In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain.
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.
Widmann
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Department of Pediatrics, National Jewish Medical Research Center, Denver, CO 80206, USA. christian.widmann@ibcm.unil.ch
Sather
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Oyer
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Johnson
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Dreskin
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D003902:Detergents; D004795:Enzyme Stability; D006801:Humans; D008239:Lysine / Q000737:chemistry; D048730:MAP Kinase Kinase Kinase 2; D048748:MAP Kinase Kinase Kinase 3; D020930:MAP Kinase Kinase Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017830:Octoxynol; D011092:Polyethylene Glycols; D012694:Serine / Q000737:chemistry; D014162:Transfection; D014633:Valine / Q000737:chemistry
D003902:Detergents; D011092:Polyethylene Glycols; D012694:Serine; D017830:Octoxynol; C010615:Nonidet P-40; D048730:MAP Kinase Kinase Kinase 2; D048748:MAP Kinase Kinase Kinase 3; D020930:MAP Kinase Kinase Kinases; C482618:MAP3K2 protein, human; C482625:MAP3K3 protein, human; D014633:Valine; D008239:Lysine