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 domain containing transmembrane and cytoplasmic domains (IRTMTPK) in insect cells, and showed that the purified IRTMTPK was highly active (Li, S. L., Yan, P.-F., Pax, I. B., and Fujita-Yamaguchi, Y. (1992) Biochemistry 31, 12455-12462). To investigate the role of COOH-terminal and acidic domains of the insulin receptor kinase, we have expressed deletion mutants IRTMTPK delta CT (delta 76 amino acids) and IRTMTPK delta Acid (delta 19 amino acids). Both enzymes were purified by a one-step method using the same immunoaffinity column as used for IRTMTPK. While Km and Vmax for prephosphorylated IRTMTPK and delta Acid mutant enzyme determined using poly(Glu, Tyr)(4:1) were similar, catalytic efficiency of the delta CT mutant enzyme was significantly lower than those of IRTMTPK and delta Acid mutant enzyme as judged by Km and Vmax. Experiments for thermostability and susceptibility to proteases revealed that Tm of delta CT mutant enzyme was 3.5 degrees C lower than that of IRTMTPK enzyme (= 33.3 degrees C) and that delta CT mutant enzyme was digested by either trypsin or Lys-C into a 28,000 core domain much faster than IRTMTPK. Activation of delta CT mutant enzyme by polylysine was less significant than that of IRTMTPK and delta Acid mutant enzyme, approximately 4-versus approximately 17-fold. These studies suggested that the COOH-terminal domain plays important roles in both catalytic efficiency and stability of the insulin receptor kinase, and that the acidic domain by itself is not responsible for kinase activation by polylysine.
Yan
P F
PF
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Li
S L
SL
Liang
S J
SJ
Giannini
S
S
Fujita-Yamaguchi
Y
Y
D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D011972:Receptor, Insulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013816:Thermodynamics
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011107:Polylysine; D011972:Receptor, Insulin; D010450:Endopeptidases