Activation of JNK31 Requires both MKK4 and MKK7: Kinetic Characterization of in Vitro Phosphorylated JNK3
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- 作者：JeanMarie Lisnock ; Patrick Griffin ; Jimmy Calaycay ; Betsy Frantz ; Janey Parsons ; Stephen J. O'Keefe ; and Philip LoGrasso
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：March 21, 2000
- 年：2000
- 卷：39
- 期：11
- 页码：3141 - 3148
- 全文大小：129K
- 年卷期：v.39,no.11(March 21, 2000)
- ISSN：1520-4995
文摘
JNK3
1 is predominantly a neuronal specific MAP kinase that is believed to require, like allMAP kinases, both threonine and tyrosine phosphorylation for maximal enzyme activity. In this study weinvestigated the in vitro activation of JNK31 by MAP kinase kinase 4 (MKK4), MAP kinase kinase 7(MKK7), and the combination of MKK4 + MKK7. Mass spectral analysis showed that MKK7 was capableof monophosphorylating JNK31 in vitro, whereas both MKK4 and MKK7 were required for bisphosphorylation and maximal enzyme activity. Measuring catalysis under Vmax conditions showed MKK4 +MKK7-activated JNK31 had Vmax 715-fold greater than nonactivated JNK31 and MKK7-activatedJNK31 had Vmax 250-fold greater than nonactivated JNK31. In contrast, MKK4-activated JNK31had no increase in Vmax compared to nonactivated levels and had no phosphorylation on the basis of massspectrometry. These data suggest that MKK7 was largely responsible for JNK31 activation and that asingle threonine phosphorylation may be all that is needed for JNK31 to be active. The steady-state rateconstants kcat, Km(GST-ATF2), and Km(ATP) for both monophosphorylated and bisphosphorylated JNK31were within 2-fold between the two enzyme forms, suggesting the addition of tyrosine phosphorylationdoes not affect the binding of ATF2, ATP, or maximal turnover. Finally, the MAP kinase inhibitor,SB203580, had an IC50 value approximately 4-fold more potent on the monophosphorylated JNK31compared to the bisphosphorylated JNK31, suggesting only a modest effect of tyrosine phosphorylationon inhibitor binding.
1 is predominantly a neuronal specific MAP kinase that is believed to require, like allMAP kinases, both threonine and tyrosine phosphorylation for maximal enzyme activity. In this study weinvestigated the in vitro activation of JNK31 by MAP kinase kinase 4 (MKK4), MAP kinase kinase 7(MKK7), and the combination of MKK4 + MKK7. Mass spectral analysis showed that MKK7 was capableof monophosphorylating JNK31 in vitro, whereas both MKK4 and MKK7 were required for bisphosphorylation and maximal enzyme activity. Measuring catalysis under Vmax conditions showed MKK4 +MKK7-activated JNK31 had Vmax 715-fold greater than nonactivated JNK31 and MKK7-activatedJNK31 had Vmax 250-fold greater than nonactivated JNK31. In contrast, MKK4-activated JNK31had no increase in Vmax compared to nonactivated levels and had no phosphorylation on the basis of massspectrometry. These data suggest that MKK7 was largely responsible for JNK31 activation and that asingle threonine phosphorylation may be all that is needed for JNK31 to be active. The steady-state rateconstants kcat, Km(GST-ATF2), and Km(ATP) for both monophosphorylated and bisphosphorylated JNK31were within 2-fold between the two enzyme forms, suggesting the addition of tyrosine phosphorylationdoes not affect the binding of ATF2, ATP, or maximal turnover. Finally, the MAP kinase inhibitor,SB203580, had an IC50 value approximately 4-fold more potent on the monophosphorylated JNK31compared to the bisphosphorylated JNK31, suggesting only a modest effect of tyrosine phosphorylationon inhibitor binding.