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 believed to require, like all MAP kinases, both threonine and tyrosine phosphorylation for maximal enzyme activity. In this study we investigated the in vitro activation of JNK3 alpha 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 capable of monophosphorylating JNK3 alpha 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 alpha 1 had Vmax 715-fold greater than nonactivated JNK3 alpha 1 and MKK7-activated JNK3 alpha 1 had Vmax 250-fold greater than nonactivated JNK3 alpha 1. In contrast, MKK4-activated JNK3 alpha 1 had no increase in Vmax compared to nonactivated levels and had no phosphorylation on the basis of mass spectrometry. These data suggest that MKK7 was largely responsible for JNK3 alpha 1 activation and that a single threonine phosphorylation may be all that is needed for JNK3 alpha 1 to be active. The steady-state rate constants kcat, Km(GST-ATF2++), and Km(ATP) for both monophosphorylated and bisphosphorylated JNK3 alpha 1 were within 2-fold between the two enzyme forms, suggesting the addition of tyrosine phosphorylation does 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 JNK3 alpha 1 compared to the bisphosphorylated JNK3 alpha 1, suggesting only a modest effect of tyrosine phosphorylation on inhibitor binding.
Lisnock
J
J
Department of Molecular Design and Diversity, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, USA.
Griffin
P
P
Calaycay
J
J
Frantz
B
B
Parsons
J
J
O'Keefe
S J
SJ
LoGrasso
P
P
D051698:Activating Transcription Factor 2; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004357:Drug Synergism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007700:Kinetics; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; D048057:Mitogen-Activated Protein Kinase 10; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007093:Imidazoles; D011725:Pyridines; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019000:Phosphotyrosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D048057:Mitogen-Activated Protein Kinase 10; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; C482559:MAP2K4 protein, human; C482569:MAP2K7 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C093642:SB 203580