Biochemical and biological analysis of Mek1 phosphorylation site mutants.
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.
Huang
W
W
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Kessler
D S
DS
Erikson
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D048369:MAP Kinase Kinase 1; D016200:Maturation-Promoting Factor / Q000378:metabolism; D008845:Microinjections; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D011518:Proto-Oncogene Proteins; D010768:Phosphoserine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016200:Maturation-Promoting Factor; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases