Activation of JNK3α1 Requires both MKK4 and MKK7: Kinetic Characterization of in Vitro Phosphorylated JNK3α1
Abstract
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.
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Tyr phosphorylation adds 25% activity by binding the C-lobe and further locking the active interlobe conformer.
“Phosphorylation of Tyr adds an additional 25% activity by binding to the C-lobe and further locking in the active interlobe conformer.”
From Structural Mechanisms of Allostery and Autoinhibition in JNK Family Kinases (2012) · cites this paper as Lisnock et al., 2000
After Thr phosphorylation, a movement makes Tyr accessible for phosphorylation by MKK7.
“Indeed, this has been shown biochemically for JNK3 because it was suggested that once Thr is phosphorylated, a movement occurred so that Tyr is now accessible for phosphorylation by MKK7 (Lisnock et al., 2000).”
From Structural Mechanisms of Allostery and Autoinhibition in JNK Family Kinases (2012) · cites this paper as Lisnock et al., 2000
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The Structure of JNK3 in Complex with Small Molecule Inhibitors
Scapin, G., Patel, S. B., Lisnock, J., Becker, J. W., & LoGrasso, P. V. (2003) · Chemistry & Biology · doi:10.1016/s1074-5521(03)00159-5 · Cited by 131 · see citing works
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Structural Mechanisms of Allostery and Autoinhibition in JNK Family Kinases
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“We prepared recombinant active JNK3α1 and JNK1β1 by incubation with active MKK4 and MKK7 (Chambers et al., 2011b; FigueraLosada and LoGrasso, 2012b; Kamenecka et al., 2009, 2010; Lisnock et al., 2000).”
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Self-citation
- Authors
- Lisnock, J., Griffin, P., Calaycay, J., Frantz, B., Parsons, J., O'Keefe, S. J., & LoGrasso, P.
- Affiliations
- Merck & Co., Inc., Rahway, NJ, USA (United States) (US)
- Departments of Molecular Design and Diversity, and Immunology and Rheumatology, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
- Corresponding author
- LoGrasso, Philip
- ORCID
- Griffin, Patrick
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2000-02-25
- Volume
- 39
- Issue
- 11
- Pages
- 3141-3148
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2240139143
- Cited by
- 79
- References
- 12
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 3 · 2023: 1 · 2022: 2 · 2021: 2 · 2020: 4 · 2018: 1 · 2017: 2 · 2016: 4 · 2015: 4 · 2013: 2 · 2012: 7
- FWCI
- 1.69
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Melanoma and MAPK Pathways
- Keywords
- MAPK activation, MKK4, MKK7, MAP kinase kinase, threonine phosphorylation, tyrosine phosphorylation, enzyme kinetics, Vmax, SB203580
- MeSH terms
- Adenosine Triphosphate, Binding Sites, Cloning, Molecular, Drug Synergism, Enzyme Activation, Enzyme Inhibitors, Humans, Imidazoles, Kinetics, Phosphorylation, Protein Kinases, Protein-Tyrosine Kinases, Pyridines, Recombinant Fusion Proteins, Transcription Factors, Mutagenesis, Insertional, Protein Serine-Threonine Kinases, Cyclic AMP Response Element-Binding Protein, Phosphotyrosine, Mitogen-Activated Protein Kinases, Mitogen-Activated Protein Kinase Kinases, Mitogen-Activated Protein Kinase 10, MAP Kinase Kinase 4, MAP Kinase Kinase 7, Activating Transcription Factor 2
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