Bell S M, Connolly D C, Maihle N J, Degen J L
Division of Basic Science Research, Children's Hospital Research Foundation, Cincinnati, Ohio 45229.
Mol Cell Biol. 1993 Sep;13(9):5888-97. doi: 10.1128/mcb.13.9.5888-5897.1993.
Urokinase-type plasminogen activator (uPA) gene transcription is increased > or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.
在鸡胚成纤维细胞（CEF）中，经蛋白酪氨酸激酶pp60v-src转化后，尿激酶型纤溶酶原激活剂（uPA）基因转录增加≥50倍。蛋白质磷酸化似乎在这些细胞的uPA基因表达中起关键作用；蛋白激酶C激活剂佛波酯与pp60v-src协同作用，可协同增加uPA mRNA，而环磷酸腺苷（cAMP）依赖性蛋白激酶激活剂（如8-溴cAMP）则抑制uPA mRNA水平。为了探究转化癌基因与uPA基因表达之间的关系，我们检测了感染特定禽逆转录病毒的CEF中的uPA mRNA水平。我们报告称，v-ras以及转化蛋白酪氨酸激酶v-src、v-yes和v-ros均能增加细胞内uPA mRNA水平。然而，用v-erbB编码的蛋白酪氨酸激酶或v-jun、v-ski或v-myc编码的核蛋白进行转化，并未显著增加uPA mRNA水平。Ras以及所有分析过的蛋白酪氨酸激酶，包括v-erbB产物，但没有一种核癌蛋白能使细胞对佛波酯诱导的uPA基因表达敏感。因此，uPA基因表达的增加并非细胞转化的简单次要结果，而是仅由一部分癌基因产物调节或共同调节的。对表达pp60v-src定点突变体的细胞进行分析表明，uPA基因的诱导依赖于蛋白酪氨酸激酶的催化活性、肉豆蔻酰化和质膜定位。然而，这些特性加在一起并不足够；src同源2结构域还需要一个额外的特征。丝氨酸磷酸化的主要位点，即丝氨酸12和17，以及自磷酸化位点酪氨酸416，对于uPA基因诱导并非必需。然而，8-溴cAMP使pp60v-src转化细胞中的uPA mRNA减少依赖于酪氨酸416。
