Pham P T, Heydrick S J, Fox H L, Kimball S R, Jefferson L S, Lynch C J
Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033, USA.
J Cell Biochem. 2000 Sep 7;79(3):427-41. doi: 10.1002/1097-4644(20001201)79:3<427::aid-jcb80>3.0.co;2-0.
Enhanced phosphorylation of the ribosomal protein s6 kinase, p70(s6k), and the translational repressor, 4E-BP1, are associated with either insulin-induced or amino acid-induced protein synthesis. Hyperphosphorylation of p70(s6k) and 4E-BP1 in response to insulin or amino acids is mediated through the mammalian target of rapamycin (mTOR). In several cell lines, mTOR or its downstream targets can be regulated by phosphatidylinositol (PI) 3-kinase; protein kinases A, B, and C; heterotrimeric G-proteins; a PD98059-sensitive kinase or calcium; as well as by amino acids. Regulation by amino acids appears to involve detection of levels of charged t-RNA or t-RNA synthetase activity and is sensitive to inhibition by amino acid alcohols. In the present article, however, we show that the rapamycin-sensitive regulation of 4E-BP1 and p70(s6k) in freshly isolated rat adipocytes is not inhibited by either L-leucinol or L-histidinol. This finding is in agreement with other recent studies from our laboratory suggesting that the mechanism by which amino acids regulate mTOR in freshly isolated adipocytes may be different than the mechanism found in a number of cell lines. Therefore we investigated the possible role of growth factor-regulated and G-protein-regulated signaling pathways in the rapamycin-sensitive, amino acid alcohol-insensitive actions of amino acids on 4E-BP1 phosphorylation. We found, in contrast to previously published results using 3T3-L1 adipocytes or other cell lines, that the increase in 4E-BP1 phosphorylation promoted by amino acids was insensitive to agents that regulate protein kinase A, mobilize calcium, or inhibit protein kinase C. Furthermore, amino acid-induced 4E-BP1 phosphorylation was not blocked by pertussis toxin nor was it mimicked by the G-protein agonists fluoroaluminate or MAS-7. However, amino acids failed to activate either PI 3-kinase, protein kinase B, or mitogen-activated protein kinase and failed to promote tyrosine phosphorylation of cellular proteins, similar to observations made using cell lines. In summary, amino acids appear to use an amino acid alcohol-insensitive mechanism to regulate mTOR in freshly isolated adipocytes. This mechanism is independent of cell-signaling pathways implicated in the regulation of mTOR or its downstream targets in other cells. Overall, our study emphasizes the need for caution when extending results obtained using established cell lines to the differentiated nondividing cells found in most tissues.
核糖体蛋白s6激酶（p70(s6k)）和翻译抑制因子4E-BP1的磷酸化增强与胰岛素诱导或氨基酸诱导的蛋白质合成有关。p70(s6k)和4E-BP1对胰岛素或氨基酸的过度磷酸化是通过雷帕霉素的哺乳动物靶点（mTOR）介导的。在几种细胞系中，mTOR或其下游靶点可受磷脂酰肌醇（PI）3激酶、蛋白激酶A、B和C、异三聚体G蛋白、对PD98059敏感的激酶或钙以及氨基酸的调节。氨基酸的调节似乎涉及对带电t-RNA水平或t-RNA合成酶活性的检测，并且对氨基酸醇的抑制敏感。然而，在本文中，我们表明，在新鲜分离的大鼠脂肪细胞中，雷帕霉素敏感的4E-BP1和p70(s6k)调节不受L-亮氨醇或L-组氨醇的抑制。这一发现与我们实验室最近的其他研究一致，表明氨基酸调节新鲜分离脂肪细胞中mTOR的机制可能与许多细胞系中的机制不同。因此，我们研究了生长因子调节和G蛋白调节的信号通路在氨基酸对4E-BP1磷酸化的雷帕霉素敏感、氨基酸醇不敏感作用中的可能作用。我们发现，与先前使用3T3-L1脂肪细胞或其他细胞系发表的结果相反，氨基酸促进的4E-BP1磷酸化增加对调节蛋白激酶A、动员钙或抑制蛋白激酶C的试剂不敏感。此外，氨基酸诱导的4E-BP1磷酸化不受百日咳毒素的阻断，也未被G蛋白激动剂氟铝酸盐或MAS-7模拟。然而，与使用细胞系的观察结果相似，氨基酸未能激活PI 3激酶、蛋白激酶B或丝裂原活化蛋白激酶，也未能促进细胞蛋白的酪氨酸磷酸化。总之，氨基酸似乎使用一种氨基酸醇不敏感机制来调节新鲜分离脂肪细胞中的mTOR。这种机制独立于与其他细胞中mTOR或其下游靶点调节有关的细胞信号通路。总体而言，我们的研究强调，在将使用已建立细胞系获得的结果推广到大多数组织中发现的分化的非分裂细胞时需要谨慎。
