Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1.

Abstract

Many growth factors whose receptors are protein tyrosine kinases stimulate the MAP kinase pathway by activating first the GTP‐binding protein Ras and then the protein kinase p74raf‐1. p74raf‐1 phosphorylates and activates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf‐1. This represents the first characterization of sites phosphorylated by this proto‐oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A ‘kinase‐dead’ MAPKK1 mutant was phosphorylated at the same residues as the wild‐type enzyme, establishing that both sites are phosphorylated directly by p74raf‐1, and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf‐1 was low, indicating that phosphorylation of one of these sites is rate limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation.

Dissecting yeast Hog1 MAP kinase pathway using a chemical genetic approach

“…GST-Shm2 was purified from a yeast strain as described above and its enzymatic activity was measured as described earlier [33] with minor modifications. To measure the effect of either Hog1-as1 or p38 kinases, Shm2 enzyme was pre-incubated with each kinase in the presence of either 1 mM PE-ATP (for Hog1-as1) or 1 mM ATP (for p38) for 30 min at RT before adding to the reaction mixture.…”

Cot protooncoprotein activates the dual specificity kinases MEK-1 and SEK-1 and induces differentiation of PC12 cells

“…Samples were resuspended in 30 ml of kinase bu er containing 5 mCi [g-32 P]ATP (3000 Ci/mM, Amersham) and 4 mg of recombinant K97M kinase dead MEK mutant protein (MEK-kinase assay), 4 mg of recombinant K167R kinase dead GST-SEK mutant protein or a kinasedead ERK mutant protein and incubated at 308C for 15 min. A coupled assay was performed in the presence of 2 mg GST-MEK and 2 mg of the kinase-dead ERK mutant (Robbins et al, 1993;Alessi et al, 1994). Samples were resuspended in 30 ml SDS ± PAGE loading bu er (60 mM Tris/HCl, pH 6.8, 10% (v/v) glycerol, 3% (w/v) SDS, 5% (v/v) b-mercaptoethanol, 0.005% (w/v) bromphenol blue).…”

Post-transcriptional silencing of RET occurs, but is not required, during raf-1 mediated differentiation of medullary thyroid carcinoma cells

“…Activation of MEK1/2 was measured by Western analysis and an in vitro kinase assay. MEK1/2 is activated by phosphorylation of two serine residues within kinase subdomain VIII (Alessi et al, 1994;Zheng and Guan, 1994). An antibody speci®c for the phosphoserine form of MEK1/2 demonstrated high levels of activated MEK1/2 in b-estradiol di erentiated TT:DRaf-1:ER cells compared to very low levels of MEK1/2 activity in control cells (Figure 1a).…”

“…Coupled in vitro kinase assays (Alessi et al, 1994) were performed in order to con®rm that phosphorylation of MEK1/2 correlated with activation of the kinase properties of these proteins. Protein lysates were collected from TT:DRaf-1:ER cells treated for 48 h with b-estradiol or ethanol.…”