Guy P M, Carraway K L, Cerione R A
Department of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853-6401.
J Biol Chem. 1992 Jul 15;267(20):13851-6.
The rat neu oncogene product is a member of the epidermal growth factor (EGF) receptor subgroup of the superfamily of growth factor receptor tyrosine kinases. The oncogenic activation of the neu protein occurs by a point mutation within its transmembrane region which results in an increase in its tyrosine kinase activity. Using three different forms of neu expressed in insect cells via baculovirus infection, we have examined the biochemical differences between the normal and transforming forms of neu and investigated the role of the transmembrane domain in its tyrosine kinase activity. One form of neu which was expressed in insect cells consisted of the complete tyrosine kinase domain but lacked the extracellular and transmembrane regions (designated NTK). The other two forms consisted of the tyrosine kinase domain, the transmembrane domain, and 40 amino acids of the extracellular domain. One of these transmembrane forms of neu contained the normal valine residue at position 664 within the transmembrane region (MS-N), while the other contained the oncogenic glutamic acid residue at this position (MS-T). Direct comparisons of NTK, MS-N, and MS-T have shown that the NTK protein is capable of the highest extents of both autophosphorylation activity and the tyrosine phosphorylation of exogenous substrate, suggesting that the presence of the transmembrane region of neu suppresses the tyrosine kinase activity of this receptor. In addition, we have found that the oncogenic point mutation within the transmembrane region stimulates the tyrosine kinase activity of the neu protein by allowing it to more effectively utilize Mg2+. Overall, the results of these studies suggest that the valine to glutamic acid substitution at position 664 may at least partially relieve a negative constraint imparted by the membrane-spanning domain on the tyrosine kinase activity of neu and enables a more effective use of Mg2+ in the catalysis of tyrosine phosphorylation of exogenous substrates.
大鼠neu癌基因产物是生长因子受体酪氨酸激酶超家族中表皮生长因子（EGF）受体亚组的成员。neu蛋白的致癌激活是由其跨膜区域内的一个点突变引起的，该突变导致其酪氨酸激酶活性增加。通过杆状病毒感染在昆虫细胞中表达三种不同形式的neu，我们研究了正常形式和转化形式的neu之间的生化差异，并研究了跨膜结构域在其酪氨酸激酶活性中的作用。在昆虫细胞中表达的一种neu形式由完整的酪氨酸激酶结构域组成，但缺少细胞外和跨膜区域（称为NTK）。另外两种形式由酪氨酸激酶结构域、跨膜结构域和细胞外结构域的40个氨基酸组成。这些neu的跨膜形式之一在跨膜区域的第664位含有正常的缬氨酸残基（MS-N），而另一种在该位置含有致癌的谷氨酸残基（MS-T）。对NTK、MS-N和MS-T的直接比较表明，NTK蛋白具有最高程度的自磷酸化活性和对外源底物的酪氨酸磷酸化能力，这表明neu跨膜区域的存在抑制了该受体的酪氨酸激酶活性。此外，我们发现跨膜区域内的致癌点突变通过使neu蛋白更有效地利用Mg2+来刺激其酪氨酸激酶活性。总体而言，这些研究结果表明，第664位缬氨酸到谷氨酸的取代可能至少部分缓解了跨膜结构域对neu酪氨酸激酶活性施加的负性限制，并使在催化外源底物酪氨酸磷酸化过程中能更有效地利用Mg2+。
