Biochemical properties of the autophosphorylation of RLK5, a receptor-like protein kinase from Arabidopsis thaliana.
The RLK5 gene of Arabidopsis thaliana encodes a novel receptor-like protein kinase. DNA sequence analysis suggests that the RLK5 protein contains an extracellular domain that has 21 tandemly repeated leucine-rich motifs linked, via a transmembrane hydrophobic region, to a protein kinase catalytic domain that is related to the serine/threonine family of protein kinases. To study the intrinsic biochemical properties of this protein kinase we have expressed the catalytic domain as two different recombinant fusion proteins in Escherichia coli. Both hybrid proteins have similar kinetic properties, autophosphorylate on serine and threonine residues and have significantly greater activity in the presence of Mn2+ than Mg2+. A lysine to glutamic acid substitution in the catalytic domain of RLK5 results in the catalytically inactive protein RLK5(Cat)K711E. The active RLK5 protein can phosphorylate the inactive K711E protein and the K711E protein can partially inhibit the autophosphorylation of RLK5. Tryptic cleavage of the autophosphorylated proteins followed by two-dimensional thin layer electrophoresis indicates that several sites in the catalytic domain are phosphorylated.
Horn
M A
MA
Division of Biological Sciences, University of Missouri, Columbia 65211.
Walker
J C
JC
D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D004586:Electrophoresis; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D008345:Manganese; C089427:RLK5 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases; D008274:Magnesium