IDENTIFICATION OF MUTATIONS IN THE CODING SEQUENCE OF THE PROTOONCOGENE C-KIT IN A HUMAN MAST-CELL LEUKEMIA-CELL LINE CAUSING LIGAND-INDEPENDENT ACTIVATION OF C-KIT PRODUCT
IDENTIFICATION OF MUTATIONS IN THE CODING SEQUENCE OF THE PROTOONCOGENE C-KIT IN A HUMAN MAST-CELL LEUKEMIA-CELL LINE CAUSING LIGAND-INDEPENDENT ACTIVATION OF C-KIT PRODUCT
复制标题
DOI：
10.1172/jci116761
复制
发表时间：
1993-10-01
影响因子：
15.9
通讯作者：
KANAKURA, Y
中科院分区：
文献类型：
--
作者：
FURITSU, T;TSUJIMURA, T;KANAKURA, Y
c-kit原癌基因编码受体酪氨酸激酶。已知c-kit配体、干细胞因子（SCF）与c-kit受体（c-kitR）的结合可激活c-kitR酪氨酸激酶，从而导致c-kitR在酪氨酸上的自磷酸化，并导致c-kitR与底物如磷脂酰肌醇3-激酶（PI 3 K）缔合。在人肥大细胞白血病细胞系HMC-1中，发现c-kitR在没有添加SCF的情况下在酪氨酸上组成性磷酸化、活化并与PI 3 K相关。用逆转录后PCR（RT-PCR）方法检测HMC-1细胞中SCF mRNA转录本的表达，结果表明c-kitR的组成性激活不依赖于配体。HMC-1细胞c-kit基因由一个正常的野生型等位基因和一个突变型等位基因组成，该突变型等位基因导致细胞内氨基酸由Gly-560替换为瓦尔，由瓦尔-816替换为Asp。这两个突变区域的氨基酸序列在小鼠、大鼠和人c-kit中是完全保守的。为了确定这些突变在组成型活化中的因果作用，通过定点诱变构建编码Gly-559和/或瓦尔-814（对应于人Gly-560和/或瓦尔-816）的鼠c-kit突变体，并在人胚肾细胞系293 T细胞中表达。在转染的细胞中，c-kitR（Gly-559，瓦尔-814）和c-kitR（瓦尔-814）在没有SCF的情况下在酪氨酸上被大量磷酸化并在免疫复合物激酶反应中被激活，而c-kitR（Gly-559）或野生型c-kitR的酪氨酸磷酸化和激活分别是适度的或很少的。这些结果表明，人c-kitR中Asp-816转化为瓦尔可能是一种激活突变，并负责HMC-1细胞中c-kitR的组成性激活。
The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.