Mitotic Raptor Promotes mTORC1 Activity, G₂/M Cell Cycle Progression, and Internal Ribosome Entry Site-Mediated mRNA Translation

Abstract

The mTOR signaling complex integrates signals from growth factors and nutrient availability to control cell growth and proliferation, in part through effects on the protein-synthetic machinery. Protein synthesis rates fluctuate throughout the cell cycle but diminish significantly during the G 2 /M transition. The fate of the mTOR complex and its role in coordinating cell growth and proliferation signals with protein synthesis during mitosis remain unknown. Here we demonstrate that the mTOR complex 1 (mTORC1) pathway, which stimulates protein synthesis, is actually hyperactive during mitosis despite decreased protein synthesis and reduced activity of mTORC1 upstream activators. We describe previously unknown G 2 /M-specific phosphorylation of a component of mTORC1, the protein raptor, and demonstrate that mitotic raptor phosphorylation alters mTORC1 function during mitosis. Phosphopeptide mapping and mutational analysis demonstrate that mitotic phosphorylation of raptor facilitates cell cycle transit through G 2 /M. Phosphorylation-deficient mutants of raptor cause cells to delay in G 2 /M, whereas depletion of raptor causes cells to accumulate in G 1 . We identify cyclindependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways as two probable mitosisregulated protein kinase pathways involved in mitosis-specific raptor phosphorylation and altered mTORC1 activity. In addition, mitotic raptor promotes translation by internal ribosome entry sites (IRES) on mRNA during mitosis and is demonstrated to be associated with rapamycin resistance. These data suggest that this pathway may play a role in increased IRES-dependent mRNA translation during mitosis and in rapamycin insensitivity.

Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein of the rDNA transcription machinery in nucleoli and chromosomal nucleolus organizer regions (NORs)

“…In prophase, the RNA . 7 As mentioned above, AMPK has been shown to directly phosphorylate Raptor at Ser 722 . 13 In this regard, we have previously reported that the active form of AMPK directly binds the mitotic apparatus and travels from centrosomes to the spindle midzone during mitosis.…”

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

“…Given this complexity, ULK1-mediated Raptor phosphorylation may be an epiphenomenon. As Raptor is phosphorylated by many positive and negative signaling inputs, [20][21][22][23][24][25][26] it is likely that the Raptor phosphorylation events observed by either overexpression or knockdown of ULK1 contribute to the regulation of mTORC1. It is highly probable that other factors will contribute to this negative feedback mechanism, which could include phosphorylation as playing an important role in autophagy induction.…”

“…It is known that phosphorylation of Raptor by RSK, 21 ERK1/2, 26 AMPK 20 or during mitosis 23 can modulate the activity of mTORC1. Therefore, to determine whether ULK1-mediated phosphorylation of Raptor could modulate mTORC1 signaling, we analyzed the activity of the downstream substrate S6K1 and the phosphorylation of 4E-BP1 in the presence or absence of ULK1 overexpression.…”

“…[20][21][22][23][24][25][26] To explore which sites were being phosphorylated in response to ULK1 overexpression, we employed a series of phospho-specific Raptor antibodies, directed towards the sites highlighted in Figure 2A. ULK1 promoted in vivo phosphorylation of Ser696, Thr706, Ser792, Ser855, Ser859 and Ser863 and weakly promoted the phosphorylation of Ser877 (Fig.…”

“…In other studies, Raptor phosphorylation by multiple positive and negative inputs has not been found to affect mTORC1 formation or substrate binding. [21][22][23][24][25][26] To examine mTORC1 formation, we analyzed the amount of endogenous mTOR and mLST8 bound to HA-Raptor expressed in the presence or absence of V5-ULK1. ULK1 overexpression had no significant effect on mTOR/Raptor/mLST8 protein association within mTORC1 (Fig.…”