The main objective of this project is to analyze growth stimulatory and inhibitory factor induced modulation of phosphoprotein expression in cultured rat liver cells in an attempt to delineate receptor mediated signaling pathways. Low passage unsynchronized rat liver epithelial (RLE) cells were prelabeled with 32-P-ortho- phosphate and treated with TGF-beta1 (5 ng/ml) for 15 and 60 minutes. Fifteen minutes after treatment with TGF-beta1, the expression of the phosphorylated isoforms of two polypeptides, 2 (pI 5.00/85 kDa) and 3 (4.90/84 kDa) were markedly decreased but returned to constitutive levels after 60 minutes. In order to address the question of whether the observed decrease in the expression of phosphoproteins 2 and 3 was the result of specific modulation of phosphorylation pathways or was the result of a transient decrease in the synthesis of either 2 or 3, a """"""""triple labeling"""""""" technique was developed. Analysis of polypeptides 2 and 3, utilizing this technique, indicated a marked decrease in the expression of poly-peptide 3 as a result of either a decreased synthesis or increased degradation of polypeptide 3 as a result of TGF-beta1 treatment. The decreased phosphorylation of polypeptide 2, on the other hand, may be the result of an activation of a specific TGF-beta1 phosphatase(s). Previous work has demonstrated that TGF-beta1 mediates its growth inhibitory action in the late G1 stages of the cell cycle; therefore, RLE cells were synchronized with lovastatin/mevalonate and, at various times during the G1 phase, 32- P-orthophosphate prelabeled cells were treated for 15 and 60 minutes with TGF-beta1 (5 ng/ml). At 7 and 8 hours there was a marked increase in the phosphorylation of two polypeptides (pI 7.00/22 kDa and 7.10/20 kDa) after 15 minutes treatment with TGF-beta1; both returned to constitutive levels after 60 minutes treatment. Similar treatment of RLE cells with TGF-beta1 either earlier (6 hours) or later (9, 10 or 11 hours) in the cell cycle had no effect on the expression of these polypeptides. Results to date indicate that TGF-beta1 induces rapid and transient modulation of specific subsets of polypeptides some of which may be involved in the growth inhibitory effects of TGF-beta1.
