MITOGENIC FUNCTION OF THE V-ABL TYROSINE KINASE

Abelson白血病病毒的癌基因v-able编码细胞内的 膜相关非受体酪氨酸激酶。 我们已经证明， v-abl酪氨酸激酶可刺激已建立的 造血细胞或成纤维细胞系。 v-abl酪氨酸激酶 需要持续的活性来消除生长因子 依赖这些细胞。 因为正常的促有丝分裂刺激是 由受体相关酪氨酸激酶激活，被广泛接受 致癌酪氨酸激酶通过连续的 刺激促有丝分裂途径。 这一假设得到了 然而，许多相关的证据表明， 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， 有了一个重要发现 我们发现， V-ABL酪氨酸激酶引起的细胞增殖依赖于 细胞背景 我们已经分离出两种稳定的NIH 3 T3细胞亚型。 在“阳性”3 T3细胞中，v-abl酪氨酸激酶的表达 导致血清需求的消除并且V-ABL诱导c-Fos， c-jun和c-myc基因。 另一方面，在“阴性”细胞中， 酪氨酸激酶不激活血清诱导基因， 生长停滞 “阳性”表型占优势。 我们的结果 证明v-abl酪氨酸激酶需要一个 3 T3细胞功能转化细胞。 这两种变异细胞系 提供了一个独特的系统来研究V-ABL酪氨酸 激酶刺激有丝分裂。 我们将研究分子基础 通过实现以下具体目标，加强合作职能： 1. 定义c-fos和c-jun启动子中的顺式元件， 确定在细胞中对v-abl酪氨酸激酶的不同反应， 阳性和阴性3和3细胞。 2. 识别和表征与蛋白质相互作用的蛋白质因子， “积极”和“消极”中的差异响应元件 细胞 3. 调查“阳性”和“阴性”细胞的反应性 编码促有丝分裂信号成分的其他癌基因 转导途径 4. 确定磷酸酪氨酸和V-abl的SH 2结构域在 有丝分裂的调节。 5. 鉴定和表征与v-abl相互作用的蛋白质， “阳性”和“阴性”细胞。 将使用蛋白亲和基质 为了从两种细胞中分离出显示差异相互作用的蛋白质， 类型 6. “阳性”中显性协作基因的克隆 细胞功能选择。 DNA转染以及微- 将采用注射方法来引入来自所述细胞的基因或mRNA。 阳性细胞进入阴性细胞，并选择合作 与适当的化验一起起作用。

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.