Phospholipase D Regulates Myogenic Differentiation through the Activation of Both mTORC1 and mTORC2 Complexes

Abstract

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKC␣. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.The mammalian target of rapamycin (mTOR) 2 is a serine/ threonine protein kinase that integrates signals provided by growth factors, nutrient availability, energy levels, or redox status to adapt protein synthesis and major cell functions such as growth, proliferation, and survival to the physiological conditions (1). It exists in two complexes, mTORC1 and mTORC2, that are differentially regulated, have distinct effector substrates, and are differentially sensitive to rapamycin, a bacterial macrolide endowed with anti-proliferative and immunosuppressant activities. Whereas activity of mTORC1 complex is highly sensitive to acute treatment by nanomolar concentrations of rapamycin, only prolonged rapamycin treatment is able to induce mTORC2 disruption and inactivation (2-4).A recently identified regulatory signal impacting on mTOR activity is the production of phosphatidic acid (PA) by phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (5, 6). PLD, which can be activated by a variety of hormones, growth factors, and cytokines, is present in most tissues under two isoforms, PLD1 and PLD2, endowed with different properties, regulations, and functions (7). Phosphatidic acid has been shown to specifically bind to mTOR protein on the FRB domain, a regulatory site also responsible for the binding of rapamycin in complex with protein FKBP12. The protein/ phospholipid interaction causes the activation of mTOR kinase, and rapamycin has been proposed to exert its inhibitory effects on mTOR by competing with PA and blocking PA-mediated activation (8 -10).Signaling by mTOR has become a topic of particular interest in the field of skeletal muscle biology due to the critical involvement of this kinase in muscle remodeling. Thus, muscle hypertrophy induced by exercise has been shown to involve mTOR signaling (11-13). Rapamycin inhibits...

Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1)

“…When myoblasts are maximally differentiated in culture, it may be difficult to observe further increase of differentiation upon knockdown of a negative regulator. During the preparation of this manuscript, Jaafar et al also reported a negative role of mTORC1 in vasopressin-induced differentiation of rat L6 myoblasts (36), in agreement with our findings in C2C12 cells.…”

Role of the mTORC1 Complex in Satellite Cell Activation by RNA-Induced Mitochondrial Restoration: Dual Control of Cyclin D1 through MicroRNAs

“…Moreover, in contrast to the opposite effects of mTORC and Raptor on in vitro differentiation (19,20), we observed that KD of mTORC and Raptor have identical effects on early satellite cell differentiation in vivo, indicating that the mTORC1 kinase, rather than the individual components, mediates the timing of satellite cell activation. One explanation of these discrepancies is that the microenvironment of the myoblasts at the injury site being different from that in culture, energy-sensing mechanisms differ in their contribution to the differentiation program.…”

“…Rapamycin inhibits the proliferation of primary myoblasts in an S6K-independent manner (14), as well as the differentiation of cultured myoblasts to myotubes upon serum deprivation in vitro (15)(16)(17), but the inhibition can be rescued by a rapamycin-resistant mTORC through a process that involves insulin-like growth factor II (IGFII) but does not require its kinase activity (17,18). Knockdown (KD) of mTORC in myoblasts inhibits their differentiation to myotubes in vitro, but unexpectedly, knockdown of Raptor has the opposite effect (19,20); the role of Rictor, a subunit of the mTORC2 complex, is uncertain, with reports of either inhibition of differentiation (20) or no effect (19) of Rictor KD. From these studies, it is apparent that mTORC can regulate cell growth and division in vitro, that the effect on growth requires S6K whereas that on myoblast division does not, indicating the presence of additional (unknown) mTORC targets, and finally, that mTORC and Raptor regulate the differentiation of cultured myoblasts to myotubes in different ways that are still unexplained.…”

An In Vitro TORC1 Kinase Assay That Recapitulates the Gtr-Independent Glutamine-Responsive TORC1 Activation Mechanism on Yeast Vacuoles

“…Vps34 is necessary for the TORC1 amino acid respon- siveness in mammals (37)(38)(39). The involvement of Vps34 in Drosophila melanogaster is contradicted by the observation that the Vps34 null mutant does not have altered TORC1 activity (40).…”

“…In mammals, Vps34 is suggested to activate TORC1 by recruiting phospholipase D (PLD) to the lysosomal membrane (37)(38)(39). Lysosome-recruited PLD generates phosphatidic acid (PA) on the membranes, which is required for TORC1 activation.…”