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08:05
Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Distinct patterns of activation-dependent changes in conformational mobility between ERK1 and ERK2
Adam Y Ring1, Kevin M Sours, Thomas Lee
1Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309.
International Journal of Mass Spectrometry
|July 26, 2011
Summary
MAP kinase ERK1 and ERK2 share similar structures but differ in activation mechanisms. Hydrogen/deuterium exchange mass spectrometry (HX-MS) reveals distinct regulation of hinge flexibility and interdomain closure between ERK1 and ERK2.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Hydrogen/deuterium exchange mass spectrometry (HX-MS) measures protein conformational mobility.
- Changes in mobility can impact protein function, even without observable structural changes.
- Previous studies suggested ERK2 activation involves increased hinge flexibility and interdomain closure.
Purpose of the Study:
- To investigate the role of hinge flexibility and interdomain closure in the activation mechanism of MAP kinase ERK1.
- To compare the activation mechanisms of ERK1 with the closely related ERK2.
- To determine if ERK1 activation also involves modulation of hinge flexibility.
Main Methods:
- Utilized hydrogen/deuterium exchange measurements by mass spectrometry (HX-MS) to assess conformational mobility in ERK1.
- Analyzed HX patterns upon activation to identify changes in flexibility.
- Performed HX-MS measurements of nucleotide binding to infer domain closure states.
Main Results:
- ERK1 and ERK2 exhibit similar overall HX patterns, consistent with their high sequence identity and similar tertiary structures.
- Unlike ERK2, ERK1 activation did not show altered HX at the hinge region.
- HX-MS indicated domain closure in both inactive and active forms of ERK1, suggesting a different regulatory mechanism than ERK2.
Conclusions:
- ERK1 and ERK2, despite structural similarities, employ distinct mechanisms for regulating enzyme function.
- ERK1 appears to maintain domain closure in both active and inactive states, differing from ERK2's mechanism involving hinge flexibility modulation.
- These findings highlight differences in interdomain interaction regulation between closely related MAP kinases.
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