MITOGENIC FUNCTION OF THE V-ABL TYROSINE KINASE

Abelson白血病病毒的癌基因v-able编码一个细胞内的 膜相关非受体酪氨酸激酶。我们已经证明了 V-ABL酪氨酸激酶可刺激已建立的细胞增殖 造血系或成纤维细胞系。V-ABL酪氨酸激酶 需要持续不断的活动来消除生长因子 这些细胞的依赖性。因为正常的促有丝分裂刺激 它被受体相关的酪氨酸激酶激活，被广泛接受 致癌酪氨酸激酶通过连续的 有丝分裂途径的刺激。这一假设得到了 然而，许多相关证据表明， 致癌酪氨酸激酶转化细胞的机制尚不清楚。我们有 一直在研究v-ABL酪氨酸激酶在有丝分裂中的作用 有了一个重要的发现。我们已经发现，人的刺激 V-ABL酪氨酸激酶诱导的细胞增殖依赖于 蜂窝环境。我们已经分离出两种稳定的NIH3T3细胞亚型。 在“阳性”的3T3细胞中，v-ABL酪氨酸激酶的表达 导致血清需求量的减少和v-ABL诱导c-fos， C-jun和c-myc基因。另一方面，在“阴性”细胞中，v-abl 酪氨酸激酶不会激活血清诱导基因，但会导致 增长停滞。“阳性”表型占主导地位。我们的结果 证明v-ABL酪氨酸激酶需要一种 3T3细胞具有转化细胞的功能。这两个变异的细胞系 提供了一种独特的系统来研究v-ABL酪氨酸的机制 激活剂能刺激有丝分裂。我们将研究分子基础 通过追求以下具体目标来实现协作职能： 1.定义c-fos和c-jun启动子中的顺式元素 确定v-ABL酪氨酸激酶在脑内的差异反应 阳性和阴性的3&3个细胞。 2.确定和表征与蛋白质相互作用的蛋白质因子 “积极”和“消极”中的不同反应要素 细胞。 3.调查“阳性”和“阴性”细胞的反应性 与编码有丝分裂信号成分的其他癌基因有关 转导途径。 4.确定磷酸化酪氨酸和v-ABL的SH2结构域在蛋白合成中的作用 有丝分裂的调控。 5.鉴定和鉴定与v-ABL相互作用的蛋白质 “阳性”和“阴性”细胞。将使用蛋白质亲和力矩阵 从两个细胞中分离出显示不同相互作用的蛋白质 类型。 6.“积极”中显性作用协同基因的克隆 按功能选择的单元格。DNA转染法和微孔法 将采用注射方法将基因或信使核糖核酸从 正向单元格变为负向单元格，并选择进行协作 用适当的化验方法起作用。

The oncogene of Abelson Leukemia Virus, v-able, encodes an intracellular membrane-associated non-receptor tyrosine kinase. We have shown that the v-abl tyrosine kinase can stimulate the proliferation of established lines of hemopoietic or fibroblastic cells. The v-abl tyrosine kinase activity is required continuously to abrogate the growth factor dependence of these cells. Because normal mitogenic stimulation is activated by receptor-associated tyrosine kinases, it is widely accepted that oncogenic tyrosine kinases transform cells by the continuous stimulation of the mitogenic pathway. This hypothesis is supported by many correlative evidence, however, the precise mechanism by which oncogenic tyrosine kinase transforms cells is not understood. We have been studying the v-abl tyrosine kinase function in mitogenesis and have made an important finding. We have discovered that the stimulation of cell proliferation by the v-abl tyrosine kinase is dependent on the cellular context. We have isolated two stable subtypes of NIH3T3 cells. In the "positive" 3T3 cells, expression of the v-abl tyrosine kinase leads to the abrogation of serum requirement and v-abl induces the c-fos, c-jun and c-myc genes. In the "negative" cells, on the other hand, v-abl tyrosine kinase does not activate the serum-inducible genes but causes growth arrest. The "positive" phenotype is dominant. Our results demonstrate that v-abl tyrosine kinase requires the collaboration of a 3T3 cellular function to transform cells. These two variant cell lines offer an unique system to study the mechanism by which v-abl tyrosine kinase stimulates mitogenesis. We will investigate the molecular basis of the collaborating function by pursuing the following specific aims: 1. Define the cis-elements in the c-fos and c-jun promoters that determine the differential response to v-abl tyrosine kinase in the positive and the negative 3&3 cells. 2. Identify and characterize the protein factors which interact with the differential response elements in the "positive" and the "negative" cells. 3. Investigate the responsiveness of the "positive" and "negative" cells to other oncogenes which encode components of the mitogenic signal transduction pathway. 4. Determine the role of phosphotyrosine and the SH2 domain of v-abl in the modulation of mitogenesis. 5. Identify and characterize proteins which interact with v-abl in the "positive" and "negative" cells. Protein-affinity matrix will be used to isolate proteins that show differential interaction from the two cell types. 6. Cloning of the dominant-acting collaboration gene in the "positive" cells by a functional selection. DNA transfection as well as micro- injection methods will be employed to introduce genes or mRNA from the positive cell into the negative cells and select for the collaborating function with the appropriate assays.