As can be seen in Figure 1C, no decrease in the PDGF BB induced S6 phos phoryla

To further clarify the role of Erk1 2 in mTORC1 signaling after prolonged PDGF BB treatment, we performed a time course experiment stimulating cells for up to 4 h. We found that ARN-509 臨床試験 only the rapid, initial induction of S6 phosphorylation was inhibited by CI 1040, whereas the S6 phosphorylation reached almost the same level in cells treated with CI 1040 as in vehicle treated cells after longer time periods of PDGF BB stimulation. The PDGF BB induced Erk1 2 phosphorylation was not dependent on mTORC2, mTORC1, PKCs, or the presence of Ca2. In summary, PDGF BB induced Erk1 2 activity is only important for the early onset of mTORC1 mediated phosphorylation of S6. Furthermore, neither mTORC1 nor mTORC2 are needed for PDGF BB induced Erk1 2 activation.<br><br> Role of mTOR signaling in PDGF BB induced cellular responses Next, we wanted to elucidate the functional conse quences of interfering with mTOR signaling for PDGF BB mediated cellular responses, i. e. survival, migration and proliferation. To this end, we used the Rictor null cells which lack a functional mTORC2 complex, AUY922 臨床試験 as well as long term treatment with rapamycin to inhibit both mTORC1 and 2. We found that serum starvation induced caspase 3 cleavage, which could be rescued by addition of PDGF BB in control cells, but not in Rictor null cells, suggesting a role of mTORC2 in promoting cell survival in response to PDGF BB. In ac cordance with a recent report we could confirm that Rictor null cells have increased rate of apoptosis compared to control MEFs.<br><br> Interestingly, in these cells the apoptosis could not be modulated by ALK 阻害剤 either serum levels or addition of PDGF, despite the reduction of caspase 3 cleavage observed in control MEFs in the presence of PDGF. The reasons of these findings remain to be elucidated. In contrast, the migratory response was not affected by loss of the mTORC2 complex. As expected, downregulation of both mTORC1 and 2 by rapamycin strongly inhibited PDGF BB promoted DNA synthesis in NIH3T3 cells. Unfortunately, we were not able to analyze the proliferation of Rictor null cells in response to PDGF BB, since neither control nor knock out cells responded to PDGF BB in the prolif eration assay. Furthermore, long term treatment with rapamycin did not affect the PDGF BB induced migration of NIH3T3 cells. In conclusion, PDGF BB signaling through mTORC2 is important for the ability of PDGF BB to suppress starvation induced cleavage of caspase 3, but not for chemotaxis.<br><br> Complete inhibition of mTOR signaling by rapamycin abolished the ability of PDGF BB to promote cell proliferation. Discussion Akt is an important kinase mediating survival signaling, which is regulated by phosphorylation on Thr308 by PDK1 and on Ser473 by several other kinases. A large number of kinases have been proposed to perform the Ser473 phosphorylation. In the present study, we showed that phosphorylation of Akt on Ser473 in response to PDGF BB was critically dependent on the mTORC2 complex since the phosphorylation was strongly repressed in Rictor null cells. Consistently, prolonged treatment with rapamycin that downregulates both mTORC1 and 2, inhibited the PDGF BB induced phos phorylation on Ser473, whereas short term rapamycin treatment which only inhibits mTORC1, did not.