Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation.
The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene encodes a receptor-like protein kinase that is transiently expressed during embryogenesis. To determine the intrinsic biochemical properties of the AtSERK1 protein, we have expressed the intracellular catalytic domain as a glutathione S-transferase fusion protein in Escherichia coli. The AtSERK1-glutathione S-transferase fusion protein mainly autophosphorylates on threonine residues (K(m) for ATP, 4 x 10(-6) m), and the reaction is Mg(2+) dependent and inhibited by Mn(2+). A K330E substitution in the kinase domain of AtSERK1 abolishes all kinase activity. The active AtSERK1(kin) can phosphorylate inactive AtSERK1(K330E) protein, suggesting an intermolecular mechanism of autophosphorylation. The AtSERK1 kinase protein was modeled using the insulin receptor kinase as a template. On the basis of this model, threonine residues in the AtSERK1 activation loop of catalytic subdomain VIII are potential targets for phosphorylation. AtSERK1 phosphorylation on myelin basic protein and casein showed tyrosine, serine, and threonine as targets, demonstrating that AtSERK1 is a dual specificity kinase. Replacing Thr-468 with either alanine or glutamic acid not only obliterated the ability of the AtSERK1 protein to be phosphorylated but also inhibited phosphorylation on myelin basic protein and casein, suggesting that Thr-468 is essential for AtSERK-mediated signaling.
Shah
K
K
Laboratory of Molecular Biology, Wageningen University and Research Center, 6703 HA Wageningen, The Netherlands. kshah@helix.mgh.harvard.edu
Vervoort
J
J
de Vries
S C
SC
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism*
D029681:Arabidopsis Proteins; D013912:Threonine; D011494:Protein Kinases; C440035:SERK1 protein, Arabidopsis