Mutagenesis of a Lotus japonicus GSK3β/Shaggy-like kinase reveals functionally conserved regulatory residues

The glycogen synthase kinases 3 family (GSK3s/SKs; serine/threonine protein kinases) is conserved throughout eukaryotic evolution from yeast to plants and mammals. We studied a plant SK kinase from Lotus japonicus (LjSK1), previously implicated in nodule development, by enzyme kinetics and mutagenesis studies to compare it to mammalian homologues. Using a phosphorylated peptide as substrate, LjSK1 displays optimum kinase activity at pH 8.0 and 20 degrees C following Michaelis-Menten kinetics with K-m and V-max values of 48.2 mu M and 111.6 nmol/ min/mg, respectively, for ATP. Mutation of critical residues, as inferred by sequence comparison to the human homologue GSK3 beta and molecular modeling, showed a conserved role for Lys167, while residues conferring substrate specificity in the human enzyme are not as significant in modulating LjSK1 substrate specificity. Mutagenesis studies also indicate a regulation mechanism for LjSK1 via proteolysis since removal of a 98 residue long N-terminal segment increases its catalytic efficiency by almost two-fold. In addition, we evaluated the alteration of LjSK1 kinase activity in planta, by overexpressing the mutant variants in hairy-roots and a phenotype in nodulation and lateral root development was verified.

Automated summary

The study investigated a plant SK kinase (LjSK1) and found that it displayed optimal kinase activity at pH 8.0 and 20 degrees C, showing some differences compared to mammalian homologues. Mutagenesis studies revealed some conservation and regulatory mechanisms of LjSK1.