To see if the absence of Rictor affects PLCfunction, we analyzed

To see if the absence of Rictor affects PLCfunction, we analyzed the ability of PDGF BB to stimulate PLCphosphorylation. Surprisingly, we found that in Rictor null cells the PLCphosphorylation was abolished and similar to what was seen for PKC, the total protein level was slightly reduced. The mechanism for the reduced PLCprotein level is unclear, but in the case of PKC it has been demonstrated that mTOR mediated phosphorylation is important for protein sta bility. To conclude, inhibition of PLCor Ca2 chelation resulted in decreased PDGF BB induced phosphory lation of Akt on Ser473, but did not affect phosphory lation on Thr308. In contrast, the presence of PLCprotein was needed for the phosphorylation on Thr308. Furthermore, we found that Rictor null cells, which have defective PDGF BB induced Akt Ser473 phosphory lation, are impaired in PLC PKC signaling.

However, treatment overnight with PMA inhibited Akt phospho rylation on both Ser473 and Thr308. These findings suggest Inhibitors,Modulators,Libraries that Thr308 is phosphorylated by a kinase that is downregulated by PMA treatment Inhibitors,Modulators,Libraries and thus normally regulated by DAG, possibly a novel PKC isoforms that requires DAG but not Ca2. Overnight treatment with PMA did not affect PDK 1 phosphorylation and neither did PDGF BB treatment. In contrast, Inhibitors,Modulators,Libraries phosphorylation of Akt on Ser473 is dependent on PLC 1 activity, Ca2, DAG and the conventional PKCs. PDGF BB induced Erk12 MAP kinase signaling is important for the kinetics of S6 phosphorylation In addition to Akt, MAP kinase pathways have been linked to mTOR signaling.

We found that the selective Inhibitors,Modulators,Libraries Mek12 inhibitor CI 1040 completely blocked Erk12 phosphorylation and Inhibitors,Modulators,Libraries reduced S6 phosphory lation, primarily after 15 min of stimulation, but had no effect on Akt phosphorylation. Thus, Erk12 may contribute to mTORC1 activation at early stages of signaling, as previously noted. To further clarify the role of Erk12 in mTORC1 signaling after prolonged PDGF BB treatment, we performed a time course experiment stimulating cells for up to 4 h. We found that 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 Erk12 phosphorylation was not dependent on mTORC2, mTORC1. PKCs, or the presence of Ca2.

In summary, PDGF BB induced Erk12 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 Erk12 activation. Role of mTOR signaling in PDGF BB induced cellular responses things 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.

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