TY - JOUR
T1 - Autophosphorylation activates the soluble cytoplasmic domain of the insulin receptor in an intermolecular reaction
AU - Cobb, M. H.
AU - Sang, B. C.
AU - Gonzalez, R.
AU - Goldsmith, E.
AU - Ellis, L.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1989
Y1 - 1989
N2 - The cytoplasmic protein-tyrosine kinase domain of the insulin receptor (residues 959-1355) has been expressed as a soluble protein in Sf9 insect cells via a Baculovirus expression vector (Ellis, L., Levitan, A., Cobb, M.H., and Ramos, P. (1988) J. Virol. 62, 1634-1639). The purified protein is a monomer as judged by its behavior in sucrose gradients and on gel filtration in the presence or absence of protamine. The initial rate of autophosphorylation using 3 mM MgCl2 is increased 20-30-fold by protamine. A maximum of 4-5 mol of phosphate are incorporated per mol of enzyme. The activity of the enzyme as a function of phosphorylation state was studied for three substrates: a synthetic dodecapeptide derived from the sequence of the major autophosphorylation site in the insulin receptor, poly(Glu,Tyr), 4:1, and histone 2B. Autophosphorylation of the protein to a stoichiometry of 4-5 mol of phosphate/mol increases its enzymatic activity as much as 200-fold; a 30-fold increase in activity occurs upon addition of 1 mol of phosphate/mol. The activities of unphosphorylated enzyme with the three substrates are 3.4, 2.3, and 0.44 nmol/min/mg, respectively. The activities of the autophosphorylated enzyme with the three substrates are 175, 274, and 45 nmol/min/mg, respectively. Exposure of the autophosphorylated enzyme to ADP results in a loss of phosphate from the enzyme which is associated with a decrease in enzymatic activity. Autophosphorylation of the kinase in the presence or absence of protamine displays a marked dependence on enzyme concentration. Furthermore, the rate of autophosphorylation decreases as the viscosity of the solution increases. Taken together, these data suggest that phosphorylation occurs via an intermolecular reaction.
AB - The cytoplasmic protein-tyrosine kinase domain of the insulin receptor (residues 959-1355) has been expressed as a soluble protein in Sf9 insect cells via a Baculovirus expression vector (Ellis, L., Levitan, A., Cobb, M.H., and Ramos, P. (1988) J. Virol. 62, 1634-1639). The purified protein is a monomer as judged by its behavior in sucrose gradients and on gel filtration in the presence or absence of protamine. The initial rate of autophosphorylation using 3 mM MgCl2 is increased 20-30-fold by protamine. A maximum of 4-5 mol of phosphate are incorporated per mol of enzyme. The activity of the enzyme as a function of phosphorylation state was studied for three substrates: a synthetic dodecapeptide derived from the sequence of the major autophosphorylation site in the insulin receptor, poly(Glu,Tyr), 4:1, and histone 2B. Autophosphorylation of the protein to a stoichiometry of 4-5 mol of phosphate/mol increases its enzymatic activity as much as 200-fold; a 30-fold increase in activity occurs upon addition of 1 mol of phosphate/mol. The activities of unphosphorylated enzyme with the three substrates are 3.4, 2.3, and 0.44 nmol/min/mg, respectively. The activities of the autophosphorylated enzyme with the three substrates are 175, 274, and 45 nmol/min/mg, respectively. Exposure of the autophosphorylated enzyme to ADP results in a loss of phosphate from the enzyme which is associated with a decrease in enzymatic activity. Autophosphorylation of the kinase in the presence or absence of protamine displays a marked dependence on enzyme concentration. Furthermore, the rate of autophosphorylation decreases as the viscosity of the solution increases. Taken together, these data suggest that phosphorylation occurs via an intermolecular reaction.
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M3 - Article
C2 - 2808393
AN - SCOPUS:0024450708
SN - 0021-9258
VL - 264
SP - 18701
EP - 18706
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 31
ER -