TGF-BETA1-MEDIATED SIGNAL TRANSDUCTION PATHWAYS AND GROWTH REGULATION

先前的工作已经证明两个多肽(pI 7.00/22 kDa和

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.