Identification and characterization of an auto-activating MEK kinase from bovine brain: phosphorylation of serine-298 in the proline-rich domain of the mammalian MEKs.
Mitogen-activated protein kinase kinases (MKKs or MEKs) are dual specificity tyrosine/threonine protein kinases that are activated by phosphorylation at two closely spaced serine residues (serines-218 and -222) by the c-mos and raf proto-oncogenes. This double phosphorylation is both necessary and sufficient for MEKs to activate the MAP kinase enzymes in vitro. The specificity or regulation of in vivo signaling to the mammalian MEKs (MEK1 and MEK2) was recently reported also to involve the differential phosphorylation of a proline-rich peptide located between the MEK kinase-subdomains IX and X. Here we report the purification and characterization of an auto-activating protein kinase from bovine brain that phosphorylates serine-298 of the MEK1 and MEK2 proline-rich insert peptides. The auto-activation of the MEK-S298 peptide kinase is the result of an intermolecular phosphorylation event that can be prevented by the peptide substrates. The inactive kinase migrates on gel filtration as a 90 kDa protein, and after activation as a 43 kDa phosphoprotein. Incorporation of 32P[phosphate] into 40-42 kDa proteins on SDS-PAGE parallels the activation of the enzyme, and dephosphorylation by protein phosphatase 2Ac reverses the activation. SDS-PAGE renaturation assays show that the 40 kDa protein has the capacity to autophosphorylate, and exhibits kinase activity towards myelin basic protein after activation. Phosphorylation of purified bovine brain MEK or recombinant MEK1 by the auto-activated kinase does not activate the enzyme, and does not interfere with the in vitro raf-mediated MEK activation. We conclude that still unknown kinases may control the MAP kinase pathway by targeting MEK.
Wang
J
J
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Belgium.
Vantus
T
T
Merlevede
W
W
Vandenheede
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011392:Proline / Q000032:analysis*; D011489:Protein Denaturation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity
D010768:Phosphoserine; D011392:Proline; D017346:Protein Serine-Threonine Kinases