In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain

Détails

ID Serval
serval:BIB_56156C81B40F
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain
Périodique
Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology
Auteur(s)
Widmann  C., Sather  S., Oyer  R., Johnson  G. L., Dreskin  S. C.
ISSN
0167-4838
ISSN-L
1879-2588
Statut éditorial
Publié
Date de publication
05/2001
Peer-reviewed
Oui
Volume
1547
Numéro
1
Pages
167-73
Notes
Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. --- Old month value: May 5
Résumé
MEKK2 and MEKK3 are mitogen-activated protein kinase kinase kinases (MAP3 kinases) of 70 and 71 kDa respectively that are markedly homologous (94%) in their kinase domains. Both MEKK2 and MEKK3 are able to activate the Jun kinase pathway in vivo. However, following routine immunoprecipitation in Triton X-100, MEKK2 but not MEKK3 is able to effectively phosphorylate both SEK-1 and MEK-1 and to undergo autophosphorylation. Unexpectedly, both MEKK2 and MEKK3 are functional in an in vitro kinase assay when cells are solubilized with the closely related detergent, NP-40. Given the high homology between these kinases, we set out to relate this differential sensitivity to Triton X-100 to differences in primary structure. A set of chimeric molecules were generated and the loss of activity in Triton X-100 mapped to kinase domain II/III and specifically to serine 390 of MEKK3 and valine 384 of MEKK2, residues immediately N-terminal to the active site lysine. Mutation of serine 390 of MEKK3 to a valine (as is found in MEKK2) conferred catalytic activity to MEKK3 in Triton X-100 whereas the reciprocal alteration of valine 384 of MEKK2 to a serine conferred lack of activity in Triton X-100 to MEKK2. Search of the protein database identified only three kinases, MEKK3, Pbs2p and Dd-PKI, with a serine or threonine at this site. The presence of a serine or threonine adjacent to the active site lysine in protein kinases is rare and, in MEKK3, results in detergent instability.
Mots-clé
Amino Acid Sequence Binding Sites Catalysis Cell Line *Detergents Enzyme Stability Humans Lysine/chemistry MAP Kinase Kinase Kinase 2 MAP Kinase Kinase Kinase 3 MAP Kinase Kinase Kinases/biosynthesis/*chemistry/genetics Molecular Sequence Data Mutagenesis, Site-Directed Octoxynol Polyethylene Glycols Serine/chemistry Transfection Valine/chemistry
Pubmed
Web of science
Création de la notice
24/01/2008 15:43
Dernière modification de la notice
20/08/2019 15:10
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