Isoflavonoids are important for human health and for stress resistance in plants. To increase the production of isoflavonoids in plants, an understanding of the biosynthetic pathway is critical. One of the key steps in the isoflavonoid pathway is the formation of chalcone by CHS8, which is regulated by GmMYB176 (Yi et al., 2010b). GmMYB176 interacts with 14-3-3 proteins, and serine-29 in GmMYB176 is critical for the interaction (Li et al., 2012). It is hypothesized that GmMYB176 is phosphorylated at serine-29 by a specific protein kinase. GmMYB176 interacting protein kinases were identified in a co- immunoprecipitation experiment and are being characterized.
From the in silico analysis, it was known that Gm02PK carries the least potential to be an active kinase, as it lacks three critical residues for phosphorylation. The remaining candidate protein kinases have critical residues conserved, and thus likely have the ability to phosphorylate its substrate. Furthermore, these seven candidate protein kinases show interaction with GmMYB176 in the nucleus in planta. The subcellular localization study revealed that Gm08PK.1 and Gm08PK.2 localize to the nucleus while Gm02PK, Gm04PK and Gm14PK localize to the endoplasmic reticulum. The other protein kinases Gm17PK and Gm17PPD localize to the plasma membrane and to the plastid, respectively. It is not known what molecular mechanism(s) is responsible for the observation that GmPKs localizing to different compartments of the cell still interact with GmMYB176 in the nucleus.
As Gm08PK.1 and Gm08PK.2 localize to the nucleus, and show interaction with the GmMYB176, they are selected to test if they are GmMYB176-specific protein kinase. Recombinant Gm08PK.1/Gm08PK.2 proteins were produced and purified. The purified recombinant protein, Gm08PK.1/Gm08PK.2 was used in conjunction with GmMYB176 peptide (SMRKSVSMNNLSQY) in the in vitro kinase assay. Both of the candidate protein kinases showed comparatively high optical density than negative control in the in vitro kinase assays. This result suggests that both candidate protein kinases phosphorylate GmMYB176 in vitro. However, experiments with additional controls should be conducted to conclude Gm08PK.1 or Gm08PK.2 phosphorylates GmMYB176 in vitro. Furthermore, if phosphorylation in GmMYB176 is occurring in the kinase reactions, it is essential to
identify serine residue that is phosphorylated in the GmMYB176 peptide. It is possible that Gm08PK.1/Gm08PK.2 phosphorylate serine residue in GmMYB176 other than serine-29. In that case, in order to identify the GmMYB176-specific protein kinase that phosphorylates at serine-29, in vitro kinase assay should be performed with each remaining candidate protein kinases.
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