Self-activation of serine/threonine kinase AfsK on autophosphorylation at threonine-168.
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 AfsK deltaC 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 AfsK deltaC 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.
Tomono
Ayami
A
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Mashiko
Mari
M
Shimazu
Tadahiro
T
Inoue
Hirotaka
H
Nagasawa
Hiromichi
H
Yoshida
Minoru
M
Ohnishi
Yasuo
Y
Horinouchi
Sueharu
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005798:Genes, Bacterial; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D048372:Streptomyces coelicolor / Q000378:metabolism*; D013379:Substrate Specificity; D013912:Threonine; D014157:Transcription Factors / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C066581:AfsR protein, Streptomyces coelicolor; D013912:Threonine; D017346:Protein Serine-Threonine Kinases