摘要：

Menadione formerly known as vitamin K3 is a synthetic derivative of naphthoquinone that has been widely used to study superoxide stress in mammalian system. 1 Menadione has also attracted attention because of its significant antican-cer activity in vitro 2 and in vivo. 3 Since the last decade, the cell growth inhibitory effect of this compound has been attributed to two principle mechanisms: oxidative stress 4 and arylation of cellular thiol. 5 Recent study has shown differences at the level of cell cycle arrest between the response of cells to hydrogen per-oxide and superoxide stress. 1f Thus, hydrogen peroxide and menadione treatment cause cells to arrest in G2 and G1, respectively. However, in the experiment reported by others, superoxide dismutase did not antagonize the growth inhibi-tory effects by menadione, suggesting that cell cycle arrest is not mainly due to generation of superoxide anion radical. 5c Another possible mechanism of toxicity of menadione is inhibition of sulfhydryl-dependent protein. Indeed, menadi-one has been proposed to inhibit cdc25 phosphatase. 6 Work from our laboratory has also shown that cdc25A phos-phatase was inactivated by menadione and that the loss of enzyme activity was due to the modification of the active site. 7 Cdc25 exists as three human homologues termed cdc25A, -B, and -C. 8 Since cdc25A is predominantly expressed in G1, 9 in contrast to cdc25B and cdc25C, which are mostly expressed in G2-M, 10 it is of interest to know whether the inactivation by menadione on cdc25A phos-phatase is specific. Therefore, we decided to determine the effect of menadione on p-nitrophenyl phosphate (p-NPP) hydrolysis catalyzed by cdc25B and -C phosphatases. To produce the cdc25B and -C phosphatases, standard molecular cloning procedures were followed. Reverse tran-scriptase PCR on total RNA from human HeLa cells with primers 11 produced the expected 1719-bp and 1440-bp prod-uct for cdc25B and -C, respectively. These fragments were inserted into BamHI and HindIII sites of pGEX-KG, an expression vector encoding GST. The resultant plasmids were introduced into the Escherichia coli BL21 strain and the GST-cdc25B and -C proteins were produced and puri-fied to apparent homogeneity as described previously. 12 Next purified phosphatases were incubated with the chro-mogenic substrate, 40 mM p-nitrophenyl phosphate in 20 mM Tris (pH 8.0), 1mM EDTA, and 0.2 mM DTT at 37.0 ± 0.1 o C. However, the result indicates that menadione also inactivates the cdc25B and -C phosphatases as well as cdc25A phosphatase (Table 1). Since evaluation of the rate of enzyme inactivation by menadione using the method of Kitz and Wilson 13 shows the similar range of K i and k inact , we conclude that there is no significant selectivity associated with the inactivation of different cdc25 phosphatases. Cdc25 phosphatases are dual-specificity phosphatases which dephosphorylate the phosphotyrosine residue as well as phosphoserine and phosphothreonine residues. Therefore, we subsequently tested whether or not menadione affects other phosphatases and found out to be that protein tyrosine phosphatase such as LAR, PTP1B, and Yersinia PTP, and protein-serine/threonine phosphatase such as PP1 and PP2A (up to 50 M) were not inactivated by menadione (data not shown). Protein tyrosine phosphatases have an 11-residue sequence motif that contains the catalytically essential Cys and Arg residue and that is also shared by cdc25 phos-phatases. Aside from the sequence His-Cys-(X)5-Arg, cdc25 phosphatases have no sequence homology with the catalytic domains of other protein tyrosine phosphatases or serine/ threonine phosphatases. Crystallographic investigation has also revealed that the active site of cdc25 phosphatases is extremely shallow in contrast to other non dual-specificity phosphatases. 14 We believe that these structural features may control the inactivation specificity between the dual-and non dual-specificity phosphatases by menadione, although menadione did not selectively inactivate the different cdc25 homologs. Acknowledgment. The author

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