Crosstalk Signaling Between cAMP and mTORC1

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. G-protein-coupled receptors (GPCRs) paired to Gs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA), which phosphorylates Raptor at Ser 791 resulting in potent mTORC1 inhibition. We identified a novel mTORC1-interacting protein called A-kinase anchoring protein 8L (AKAP8L). Using biochemical assays, we found that the N-terminal region of AKAP8L binds to mTORC1 in the cytoplasm. Importantly, loss of AKAP8L decreased mTORC1-mediated processes such as translation, cell growth and cell proliferation. AKAPs anchor protein kinase A (PKA) through PKA regulatory subunits, and we show that AKAP8L can anchor PKA through regulatory subunit I (RI). Full-length AKAP8L restored mTORC1-regulated biology, whereas AKAP8L missing the N-terminal region that confers interaction with mTORC1 did not. Additionally, we have shown that H89 (N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new unknown target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling. Lastly, we identified A-kinase anchoring protein 13 (AKAP13) as a crucial scaffold involved in GPCR-Gs signaling to mTORC1. AKAP13 potently enhances Raptor Ser 791 phosphorylation and inhibits mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, AKAP13 is unable to supress mTORC1 activity. AKAP13 mediates mTORC1-induced cell proliferation, cell size and colony formation. Interestingly, AKAP13 expression inversely correlates with mTORC1 activation and positively correlates with overall lung adenocarcinoma patient survival. Our results place the GPCR-Gas signaling pathway to mTORC1 as a potential target that may be beneficial for human diseases with hyperactivated mTORC1.