Abstract
A Hanks-type protein kinase AfsK autophosphorylates on threonine residue(s) and phosphorylates AfsR, a global regulator for secondary metabolism in Streptomyces coelicolor A3(2). Mass spectrometry of a tryptic digest of the autophosphorylated form of AfsKΔC corresponding to the kinase catalytic domain (Met-1 to Arg-311) of AfsK, together with subsequent site-directed mutagenesis of the candidate amino acids, identified threonine-168 as a single autophosphorylation site. Threonine-168 is located in the activation loop that is known for some Ser/Thr kinases to modulate kinase activity on phosphorylation of one or more threonine residues within the loop. Consistent with this, mutant T168D, in which Thr-168 was replaced by Asp, became a constitutively active kinase; it phosphorylated AfsR to the same extent as AfsKΔC produced in and purified from Escherichia coli cells during which a considerable population of it had been already phosphorylated intermolecularly. All these findings show that autophosphorylation or intermolecular phosphorylation of threonine-168 in AfsK accounts for the self-activation of its kinase activity.
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Tomono, A., Mashiko, M., Shimazu, T. et al. Self-activation of Serine/Threonine Kinase AfsK on Autophosphorylation at Threonine-168. J Antibiot 59, 117–123 (2006). https://doi.org/10.1038/ja.2006.18
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DOI: https://doi.org/10.1038/ja.2006.18
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