TGF-BETA1-MEDIATED SIGNAL TRANSDUCTION PATHWAYS AND GROWTH REGULATION

以前的工作已经证明了两个多肽(等电点7.00/22 kDa和 7.10/20 kDa)表现出快速(在15分钟内)和瞬时增加 体外暴露于转化生长因子后的磷酸化- β1(转化生长因子β1)(5 ng/ml)。为了刻画...的性质 转化生长因子-β1诱导的同步化大鼠肝上皮细胞 用32-P-正磷酸盐预先标记RLE细胞4小时，然后 用转化生长因子-β1(5 ng/ml)处理15、30、60min。 总磷酸化蛋白裂解物的免疫沉淀法 磷酸酪氨酸(p-Tyr)抗体，然后是二维 免疫沉淀物的聚丙烯酰胺凝胶电泳(2D-PAGE) 揭示了蛋白质磷酸化的许多数量差异。 其中包括两组明显相关的多肽的增加。 (A和C)，减少为两个单一(45 kDa/Pi 6.0和55 kDa/6.0) 两组(B组和D组)均有不同程度降低。多肽A组分 在4个多肽(97-100 kDa/等电点6.1-6.5)中，其磷酸化是 转化生长因子-β1在15分钟内升高，然后迅速恢复到基础水平 多肽C(4)(66-68 kDa/pI 6.7-7.0) 在15分钟增加，在30分钟保持升高，然后返回 60min时的本构水平。多肽B(5)(66-70 kDa/等电点5.6-5.8)， 然而，在15分钟时下降，在30分钟时几乎检测不到，然后 在60分钟内升至基础水平。蛋白的磷酸化 多肽D(22-24 kDa/等电点4.8-4.9)在15分钟内不受影响，降低 30分钟时恢复到基础水平，60分钟时恢复到基础水平。这是很有趣的 注意，在培养的小鼠中，多肽D显著增加 表皮生长因子刺激后的肝细胞。这 转化生长因子-β1介导的磷酸化/时程差异 特定磷酸酪氨酸蛋白的去磷酸化非常耐人寻味， 表明一系列逐步的蛋白质磷酸化/- 去磷酸化可能在转化生长因子-β1诱导的信号转导过程中起作用 转导。

Previous work has demonstrated that two polypeptides (pI 7.00/22 kDa and 7.10/20 kDa) exhibit a rapid (within 15 min) and transient increase in phosphorylation following in vitro exposure to transforming growth factor- beta-1 (TGF-beta1) (5 ng/ml). In an attempt to characterize the nature of the TGF-beta1 induced phosphorylation, synchronized rat liver epithelial (RLE) cells were prelabeled with 32-P-orthophosphate for 4 hr and then treated with TGF-beta1 (5 ng/ml) for 15, 30, and 60 min. Immunoprecipitation of total phosphorylated protein lysates with anti- phosphotyrosine (p-Tyr) antibody followed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of the immunoprecipitates revealed numerous quantitative differences in protein phosphorylation. These included increases in two groups of apparently related polypeptides (A and C) and decreases in two single (45 kDa/pI 6.0 and 55 kDa/6.0) polypeptides and decrease in two groups (B and D). Polypeptides A consist of 4 polypeptides (97-100 kDa/pI 6.1-6.5) whose phosphorylation is increased within 15 min of TGF-beta1 and then rapidly return to basal levels within 30 min, while polypeptides C (4) (66-68 kDa/pI 6.7-7.0) increase at 15 min, remain elevated at 30 min and then return to constitutive levels at 60 min. Polypeptides B (5) (66-70 kDa/pI 5.6-5.8), however, decrease at 15 min, are almost undetectable at 30 min, and then increase to basal levels within 60 min. The phosphorylation of polypeptides D (22-24 kDa/pI 4.8-4.9) is unaffected at 15 min, decreases at 30 min and returns to basal levels at 60 min. It is interesting to note that polypeptides D are markedly increased in cultured mouse hepatocytes following stimulation with epidermal growth factor. This differential time course of TGF-beta1 mediated phosphorylation/ dephosphorylation of specific phosphotyrosine protein is very intriguing, suggesting that a stepwise series of protein phosphorylations/- dephosphorylations may be operative during TGF-beta1 induced signal transduction.