식물추출물에 의한 VHR의 활성 저해 효과 및 DUSP28의 특성분석

Abstract

Dual-specificity protein phosphatases (DUSPs) constitutes a family of protein phosphatase characterized by the ability to dephosphorylate phospho-tyrosyl and phospho-seryl/threonyl residues. Most of DUSPs are involved in regulation of cell survival and differentiation. The full-length Vaccinia H1-related phosphase (VHR) gene was amplified by PCR using the human cDNA. The amplified PCR product was subcloned into the NdeI - BamHI site of the pET28a(+) vector. BL21(DE3) E. coli cells harboring the VHR gene were grown at 18℃, and the protein expression was induced with 0.1mM IPTG for 16 hour. The His-tagged VHR protein was purified by nickel-affinity chromatography. The inhibitory effects of the 82 plant extracts on the VHR activity were measured using the p-nitrophenylphosphate (p-NPP) as a substrate. Empetrum nigrum L. var. japonicum K. Koch, Geranium nepalense subsp. thunbergii, Halorrhagis micrantha R. Br, Oenothera biennis L., and stems of Platycarya strobilacea Siebold et Zuccarini has strong inhibitory effects on the VHR activity. The serum-starved 293T cells were incubated with the plant extracts (0.02mg/ml) such as Empetrum nigrum L. var. japonicum K. Koch, Geranium nepalense subsp. thunbergii, Halorrhagis micrantha R. Br., Oenothera biennis L, and Oenothera biennis L for 1 hour and treated with the epidermal growth factor (EGF).The cell lyastes were prepared, and the levels of phosphorylated ERKs were determined by Western blotting using antibodies against phospho-p42/p44 ERK and p42/p44 ERK. Halorrhagis micrantha R. Br showed little increase in the phosphorylation of ERK in 293T cells, whereas Empetrum nigrum L. var. japonicum K. Koch, Geranium nepalense subsp. thunbergii, Platycarya strobilacea Siebold et Zuccarini, and Oenothera biennis L enhanced the phosphorylation of ERKs at 5 minute. Among the extracts tested, Platycarya strobilacea Siebold et Zuccarini showed the strongest accumulation of the phosphorylated ERKs at 5 minute, and a strong band of the phospho-p42/p44 ERK was detected after 1 hour, compared to those of the control and the other extracts. Although the extract of Platycarya strobilacea Siebold et Zuccarini has been used for various medicinal purposes, the inhibitory effect on phosphatase has not yet been reported. Our results demonstrate that No.56 is a potent candidate for the development of a VHR inhibitor. Next, a human dual-specificity protein phosphatase, DUSP28, was isolated from a human kidney cDNA. The recombinant protein was successfully produced in E. coli and showed a sufficient phosphatase activity toward DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate). Various phosphatase inhibitors and divalent metals were tested for their effects on the DUSP28 phosphatase activity. As results, Zn2+ was found to strongly inhibit DUSP28 phosphatase activity, suggesting DUSP28 is involved Zn-related signal transduction pathway. Furthermore, the DUSP28 protein preferred phospho-tyrosyl residues to phosphor-threonyl residues, implying its physiological roles in cellular process.