Reversibility of the epidermal growth factor receptor self-phosphorylation reaction. Evidence for formation of a high energy phosphotyrosine bond.
The reversibility of the epidermal growth factor (EGF) receptor self-phosphorylation reaction was studied using highly purified receptor from A431 human epidermoid carcinoma cells. Self-phosphorylation is inhibited by the reaction product ADP in a dose-dependent manner exhibiting an IC50 approximately 2 microM. In addition, phosphorylated EGF receptor can be rapidly dephosphorylated in the presence of ADP. The dephosphorylation reaction results in equimolar production of ATP and loss of phosphate from the receptor. The reverse reaction is dependent on time and ADP exhibiting a t1/2 of 15 s and a Km(ADP) = 0.40 +/- 0.14 microM. The dephosphorylation reaction can be effectively inhibited by an exogenous peptide substrate for the forward reaction, i.e., the src-peptide (a synthetic peptide corresponding to one of the self-phosphorylation sites in p60v-src). This suggests that the dephosphorylation reaction is intrinsic to the EGF receptor. The equilibrium constant, K, for the self-phosphorylation reaction was estimated to be 0.5-1.6 using kinetic and reactant/product concentration analyses. Assuming that the standard free energy change, delta G0, for ATP hydrolysis is -9.5 kcal/mol, an observed delta G0 for hydrolysis of the EGF receptor phosphotyrosine bond was calculated to be -9 to -10 kcal/mol. These results indicate that the EGF receptor self-phosphorylation reaction, which appears important in the regulation of EGF receptor function, is readily reversible and that the phosphotyrosine bond formed by this reaction is of relatively high energy.
Hubler
L
L
Department of Physiological Chemistry, University of Wisconsin-Madison 53706.
Gill
G N
GN
Bertics
P J
PJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D019000:Phosphotyrosine; D013816:Thermodynamics; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D019000:Phosphotyrosine; D014443:Tyrosine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D066246:ErbB Receptors