Thermal inactivation of the protein tyrosine kinase of the epidermal growth factor receptor.
It has been shown previously that the EGF-stimulable protein tyrosine kinase in a membrane preparation from A431 cells was inactivated by heat shock (45 degrees C), under conditions where EGF binding was unaffected [Carpenter et al. (1979) J. Biol. Chem. 254, 4884-4891]. A later study found that in intact cells, the protein tyrosine kinase of the EGF receptor was insensitive to heat shock [Liu & Carpenter (1992) Biochem. J. 286, 541-547]. We have extended these previous studies to better understand the thermal stability of the protein tyrosine kinase of the EGF receptor. We have measured the rate of inactivation of the kinase in membrane vesicles in the physiological to heat shock temperature range (37-45 degrees C). At 45 degrees C, the protein tyrosine kinase is rapidly inactivated with a rate of approximately 0.14 min-1. There is, however, protection against inactivation by incubation of the EGF receptor with AMPPNP, a hydrolysis-resistant ATP analog. At 45 degrees C, the rate of inactivation of nucleotide-bound receptor is an order of magnitude lower than the rate of inactivation of unoccupied receptor. Analysis of the temperature dependence of inactivation between 37 and 45 degrees C yields an activation energy, E(a), of 42 kcal/mol, an activation Gibbs free energy, delta G(++), between 23 and 22 kcal/mol, an activation enthalpy, delta H(++), of 42 kcal/mol, and an activation entropy, delta S(++), of 60 cal/(K.mol). The signs and magnitudes of the thermodynamic parameters suggest that inactivation is more likely due to some local reorganization within the kinase domain than to a simple chemical process. Further, the kinetic data show that the receptor is stabilized against inactivation by binding adenine nucleotide.
Stein
R A
RA
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Staros
J V
JV
D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D002151:Calorimetry; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010766:Phosphorylation; D013816:Thermodynamics; D014407:Tumor Cells, Cultured
D000266:Adenylyl Imidodiphosphate; D004815:Epidermal Growth Factor; D066246:ErbB Receptors