Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, Koshimizu U, Sugahara H, Butterfield J H, Ashman L K, Kanayama Y
Second Department of Internal Medicine, Osaka University Medical School, Suita, Japan.
J Clin Invest. 1993 Oct;92(4):1736-44. doi: 10.1172/JCI116761.
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.
原癌基因c-kit编码一种受体酪氨酸激酶。已知c-kit配体即干细胞因子（SCF）与c-kit受体（c-kitR）结合可激活c-kitR酪氨酸激酶，进而导致c-kitR在酪氨酸位点发生自身磷酸化，并使c-kitR与诸如磷脂酰肌醇3激酶（PI3K）等底物相结合。在人肥大细胞白血病细胞系HMC-1中，发现c-kitR在酪氨酸位点持续磷酸化、被激活，且在未添加SCF的情况下与PI3K相结合。经逆转录聚合酶链反应（RT-PCR）分析，未检测到HMC-1细胞中SCF mRNA转录本的表达，这表明c-kitR的持续激活不依赖配体。对c-kit cDNA的整个编码区进行测序发现，HMC-1细胞的c-kit基因由一个正常的野生型等位基因和一个突变等位基因组成，该突变等位基因有两个点突变，导致细胞内第560位甘氨酸被缬氨酸取代，第816位缬氨酸被天冬氨酸取代。在所有小鼠、大鼠和人的c-kit中，这两个突变区域的氨基酸序列完全保守。为了确定这些突变在持续激活中的因果作用，通过定点诱变构建了编码与人类第560位甘氨酸和/或第816位缬氨酸相对应的第559位甘氨酸和/或第814位缬氨酸的小鼠c-kit突变体，并在人胚肾细胞系293T细胞中表达。在转染细胞中，在没有SCF的情况下，c-kitR（第559位甘氨酸，第814位缬氨酸）和c-kitR（第814位缬氨酸）在免疫复合物激酶反应中均大量在酪氨酸位点磷酸化并被激活，而c-kitR（第559位甘氨酸）或野生型c-kitR的酪氨酸磷酸化和激活程度分别适中或很低。这些结果表明，人c-kitR中第816位天冬氨酸向缬氨酸的转变可能是一个激活突变，并且是HMC-1细胞中c-kitR持续激活的原因。
