MECHANISM OF TRANSFORMATION BY TYROSINE KINASES

我们的最终目标仍然是确定机制的meoplastic 通过酪氨酸特异性蛋白激酶的转化。 我们在此提议， 进一步验证我们的假设，即通过改变酪氨酸磷酸化， 细胞酪氨酸激酶在锚定非依赖性生长中发挥作用 转化细胞的特性。 我们观察到， 病毒，水泡性口炎病毒（VSV），选择性地获得特异性 来自锚定非依赖性细胞的酪氨酰激酶种类。 因此我们将 纯化并制备针对VSV相关酪氨酰的免疫试剂 激酶用于研究这种酶在 非贴壁依赖细胞 为了验证酪氨酰 VSV获得的激酶在锚定独立性中起作用，它将是 确定VSV获得的酶是否与酪氨酰相同， 激酶，以前已经显示出引起锚定独立性， 一些转化的细胞 为了确定酪氨酸磷酸化的特定底物蛋白 可能受到调控，我们将测试细胞酪氨酸激酶种类是否 在活性上扩增或重新定位于不依赖于细胞的细胞中。 这 将通过HPLC凝胶过滤单个种类的酪氨酰激酶， 它们的活性和亚细胞位置的定量。 我们 初步数据表明，另一种调节机制是 前列腺酸性磷酸酶对磷酸酪氨酸的去磷酸化作用。 这 将在人前列腺癌细胞中测试调节模式，其中 前列腺酸性磷酸酶活性可受雄激素调节。 为了开始了解蛋白质的酪氨酸磷酸化是如何导致 恶性生长的细胞，我们将研究一类新的酪氨酰 我们在人类细胞外组分中鉴定的激酶底物 前列腺癌细胞 我们将确定这些是否是 蛋白质并制备用于其进一步的免疫学试剂 特征化

Our ultimate goal remains to determine the mechanism of meoplastic transformation by tyrosine-specific protein kinases. We propose here to further test our hypothesis that altered tyrosine phosphorylation by cellular tyrosyl kinases functions in the anchorage independent growth property of transformed cells. We have observed that a membrane-enveloped virus, vesicular stomatitis virus (VSV), selectively acquires specific tyrosyl kinase species from anchorage independent cells. We will therefore purify and prepare immunological reagents to the VSV-associated tyrosyl kinase to be used to investigate location and amount of this enzyme in anchorage independent cells. To test the hypothesis that the tyrosyl kinase acquired by VSV functions in anchorage independence, it will be determined whether the VSV-acquired enzyme is the same as the tyrosyl kinase which has previously been shown to cause anchorage independence in some transformed cells. To determine how tyrosine-phosphorylation of specific substrate proteins may be regulated, we will test whether cellular tyrosyl kinase species are amplified in activity or relocalized in achorage independent cells. This will be by HPLC gel filtration of individual species of tyrosyl kinases and quantitations of their activities and subcellular locations. Our preliminary data suggests that another mechanism for regulation is by dephosphorylation of phosphotyrosine by prostatic acid phosphatase. This mode of regulation will be tested in human prostate carcinoma cells where prostatic acid phosphatase activity can be regulated by androgens. To begin to understand how tyrosine phosphorylation of proteins may cause malignant growth of cells, we will investigate a new class of tyrosyl kinase substrates identified by us in the extracellular fraction of human prostate carcinoma cells. We will determine whether these are secreted proteins and prepare immunological reagents to be used in their further characterization.