Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-γ1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D.
Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-γ1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D.
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DOI：
10.1186/1478-811x-11-3
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发表时间：
2013-01-11
期刊：
影响因子：
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通讯作者：
Lennartsson J
中科院分区：
文献类型：
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作者：
Razmara M;Heldin CH;Lennartsson J
哺乳动物雷帕霉素靶点（mTOR）可以在两种多蛋白复合物中找到，即mTORC 1（含有Raptor）和mTORC 2（含有Rictor）。在这里，我们研究了mTORC1和mTORC2被激活的机制及其下游靶点对血小板源性生长因子（PDGF）-BB治疗的反应。抑制磷脂酰肌醇3-激酶（PI3K）可抑制mTORC 1和mTORC 2的PDGF-BB活化。我们发现，在Rictor基因敲除小鼠胚胎成纤维细胞中，或在长期雷帕霉素处理NIH 3T3细胞后，PDGF-BB不能促进丝氨酸/苏氨酸激酶Akt中Ser473的磷酸化，而Thr308磷酸化受影响较小，表明Akt中Ser473以mTORC2依赖性方式磷酸化。Akt磷酸化的这种减少不影响S6蛋白的磷酸化，S6蛋白是mTORC 1下游的一种公认蛋白。一致地，曲西立滨，Akt通路的抑制剂，抑制PDGF-BB诱导的Akt磷酸化，而对S6磷酸化没有任何影响。因此，mTORC2似乎不位于mTORC1的上游。Rictor基因敲除细胞中磷脂酶C γ 1（PLC γ 1）和蛋白激酶C（PKC）的磷酸化水平降低，PKC α蛋白水平显著降低。干预PLC γ/Ca~（2+）/PKC通路可抑制PDGF-BB诱导的Akt磷酸化。此外，PDGF-BB诱导的mTORC1激活（通过下游S6蛋白的磷酸化测定）依赖于磷脂酶D（PLD）。Erk1/2 MAP激酶直接磷酸化并激活mTORC1;与这一发现部分一致的是，我们发现Mek1/2抑制剂延迟了PDGF-BB对S6磷酸化的反应，但不能阻断它。因此，尽管mTORC1和mTORC2都以PI3K依赖的方式激活，但需要不同的额外信号通路。mTORC1以PLD依赖性方式激活并促进S6蛋白的磷酸化，而mTORC2与PLC γ信号传导一致，促进Akt磷酸化。
Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation.