INSECT CELL-EXPRESSED P180(ERBB3) POSSESSES AN IMPAIRED TYROSINE KINASE-ACTIVITY
INSECT CELL-EXPRESSED P180(ERBB3) POSSESSES AN IMPAIRED TYROSINE KINASE-ACTIVITY
复制标题
DOI：
10.1073/pnas.91.17.8132
复制
发表时间：
1994-08-16
影响因子：
11.1
通讯作者：
CARRAWAY, KL
中科院分区：
文献类型：
--
作者：
GUY, PM;PLATKO, JV;CARRAWAY, KL
蛋白激酶在其催化结构域中共享许多高度保守或不变的氨基酸残基，这表明这些残基是激酶活性所必需的。在属于表皮生长因子（EGF）受体亚家族的受体酪氨酸激酶p180（erbB3）中，其中三个残基发生改变，表明该蛋白可能具有受损的蛋白酪氨酸激酶活性。为了验证这一假设，我们通过杆状病毒感染在昆虫细胞中表达了人EGF受体和牛p180(erbB3)，并比较了它们的自磷酸化和底物磷酸化活性。我们发现，虽然EGF受体容易经历EGF刺激的自磷酸化并催化磷酸盐进入模型底物(E(4)Y(1) n（谷氨酸和酪氨酸随机4:1共聚物）和GST-p85（谷胱甘肽s -转移酶融合蛋白与85-kDa的磷脂酰肌醇3-激酶亚基），但p180（erbB3）的自磷酸化和底物磷酸化的效率至少低2个数量级。然而，p180（erbB3）能够结合ATP类似物5'-对氟磺酰基苯甲酰腺苷，这表明观察到的激酶活性缺乏可能不是由于昆虫细胞表达的无功能或变性受体。基于这些结果，我们提出p180（erbB3）具有受损的内在酪氨酸激酶活性。
Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180(erbB3), a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180(erbB3) in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E(4)Y(1))(n) (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180(erbB3) autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180(erbB3) is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180(erbB3) possesses an impaired intrinsic tyrosine kinase activity.