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Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1
Fred L Robinson1, Angelique W Whitehurst, Malavika Raman
1Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
Abstract:
Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2 small middle dotMEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding alpha-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding alpha-helix G in the interaction of ERK2 with MEK1/2.
Insights
Identifying specific mutations in ERK2 (extracellular signal-regulated kinase 2) reveals key interaction sites with MEK1/2 (MAPK/ERK kinase 1/2). These findings clarify how MEK1/2 activates ERK2, impacting cell signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are crucial for signal transduction, converting external stimuli into cellular responses.
- Mitogen-activated protein kinase (MAP) kinase kinases (MEKs), specifically MEK1 and MEK2, are the sole known activators of ERK1/2 through phosphorylation.
- The precise nature of ERK2-MEK1/2 complexes and their role in signaling remain under investigation.
Purpose of the Study:
- To identify specific point mutations in ERK2 that disrupt its interaction with MEK1/2 without affecting other protein interactions.
- To elucidate the structural regions of ERK2 critical for MEK1/2 binding and activation.
Main Methods:
- A yeast two-hybrid screen was employed to identify ERK2 mutants with impaired MEK1/2 interaction.
- Mutant ERK2 proteins were analyzed for activation levels in HEK293 cells following epidermal growth factor stimulation.
- The kinase activity and subcellular localization of mutant ERK2 proteins were assessed.
Main Results:
- Specific ERK2 residues, located on the C-terminal domain surface, within or near alpha-helix G, or in the MAP kinase insert, were identified as crucial for MEK1/2 interaction.
- Mutations differentially affected interaction with MEK1 versus MEK2, and reduced ERK2 activation in response to epidermal growth factor.
- Mutant ERK2 proteins retained kinase activity and showed constitutive nuclear localization, unlike wild-type ERK2.
Conclusions:
- The MAP kinase insert and residues near alpha-helix G are essential for the interaction between ERK2 and its activators, MEK1/2.
- Disruption of the ERK2-MEK1/2 interaction impairs signal-induced activation and alters subcellular localization.
- These findings provide critical insights into the structural basis of MAP kinase regulation and signaling specificity.