MOLECULAR-CLONING, FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI, AND CHARACTERIZATION OF MULTIPLE MITOGEN-ACTIVATED-PROTEIN KINASES FROM TOBACCO

A screening of four tobacco cDNA libraries by PCR, using degenerate ol igonucleotides 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 m itogen-activated-protein kinases from other organisms. Full-length cDN As were obtained for two of these, ntf4 and ntf6, and we have previous ly reported the isolation of one of the other cDNAs, ntf3 [Wilson, C., Eller, N., Gartner, A., Vicente, O. & Heberle-Bors, E. (1993) Plant M ol. Biol. 23,543-551]. The three cDNAs, ntf3, ntf4 and ntf6, as well a s a mutated form of ntf3, were fused to the glutathione-S-transferase gene and expressed as fusion proteins in Escherichia coli. All three w ild-type recombinant proteins, with or without the glutathione-S-trans ferase 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 prote ins autophosphorylate on tyrosine residues and are recognized by antib odies prepared against mammalian mitogen-activated-protein kinases.