Molecular Cloning, Functional Expression in Escherichia coli, and Characterization of Multiple Mitogen-Activated-Protein Kinases from Tobacco

Authors


  • Note. The novel nucleotide sequence data published here have been submitted to the EMBL nucleotide sequence database and are available under the accession numbers X83880 (ntf4) and X83879 (ntf6).

O. Vicente, Vienna Biocenter, Institute of Microbiology and Genetics, Dr Bohr-Gasse 9, A-1030 Vienna, Austria
Fax:+43 1 79 515 4114.

Abstract

A screening of four tobacco cDNA libraries by PCR, using degenerate oligonucleotides corresponding to motifs conserved in mitogen-activated-protein kinases from animals and yeasts, resulted in the isolation of five different PCR fragments that showed high sequence similarity to mitogen-activated-protein kinases from other organisms. Full-length cDNAs were obtained for two of these, ntf4 and ntf6, and we have previously reported the isolation of one of the other cDNAs, ntf3 [Wilson, C., Eller, N., Gartner, A., Vicente, O. & Heberle-Bors, E. (1993) Plant Mol. Biol. 23, 543–551], The three cDNAs, ntf3, ntf4 and ntf6, as well as a mutated form of ntf3, were fused to the glutathione-S-transferase gene and expressed as fusion proteins in Escherichia coli. All three wild-type recombinant proteins, with or without the glutathione-5-transferase fragment, are capable of autophosphorylation and phosphorylate myelin basic protein, in a reaction that is more strongly supported by Mn2+ than by Mg2+, while the kinase-negative Ntf3 mutant did not show any activity. Western-blot analysis showed that the recombinant proteins autophosphorylate on tyrosine residues and are recognized by antibodies prepared against mammalian mitogen-activated-protein kinases.

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