Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.
Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated.
Lei
J
J
Department of Pathology, Emory University, Atlanta, Georgia 30322.
Tang
X
X
Chambers
T C
TC
Pohl
J
J
Benian
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins; D002148:Calmodulin-Binding Proteins; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D015801:Helminth Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D002148:Calmodulin-Binding Proteins; D015801:Helminth Proteins; D009124:Muscle Proteins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C489233:unc-22 protein, C elegans; D005982:Glutathione Transferase; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase