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 b oth fibronectin m and immunoglobulin C2 domains and, near the C termin us, a protein kinase domain with greatest homology to the catalytic do mains of myosin light chain kinases. We have expressed and purified fr om Escherichia coil twitchin's protein kinase catalytic core and flank ing sequences that do not include fibronectin III and immunoglobulin C 2 domains. The protein was shown to phosphorylate a model substrate an d 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 phosphopeptide s revealed that autophosphorylation occurred N-terminal to the catalyt ic core, predominantly at Thr-5910, with possible minor sites at Ser-5 912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of ca lcium and phosphotidylserine, but not by cAMP-dependent protein kinase . By comparing the activities of three twitchin segments, the enzyme a ppears to be inhibited by the 60-amino acid residues lying just C-term inal to the kinase catalytic core. Thus, Like a number of other protei n kinases including myosin Light chain kinases, the twitchin kinase ap pears to be autoregulated.