreover, the potentiation of AP-1 activation by HPO appears to be dependent on its function as a sulfhydryl oxidase (6).…”

ALR, the multifunctional protein

“…Az ALR "rövid" (15 kDa; 125 AA) és "hosszú" (22 kDa; 198 vagy 205 AA) formája alternatív splicing termékként keletkezik, és nemcsak méretében, hanem funkciójában is eltér egymástól. A "rövid" ALR az emlősökben extracelluláris növekedési faktorként funkcionál [19], amely különböző specifi kus sejtfelszíni receptorokkal képes kapcsolódni [20]. A "hosszú" forma viszont -az élesz-tőhomológ ERV1-hez hasonlóan -a mitokondriális intermembrán terében lokalizálódik, és az oxidatív fehérje folding folyamatában nélkülözhetetlen diszulfi d-cserereakciókban vesz részt [21,22].…”

“…A JAB1-mediált foszforiláció pedig lehetővé teszi a c-Jun/AP1 (activator protein 1) fehérjekomplex létrejöttét, amely transzkripciós faktorként funkcionál a sejtekben [41]. Az ALR és a JAB1 közötti kölcsönhatás kialakulásához az ALR Cterminális doménjén lokalizálódó konzervált CXXC motívum megléte feltétlenül szükséges [19], ez a felfedezés viszont azt sugallja, hogy a folyamat esetleg az ALR enzimes redox funkciójához köthető. Az ALR és a JAB1 fehérje kapcsolata azonban jó eséllyel nem máj-specifi kus.…”