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MEKK1 phosphorylates MEK1 and MEK2 but does not cause activation of mitogen-activated protein kinase
1Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Abstract:
A constitutively active fragment of rat MEK kinase 1 (MEKK1) consisting of only its catalytic domain (MEKK-C) expressed in bacteria quantitatively activates recombinant mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) kinases 1 and 2 (MEK1 and MEK2) in vitro. Activation of MEK1 by MEKK-C is accompanied by phosphorylation of S218 and S222, which are also phosphorylated by the protein kinases c-Mos and Raf-1. MEKK1 has been implicated in regulation of a parallel but distinct cascade that leads to phosphorylation of N-terminal sites on c-Jun; thus, its role in the MAP kinase pathway has been questioned. However, in addition to its capacity to phosphorylate MEK1 in vitro, MEKK-C interacts with MEK1 in the two-hybrid system, and expression of mouse MEKK1 or MEKK-C in mammalian cells causes constitutive activation of both MEK1 and MEK2. Neither cotransfected nor endogenous ERK2 is highly activated by MEKK1 compared to its stimulation by epidermal growth factor in spite of significant activation of endogenous MEK. Thus, other as yet undefined mechanisms may be involved in determining information flow through the MAP kinase and related pathways.
Insights
A constitutively active fragment of MEK kinase 1 (MEKK1) activates MEK1 and MEK2 in vitro and in cells. This MEKK1 fragment phosphorylates MEK1 at sites targeted by other kinases, suggesting a role in the MAP kinase pathway.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Mitogen-activated protein (MAP) kinase pathways regulate crucial cellular processes.
- The precise role of MEK kinase 1 (MEKK1) in the MAP kinase pathway has been debated.
- MEKK1 has been linked to distinct signaling cascades, including those involving c-Jun.
Purpose of the Study:
- To investigate the activity and function of a catalytic domain fragment of MEKK1 (MEKK-C).
- To determine if MEKK-C can activate MEK1 and MEK2, key components of the MAP kinase pathway.
- To explore the interaction of MEKK-C with MEK1 in vitro and in cellular contexts.
Main Methods:
- Bacterial expression and purification of the MEKK-C catalytic domain.
- In vitro kinase assays using recombinant MEK1 and MEK2.
- Yeast two-hybrid system to assess protein-protein interactions.
- Expression of MEKK1 and MEKK-C in mammalian cells.
Main Results:
- MEKK-C quantitatively activated recombinant MEK1 and MEK2 in vitro.
- MEKK-C-mediated MEK1 activation involved phosphorylation at S218 and S222.
- MEKK1 and MEKK-C expression in mammalian cells led to constitutive activation of MEK1 and MEK2.
- Despite MEK activation, ERK2 activation by MEKK1 was limited compared to epidermal growth factor stimulation.
Conclusions:
- The catalytic domain of MEKK1 is sufficient for MEK1 and MEK2 activation.
- MEKK1 directly phosphorylates MEK1, supporting its role within the MAP kinase pathway.
- Further mechanisms likely regulate information flow downstream of MEK in response to MEKK1 signaling.