THE ROLE OF COOH-TERMINAL AND ACIDIC DOMAINS IN THE ACTIVITY AND STABILITY OF HUMAN INSULIN-RECEPTOR PROTEIN-TYROSINE KINASE STUDIED BY PURIFIED DELETION MUTANTS OF THE BETA-SUBUNIT DOMAIN

We have previously expressed the human insulin receptor beta subunit d omain containing transmembrane and cytoplasmic domains (IRTMTPK) in in sect cells, and showed that the purified IRTMTPK was highly active (Li , S. L., Yan, P.-F., Pax, I. B., and Fujita-Yamaguchi, Y. (1992) Bioch emistry 31, 12455-12462). To investigate the role of COOH-terminal and acidic domains of the insulin receptor kinase, we have expressed dele tion mutants IRTMTPKDELTACT (DELTA76 amino acids) and IRTMTPKDELTAAcid (DELTA19 amino acids). Both enzymes were purified by a one-step metho d using the same immunoaffinity column as used for IRTMTPK. While K(m) and V(max) for prephosphorylated IRTMTPK and DELTAAcid mutant enzyme determined using poly(Glu, Tyr) (4:1) were similar, catalytic efficien cy of the DELTACT mutant enzyme was significantly lower than those of IRTMTPK and DELTAAcid mutant enzyme as judged by K(m) and V(max). Expe riments for thermostability and susceptibility to proteases revealed t hat T(m) of DELTACT mutant enzyme was 3.5-degrees-C lower than that of IRTMTPK enzyme (=33.3-degrees-C) and that DELTACT mutant enzyme was d igested by either trypsin or Lys-C into a 28,000 core domain much fast er than IRTMTPK. Activation of DELTACT mutant enzyme by polylysine was less significant than that of IRTMTPK and DELTAAcid mutant enzyme, ap proximately 4-versus approximately 17-fold. These studies suggested th at the COOH-terminal domain plays important roles in both catalytic ef ficiency and stability of the insulin receptor kinase, and that the ac idic domain by itself is not responsible for kinase activation by poly lysine.