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 JNK3
1 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 JNK3
1 in vitro, whereas both MKK4 and MKK7 were required for bisphosphorylation and maximal enzyme activity. Measuring catalysis under
Vmax conditions showed MKK4 +MKK7-activated JNK3
1 had
Vmax 715-fold greater than nonactivated JNK3
1 and MKK7-activatedJNK3
1 had
Vmax 250-fold greater than nonactivated JNK3
1. In contrast, MKK4-activated JNK3
1had 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 JNK3
1 activation and that asingle threonine phosphorylation may be all that is needed for JNK3
1 to be active. The steady-state rateconstants
kcat,
Km(GST-ATF2), and
Km(ATP) for both monophosphorylated and bisphosphorylated JNK3
1were 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 IC
50 value approximately 4-fold more potent on the monophosphorylated JNK3
1compared to the bisphosphorylated JNK3
1, suggesting only a modest effect of tyrosine phosphorylationon inhibitor binding.