Activation of JNK3 alpha 1 requires both MKK4 and MKK7: Kinetic characterization of in vitro phosphorylated JNK3 alpha 1

JNK3 alpha 1 is predominantly a neuronal specific MAP kinase that is believ ed to require, like all MAP kinases, both threonine and tyrosine phosphoryl ation for maximal enzyme activity. In this study we investigated the in vit ro activation of JNK3 alpha 1 by MAP kinase kinase 4 (MKK4), MAP kinase kin ase 7 (MKK7), and the combination of MKK4 + MKK7. Mass spectral analysis sh owed that MKK7 was capable of monophosphorylating JNK3 alpha 1 in vitro, wh ereas both MKK4 and MKK7 were required for bisphosphorylation and maximal e nzyme activity. Measuring catalysis under V-max,, conditions showed MKK4 MKK7-activated JNK3 alpha 1 had V-max 715-fold greater than nonactivated JN K3 alpha 1 and MKK7-activated JNK3 alpha 1 had V-max 250-fold greater than nonactivated JNK3a1. In contrast, MKK4-activated JNK3 alpha 1 had no increa se in V,ax compared to nonactivated levels and had no phosphorylation on th e basis of mass spectrometry. These data suggest that MKK7 was largely resp onsible for JNK3 alpha 1 activation and that a single threonine phosphoryla tion may be all that is needed for JNK3 alpha 1 to be active. The steady-st ate rate constants k(cat), Km(GST-ATF2) and K-m(ATP) for both monophosphory lated and bisphosphorylated JNK3al were within 2-fold between the two enzym e forms, suggesting the addition of tyrosine phosphorylation does not affec t the binding of ATF2, ATP, or maximal turnover. Finally, the MAP kinase in hibitor, SB203580, had an IC50 value approximately 4-fold more potent on th e monophosphorylated JNK3 alpha 1 compared to the bisphosphorylated JNK3 al pha 1, suggesting only a modest effect of tyrosine phosphorylation on inhib itor binding.