A commonly used inhibitor of mTOR is rapamycin. However, the two mTOR containing complexes, mTORC1 and mTORC2, have different sensitivities to rapamycin. mTORC1 is rapidly inhibited whereas mTORC2 requires prolonged rapamycin treatment. thus, short term treatment with rapamycin only inhibits mTORC1 whereas long term treatment also inhibit FTY720 162359-56-0 mTORC2. Treating cells for extended time periods with rapamycin abolished the mito genic effect of PDGF BB, suggesting that functional mTOR signaling is required for cell proliferation. In con trast, Rictor deficient cells showed a similar chemotactic response as control cells towards PDGF BB, indicating that mTORC2 is not involved in PDGF BB dependent cell migration.

this is surprising since mTORC2 has been shown to regulate cell polarity and the dynamics of the actin cytoskeleton, although no alterations in the actin cytoskeleton were observed in Rictor null MEFs. Similarly, inhibition of mTORC1 and 2 in NIH3T3 cells did not influence the chemotactic properties of these cells. mTORC2 may affect cell migration by pro moting PKC dependent Inhibitors,Modulators,Libraries phosphorylation of the focal adhesion component paxillin. However, it has previ ously been found that Inhibitors,Modulators,Libraries PDGF BB can promote paxillin phosphorylation through the JNK MAP kinase pathway, and this may relieve the absolute requirement of mTORC2 in PDGF BB mediated fibroblast migration. Conclusions The pathway from PDGFR leading to phosphorylation of Akt involves both the mTORC2 and PLC��PKC Inhibitors,Modulators,Libraries path ways. In contrast, phosphorylation of S6 downstream of mTORC1 depends on PLD activation, but is independ ent of mTORC2 and Akt signaling.

During conditions where Erk12 signaling is inhibited, the initial S6 Inhibitors,Modulators,Libraries phosphorylation is delayed. Interfering with mTOR signaling did not affect PDGF BB induced Erk12 phos phorylation. Functionally, inhibition of mTORC1 and 2 by rapamycin effectively blocked PDGF BB mediated cell proliferation. Figure 6 depicts Inhibitors,Modulators,Libraries a schematic figure of key roles of mTOR in PDGF BB induced cell signaling. Materials and methods Reagents Recombinant human PDGF BB was generously provided by Amgen. The inhibitors CI 1040, triciribine and NVP BKM120 were from Calbiochem, Cayman Chemical Company and Selleckchem, respectively. Antibodies against phosphorylated Akt phosphorylated mTOR, phosphory lated S6, cleaved caspase 3, phosphory lated Erk12 and phospho MARCKS were purchased from Cell Signaling Technology.

A B actin antibody was purchased from Sigma. A rabbit antiserum recognizing Erk was raised against a peptide corresponding to the carboxyl terminal sequence EETARFQPGYRS conjugated to KLH. The wild type control and Rictor knockout mouse em bryonic fibroblasts have been described previously and were kindly provided by Dr Mark selleck Magnuson. PLC��1 null MEFs have been described previously and were kindly provided by Dr Matilda Katan.