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Biochemical and biological analysis of Mek1 phosphorylation site mutants
W Huang1, D S Kessler, R L Erikson
1Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Abstract:
Recently, we described the constitutive activation of Mek1 by mutation of its two serine phosphorylation sites. We have now characterized the biochemical properties of these Mek1 mutants and performed microinjection experiments to investigate the effect of an activated Mek on oocyte maturation. Single acidic substitution of either serine 218 or 222 activated Mek1 by 10-50 fold. The double acidic substitutions, [Asp218, Asp222] and [Asp218, Glu222], activated Mek1 over 6000-fold. The specific activity of the [Asp218, Asp222] and [Asp218, Glu222] Mek1 mutants, 29 nanomole phosphate per minute per milligram, is similar to that of wild-type Mek1 activated by Raf-1 in vitro. Although the mutants with double acidic substitutions could not be further activated by Raf-1, three of those with single acidic substitution were activated by Raf-1 to the specific activity of activated wild-type Mek1. Injection of the [Asp218, Asp222] Mek1 mutant into Xenopus oocytes activated both MAP kinase and histone H1 kinase and induced germinal vesicle breakdown, an effect that was only partially blocked by inhibition of protein synthesis. These data provide a measure of Mek's potential to influence cell functions and a quantitative basis to assess the biological effects of Mek1 mutants in a variety of circumstances.
Insights
Constitutively active Mek1 mutants were created by mutating phosphorylation sites. These mutants, particularly double substitutions, showed significant activation, influencing oocyte maturation and cell signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Mek1 is a key kinase in the MAPK pathway.
- Phosphorylation of Mek1 at serine 218 and 222 regulates its activity.
- Understanding Mek1 activation is crucial for cell function studies.
Purpose of the Study:
- To biochemically characterize constitutively active Mek1 mutants.
- To investigate the impact of activated Mek1 on oocyte maturation.
- To establish a quantitative basis for assessing Mek1 mutant effects.
Main Methods:
- Site-directed mutagenesis to create Mek1 serine phosphorylation site mutants.
- In vitro kinase assays to determine specific activity.
- Microinjection into Xenopus oocytes to assess biological effects.
Main Results:
- Single acidic substitutions activated Mek1 10-50 fold; double substitutions activated Mek1 over 6000-fold.
- Highly activated mutants ([Asp218, Asp222] and [Asp218, Glu222]) exhibited specific activity comparable to Raf-1 activated wild-type Mek1.
- Microinjection of the [Asp218, Asp222] Mek1 mutant induced oocyte maturation (germinal vesicle breakdown) and activated MAP kinase and histone H1 kinase.
Conclusions:
- Constitutive activation of Mek1 is achievable through specific acidic substitutions at serine 218 and 222.
- Activated Mek1 can induce oocyte maturation, demonstrating its significant role in cell cycle progression.
- These findings provide a valuable tool for studying Mek1 function and its impact on cellular processes.