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 th e biochemical properties of these Mek1 mutants and performed microinje ction experiments to investigate the effect of an activated Mek on ooc yte maturation. Single acidic substitution of either serine 218 or 222 activated Mek1 by 10-50 fold. The double acidic substitutions, [Asp(2 18) Asp(222)] and [Asp(218), Glu(222)], activated Mek1 over 6000-fold. The specific activity of the [Asp(218), Asp(222)] and [Asp(218), Glu( 222)] Mek1 mutants, 29 nanomole phosphate per minute per milligram, is similar to that of wild-type Mek1 activated by Raf-1 in vitro. Althou gh the mutants with double acidic substitutions could not be further a ctivated by Raf-1, three of those with single acidic substitution were activated by Raf-1 to the specific activity of activated wild-type Me k1. Injection of the [Asp(218), Asp(222)] Mek1 mutant into Xenopus ooc ytes activated both MAP kinase and histone H1 kinase and induced germi nal vesicle breakdown, an effect that was only partially blocked by in hibition of protein synthesis. These data provide a measure of Mek's p otential to influence cell functions and a quantitative basis to asses s the biological effects of Mek1 mutants in a variety of circumstances .