Cell migration is integral to physiology, for example in development and wound repair, and in pathophysiology, such as in diseases associated with fibrosis. The kinase complex mTORC2 phosphorylates proteins of the AGC family of kinases, such as Akt and protein kinase C (PKC), and stimulates cell migration. Lysophosphatidic acid (LPA) stimulates a G protein–coupled receptor that activates Gα12 to stimulate cell migration. Gan et al. define a pathway from LPA to PKC-δ through mTORC2 that promotes the migration of fibroblasts and show that this pathway contributes to fibroblast migration during pulmonary fibrosis. LPA stimulated an early phase of Akt phosphorylation (<5 minutes) and two phases of PKC-δ phosphorylation (<5 minutes and >60 minutes). The late phase was specifically absent in cells lacking Gα12 or in cells in which the mTORC2 core component MAPKAP1 was knocked down. In an in vitro kinase assay, mTORC2 immunoprecipitated from cells treated with LPA for 120 minutes, but not from those treated for 1 minute, showed an increased activity toward PKC but did not stimulate Akt phosphorylation. LPA treatment or overexpression of Gα12 stimulated the ubiquitylation of the regulatory mTORC2 subunit PRR5L (in cells in which the proteasome was inhibited) and reduced its abundance. Mouse embryonic fibroblasts (MEFs) deficient in PRR5L and the related protein PRR5 showed increased basal PKC-δ phosphorylation and increased basal migration but failed to show stimulation of the late phase of PKC-δ phosphorylation or increased migration in response to LPA. mTORC2 immunoprecipitated from cells in which PRR5L was knocked down had increased activity toward PKC-δ in an in vitro kinase assay and enhanced binding to PKC-δ, both of which were prevented if PRR5L was added to the in vitro assays. The E3 ubiquitin ligase RFFL was identified through transcriptional microarray analysis as induced by LPA through Gα12 signaling, and RFFL promoted the ubiquitylation of PRR5L in vitro. Knockdown and pharmacological experiments showed that the induction of RFFL transcripts and PRR5L ubiquitylation by Gα12 signaling required the mitogen-activated protein kinase (MAPK) pathway involving A-Raf (MAPKKK), MEK (MAPKK), and ERK (MAPK) but did not involve the guanosine triphosphatase Ras. Constitutively active Gα12 coimmunoprecipitated with A-Raf, and recombinant Gα12 interacted with A-Raf in a GTP-dependent manner, suggesting that Gα12 may directly activate A-Raf. In a mouse model of pulmonary fibrosis, compared with wild-type mice, mice deficient in Gα12 showed less pulmonary fibrosis, which was accompanied by decreased PRRL5 abundance and increased Rffl mRNA, and ERK and PKC-δ phosphorylation. Treatment of the mice with the proteasome inhibitor Bortezomib or the mTOR inhibitor PP242 also reduced bleomycin-induced pulmonary fibrosis and PKC-δ phosphorylation. Thus, Gan et al. defined a pathway through which mTORC2-mediated cell migration is activated by receptors coupled to Gα12.
X. Gan, J. Wang, C. Wang, E. Sommer, T. Kozasa, S. Srinivasula, D. Alessi, S. Offermanns, M. I. Simon, D. Wu, PRR5L degradation promotes mTORC2-mediated PKC-δ phosphorylation and cell migration downstream of Gα12. Nat. Cell Biol. 14, 686–696 (2012). [Online Journal]