mTORC1 induces eukaryotic translation initiation factor 4E interaction with TOS-S6 kinase 1 and its activation

Abstract

Eukaryotic initiation factor (eIF4E) phosphorylation is a recognized attribute for enhanced cell growth and proliferation. Our recent data that identified eIF4E as mTORC1 substrate with ability to influence rapamycin response highlights its prospect as a mediator of mTORC1 signaling. Here, we present evidence that eIF4E phosphorylated at S209 interacts with TOS motif of S6 Kinase1 (S6K1). We show that this interaction is sufficient to overcome rapamycin sensitivity and mTORC1 dependence of S6K1. We present data to demonstrate that eIF4E-S6K1 interaction relieves S6K1 of its auto inhibition, otherwise rate limiting for its activation. We go on to identify a highly conserved sequence segment that mediates hydrophobic motif phosphorylation of S6K1 in mTORC2 rather than mTORC1 dependent manner. We also identify a novel eight amino-acid PHLPP1 (Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatase) binding region necessary to mediate S6K1 response to serum/growth factor stimulation. Finally, we show that deletion of the PHLPP1 binding region abolishes serum/growth factor stimulatory response without interfering with S6K1 regulation by mTORC1 or mTORC2. Our data identifies eIF4E as a novel TOS binding subunit that realigns mTORC1 signaling to unravel cross talk with other growth promoting signals for accomplishing S6K1 activation.

MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1

“…The observations are further endorsed by recent data that demonstrate occurrence of S6K1 phosphorylation even in absence of raptor [18]. Furthermore, we have recently identified eukaryotic translation initiation factor 4E (eIF4E) as an intermediate in transducing signals from mTORC1 onto S6K1 [19]. Therein, we demonstrate that the role of mTORC1 is restricted to engaging eIF4E with S6K1-TOS motif for relieving the auto-inhibition, due to carboxy terminal auto-inhibitory domain (CAD), to facilitate consequential HM phosphorylation and activation of S6K1.…”

“…To determine whether or not TOS motif is indispensable for mediating Thr-412 phosphorylation of S6K1 and whether or not relieving –NH2 and –COOH termini mediated auto-inhibition renders S6K1 active and independent of-the-regulation by mTORC1, we used HEK293 cells stably expressing Flag-tagged wild type S6K1 (described previously,[19]) and furthermore created lines of HEK293 cells stably expressing Flag-tagged S6K1 truncation mutant ΔNHΔCT to observe their state of Thr-412 phosphorylation in response to mTORC1 inhibition. We chose ΔNHΔCT S6K1 for the purpose because it bears truncations at TOS motif bearing -NH2 terminal domain and -COOH terminus auto-inhibitory domain (Fig 1a).…”

“…HEK293 and HEK293T cells, described previously [19], were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (Invitrogen), 50 μg/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) at 37°C with 5% CO2.…”

“…shRNA to human raptor (plasmid#1857) and rictor (plasmid#1853) were purchased from Addgene. The preparation of shRNA infected HEK293 cells have been described previously [19]…”

“…Therein, we demonstrate that the role of mTORC1 is restricted to engaging eIF4E with S6K1-TOS motif for relieving the auto-inhibition, due to carboxy terminal auto-inhibitory domain (CAD), to facilitate consequential HM phosphorylation and activation of S6K1. Altogether, these observations suggest that relieving auto-inhibition should render S6K1 active and independent of the regulation by mTORC1 and further point towards involvement of mTORC1 independent kinase in phosphorylating S6K1 at hydrophobic motif site [19].…”