Hsu C Y, Hurwitz D R, Mervic M, Zilberstein A
Rhône-Poulenc Rorer Central Research, King of Prussia, Pennsylvania 19406.
J Biol Chem. 1991 Jan 5;266(1):603-8.
The effect of autophosphorylation on the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) is not well understood. We previously demonstrated that phospholipase C-gamma physically associates with the EGF-activated EGFR, but not with a kinase-negative mutant of the EGFR, and, moreover, that only the tyrosine-phosphorylated EGFR is able to associate with phospholipase C-gamma. We have now investigated the effect of autophosphorylation on the tyrosine kinase activity of the EGFR by employing the purified kinase-active intracellular domain of the EGFR (EGFR-IC) produced by a baculovirus expression system. Synthetic peptides, including ones which contain the individual major tyrosine phosphorylation sites of phospholipase C-gamma, were used as substrates. We found that the extensively prephosphorylated EGFR-IC exhibited similar reaction kinetics to the unphosphorylated EGFR-IC when angiotensin II was used as a nonspecific substrate. In contrast there was a clear stimulation of kinase activity due to autophosphorylation of the EGFR-IC when peptides representing either the major autophosphorylation site of the EGFR or the EGFR phosphorylation sites of phospholipase C-gamma were used as substrates. However, the modes of stimulation for these peptides differed. The binding affinity (Km) for the unphosphorylated EGFR-IC for the peptide containing Tyr-771 of phospholipase C-gamma was relatively poor compared with other peptides, but improved 5-6-fold when the EGFR-IC was prephosphorylated. On the other hand, autophosphorylation improved the reaction velocity (Vm) of the phosphorylation of other peptides by 2-3-fold, with little or no increase in affinity. These results suggest that autophosphorylation of the EGFR may induce a conformational change of its kinase domain which enhances its kinase activity with exogenous substrates and may induce association with phospholipase C-gamma by increasing its affinity to a domain containing Tyr-771.
自身磷酸化对表皮生长因子受体（EGFR）酪氨酸激酶活性的影响尚未完全明确。我们之前证实，磷脂酶C-γ可与表皮生长因子激活的EGFR发生物理结合，但不能与EGFR的激酶阴性突变体结合，此外，只有酪氨酸磷酸化的EGFR才能与磷脂酶C-γ结合。我们现在利用杆状病毒表达系统产生的纯化的具有激酶活性的EGFR细胞内结构域（EGFR-IC），研究了自身磷酸化对EGFR酪氨酸激酶活性的影响。合成肽，包括含有磷脂酶C-γ各个主要酪氨酸磷酸化位点的肽，被用作底物。我们发现，当使用血管紧张素II作为非特异性底物时，广泛预磷酸化的EGFR-IC表现出与未磷酸化的EGFR-IC相似的反应动力学。相反，当使用代表EGFR主要自身磷酸化位点或磷脂酶C-γ的EGFR磷酸化位点的肽作为底物时，由于EGFR-IC的自身磷酸化，激酶活性有明显增强。然而，这些肽的刺激模式有所不同。未磷酸化的EGFR-IC对含有磷脂酶C-γ的Tyr-771的肽的结合亲和力（Km）与其他肽相比相对较差，但当EGFR-IC被预磷酸化时，亲和力提高了5-6倍。另一方面，自身磷酸化使其他肽磷酸化的反应速度（Vm）提高了2-3倍，而亲和力几乎没有增加或没有增加。这些结果表明，EGFR的自身磷酸化可能会诱导其激酶结构域的构象变化，从而增强其对外源底物的激酶活性，并可能通过增加其对含有Tyr-771的结构域的亲和力来诱导与磷脂酶C-γ的结合。
