Molecular cloning, functional expression in Escherichia coli, and characterization of multiple mitogen-activated-protein kinases from tobacco

Eur J Biochem. 1995 Oct 1;233(1):249-57. doi: 10.1111/j.1432-1033.1995.249_1.x.

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-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Western
  • Calcium-Calmodulin-Dependent Protein Kinases / genetics*
  • Calcium-Calmodulin-Dependent Protein Kinases / isolation & purification
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • DNA, Plant / genetics
  • Escherichia coli / genetics
  • Gene Expression
  • Gene Library
  • Genes, Plant
  • Genetic Vectors
  • Molecular Sequence Data
  • Plants, Toxic*
  • Polymerase Chain Reaction
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Tobacco / enzymology*
  • Tobacco / genetics*

Substances

  • DNA, Complementary
  • DNA, Plant
  • Recombinant Proteins
  • Calcium-Calmodulin-Dependent Protein Kinases

Associated data

  • GENBANK/X83879
  • GENBANK/X83880