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

Autor: Solovou TGA; Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500, Larissa, Greece., Garagounis C; Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500, Larissa, Greece., Kyriakis E; Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500, Larissa, Greece., Bobas C; Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500, Larissa, Greece., Papadopoulos GE; Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500, Larissa, Greece., Skamnaki VT; Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500, Larissa, Greece., Papadopoulou KK; Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500, Larissa, Greece. Electronic address: kalpapad@bio.uth.gr., Leonidas DD; Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500, Larissa, Greece. Electronic address: ddleonidas@bio.uth.gr.
Jazyk: angličtina
Zdroj: Phytochemistry [Phytochemistry] 2021 Jun; Vol. 186, pp. 112707. Date of Electronic Publication: 2021 Mar 13.
DOI: 10.1016/j.phytochem.2021.112707
Abstrakt: 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 °C following Michaelis-Menten kinetics with K m and Vmax values of 48.2 μM and 111.6 nmol/min/mg, respectively, for ATP. Mutation of critical residues, as inferred by sequence comparison to the human homologue GSK3β 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.
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Databáze: MEDLINE
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