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

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 G₂/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 G₂/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 G₂/M. Phosphorylation-deficient mutants of raptor cause cells to delay in G₂/M, whereas depletion of raptor causes cells to accumulate in G₁. We identify cyclin-dependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways as two probable mitosis-regulated 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.Cell growth and cell division are tightly coordinated processes required for cells to remain equal in size after division. In unicellular organisms, cell growth and proliferation are coordinated by nutrient availability, whereas their multicellular counterparts must also respond to growth factor input. Both processes lead to organismal growth as well as to increased cell number and cell mass. Cell growth and cell proliferation are also linked via the mTOR signaling pathway (16, 17). The mTOR kinase forms a distinct signaling complex (mTORC1) that participates in the coordination of nutrient and growth factor signaling. mTORC1 is composed of the kinase mTOR, the adaptor protein raptor, and the regulatory protein LST8 (25, 33, 34, 72).Accumulation of cellular proteins leads to cell growth and cell division. However, cell growth occurs only during certain phases of the cell cycle, necessitating that protein synthesis rates oscillate during cell cycling (40). In addition, in quiescent cells in G₀, protein synthesis rates are significantly reduced, whereas a select group of mRNAs maintain active translation (20, 68). During the G₁ phase, overall protein synthesis rates increase through S phase to allow cells to grow and enter another round of cell division while maintaining cell size (2, 3, 42, 45). As with G₀, entrance into mitosis (G₂/M phase) results in a global downregulation by as much as 60 to 80% of cap-dependent mRNA translation in primary, immortalized, and some transformed cells (5, 14, 29).Studies report several possible mechanisms for inhibition of protein synthesis during mitosis. Translation initiation requires the formation of an initiation factor complex known as eukaryotic translation initiation factor 4F (eIF4F), which consists of cap binding protein eIF4E, molecular scaffold protein eIF4G, and RNA helicase eIF4A. Together, they recruit ribosomes to mRNAs via bridging interactions between the 7-methyl-GTP (m⁷GTP) 5′ cap and the small 40S ribosomal subunit. Downregulation of protein synthesis during G₂/M was first ascribed to hypophosphorylation of eIF4E and the eIF4E binding proteins (4E-BPs) (5, 46). 4E-BPs are activated by hypophosphorylation, which allows them to bind and sequester eIF4E, preventing it from binding eIF4G and thereby blocking cap-dependent mRNA translation. More recently, several studies suggest that 4E-BP1, the major 4E-BP and a key target of mTORC1, is actually hyperphosphorylated (inactivated) during mitosis (26, 49). It is puzzling, then, that the phosphatidylinositol 3-kinase (PI3K)/AKT network and AKT itself (which modulate mTORC1 activity) are reportedly inactivated during late mitosis (1, 9, 22). In addition, phosphorylation of another mTORC1 target, ribosomal S6 kinase 1 (S6K1), and its activity are actually highest during G₂/M phase, consistent with elevated mTORC1 activity during mitosis (6).In this study we show that, despite repression of AKT and other activators of mTORC1 activity in mitosis, mTORC1 remains active and phosphorylates 4E-BP1 and S6K1 during G₂/M. We describe the multisite phosphorylation of raptor during mitosis, and we identify seven mitosis-specific raptor phosphorylation sites. By developing phosphomimetic and phosphorylation-deficient mutants of raptor, we show that hyperphosphorylated raptor promotes cell cycle transit through G₂/M, whereas hypophosphorylated raptor promotes transit through G₁. Raptor phosphorylation is shown to involve kinase pathways that are known to be active during mitosis, including cyclin-dependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways that are also upregulated in certain human cancers, including breast cancers. These and other findings disclose a novel regulatory network for mTORC1 that is active during mitosis, important for G₂/M progression and increased internal ribosome entry site (IRES)-dependent translation during mitosis, and indirectly associated with rapamycin resistance.