Platelet-derived growth factor (PDGF) stimulates proliferation, migration and survival of mesenchymal cells and plays a pivotal role during embryonic development and wound healing . The biologically active form of PDGF consists of disulphide-linked dimers, PDGF-AA, -AB, -BB, -CC and –DD, which bind to two structurally similar tyrosine kinase receptors, i.e. PDGFRα and PDGFRβ [2, 3]. PDGFRα binds all PDGF chains except PDGF-D, whereas PDGFRβ interacts only with PDGF B- and D-chains. The binding of the bivalent ligand induces dimerization and activation of PDGFRs, leading to autophosphorylation of tyrosine residues in the intracellular region . Thereby, several signal transduction pathways are initiated, including phosphatidylinositol 3-kinase (PI3K), the Src tyrosine kinase, phospholipase Cγ (PLC), and several mitogen-activated protein (MAP) kinase cascades.
mTOR is the mammalian ortholog of the yeast serine/threonine kinase TOR which is involved in the regulation of various cellular functions, such as initiation of translation, cell growth and proliferation, ribosome biogenesis, transcription and cytoskeletal reorganization . Dysregulation of mTOR signaling is frequently seen in cancer and has attracted attention as a therapeutic target [5, 6]. mTOR is functional in two distinct complexes, namely mTORC1 and mTORC2 . mTORC1 activity is controlled by the G-protein Rheb; Rheb-GTP promotes mTORC1 activity and the tuberous sclerosis complex 1/2 (TSC1/2) acts as a GTPase activating protein for Rheb, consequently inhibiting mTORC1 activity . Generally, mTORC1 is described as being activated by growth factors through Akt-mediated phosphorylation which inactivates the TSC1/2 complex [8–10]. In addition, the TSC1/2 complex can also be phosphorylated and inhibited by AMPK, thus allowing the cellular energy status to impact mTORC1 activity . mTORC1 is a rapamycin-sensitive complex, and includes the proteins Raptor (regulatory-associated protein of mTOR), mLST8, PRAS40 and Deptor . Raptor acts as a scaffold and thereby controls mTORC1 activity. Established functions for mTORC1 are to phosphorylate 4EBP1 and activate S6-kinase, which in turn phosphorylates the S6 protein . Phosphorylated S6 and 4EBP1 enhance protein translation. In mTORC2, mTOR occurs in a complex with Rictor (rapamycin-insensitive companion of mTOR), mLST8, mSin1, protor, Deptor and Hsp70 [14–17]. mTORC2 is primarily activated by growth factors, but the mechanism is largely unknown. It has recently been suggested that mTORC2 activation is dependent on PI3-kinase, but independent of Akt . mTORC2 is able to phosphorylate Akt on Ser473, at least in some cell types . Other substrates for mTORC2 include PKCα and paxillin . mTOR can be activated by growth factor signaling, such as by PDGF, but the roles of mTORC1 and mTORC2 in PDGF-BB-induced signal transduction have not been established.
The serine/threonine kinase Akt is activated by PDGF-BB stimulation in a PI3-kinase-dependent manner. Activation of PI3-kinase generates PIP3 that can interact with and thereby translocate Akt to the plasma membrane, where it is activated by phosphorylation on Ser473 in a hydrophobic motif and Thr308 in the activation loop of the kinase domain [19, 21, 22]. Thr308 is phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1), whereas several candidates, including mTORC2, may perform the Ser473 phosphorylation [19, 23–25]. Furthermore, the kinase responsible for the Ser473 phosphorylation may be different for different cell and receptor types. When activated, Akt transduces important survival signals that interfere with the apoptotic process, for example by inhibition of Foxo, Bad and caspase 9 [26–28].
Phoshoplipase Cγ catalyzes the hydrolysis of PIP2, thus releasing the polar head group inositol-1,4,5-trisphosphate (IP3), while diacylglycerol (DAG) remains embedded in the plasma membrane . IP3 release results in mobilization of Ca2+ from intracellular stores. Both DAG and Ca2+ participate in the activation of protein kinase C (PKC) family members, some of which require both DAG and Ca2+ (PKCα, β, γ), whereas others require only DAG (PKCδ, ε, η, θ) . In addition, there are atypical PKC isoforms (PKCζ, ι) that are regulated by other means . PLCγ is activated by direct SH2-domain-dependent interaction with activated tyrosine kinase receptors and subsequent phosphorylation [32, 33]. Another phospholipase that is activated by receptor tyrosine kinases is phospholipase D (PLD). PLD acts by hydrolyzing phosphatidylcholine generating choline and phosphatidic acid  which is required for mTORC1 activation by mitogenic factors . Regulation of PLD activity is complex and has been shown to involve small G-proteins, phosphatidylinositol 4,5-bisphosphate (PIP2), Ca2+ and kinases . PDGF has been demonstrated to promote PLD tyrosine phosphorylation and activation by a mechanism involving the production of reactive oxygen species .
In this study, we have explored the role of mTOR in the regulation of PDGF-BB signaling. We found that Rictor, and hence mTORC2, promotes the PDGF-BB-induced phosphorylation of Akt at Ser473, as well as the phosphorylation of PLCγ1 and PKCα in addition to promoting PKCα protein stability. Moreover, we show that PLD activity is important for S6 phosphorylation and that this occurs through mTORC1. However, our data suggest that S6 phosphorylation downstream of PDGFR does not rely on Akt activation. Functionally, mTOR inhibition by rapamycin suppressed PDGF-BB-mediated cell proliferation, whereas rapamycin treatment or the loss of Rictor in the mTORC2 complex had no significant impact on the chemotactic response toward PDGF-BB.