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 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-S-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.
Wilson
C
C
Vienna Biocenter, Institute of Microbiology and Genetics, University of Vienna, Austria.
Anglmayer
R
R
Vicente
O
O
Heberle-Bors
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D017343:Genes, Plant; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics*
D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases