Involvement of Heterogeneous Ribonucleoprotein F in the Regulation of Cell Proliferation via the Mammalian Target of Rapamycin/S6 Kinase 2 Pathway

Abstract

The S6 kinases (S6Ks) have been linked to a number of cellular processes, including translation, insulin metabolism, cell survival, and RNA splicing. Signaling via the phosphotidylinositol 3-kinase and mammalian target of rapamycin (mTOR) pathways is critical in regulating the activity and subcellular localization of S6Ks. To date, nuclear functions of both S6K isoforms, S6K1 and S6K2, are not well understood. To better understand S6K nuclear roles, we employed affinity purification of S6Ks from nuclear preparations followed by mass spectrometry analysis for the identification of novel binding partners. In this study, we report that in contrast to S6K1, the S6K2 isoform specifically associates with a number of RNA-binding proteins, including heterogeneous ribonucleoproteins (hnRNPs). We focused on studying the mechanism and physiological relevance of the S6K2 interaction with hnRNP F/H. Interestingly, the S6K2-hnRNP F/H interaction was not affected by mitogenic stimulation, whereas mTOR binding to hnRNP F/H was induced by serum stimulation. In addition, we define a new role of hnRNP F in driving cell proliferation, which could be partially attenuated by rapamycin treatment. S6K2-driven cell proliferation, on the other hand, could be blocked by small interfering RNA-mediated down-regulation of hnRNP F. These results demonstrate that the specific interaction between mTOR and S6K2 with hnRNPs is implicated in the regulation of cell proliferation.Regulation of cellular processes by extracellular stimuli, such as growth factors and nutrients, is an essential process in cell size and cell cycle control. The mammalian target of rapamycin (mTOR) 2 is a key regulator of cellular signaling and is highly dependent on the presence of nutrients, as well as growth factor-stimulated signaling from the phosphotidylinositol 3-kinase (PI3K) pathway. Activation of PI3K by a number of growth factor receptors leads to increased levels of phosphatidylinositol 3,4,5-triphosphate (1), a secondary messenger that recruits downstream kinases, such as 3-phosphoinositide-dependent kinase 1 (PDK1), to the plasma membrane (2). Membrane-associated PDK1 in turn phosphorylates and activates Akt at the T loop (3, 4), whereas activated Akt phosphorylation of tuberous sclerosis complex 2 (TSC2) blocks its inhibitory role in mTOR signaling (5, 6). TSC2 forms a heterodimer with TSC1, which as a complex acts as a GTPase activator toward the small GTPase, Rheb (Ras homolog enriched in brain) (7,8). Rheb associates directly with the catalytic domain of mTOR and activates it by antagonizing FKBP38, the proposed endogenous inhibitor of mTOR (9). Activated mTOR with its interacting partner protein Raptor, as part of the mTOR complex I (mTORC1) is able to phosphorylate S6Ks (at Thr 389 in S6K1 and Thr 388 in S6K2) (10). This early phosphorylation event is required for subsequent phosphorylation by PDK1 of Thr 229 and Thr 228 in the activation loops of S6K1 and S6K2, respectively (11). Phosphorylation by both mTORC1 and PDK1 leads to S6K activation, resul...

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“…Interestingly, recent studies have shown that S6K2 is constitutively associated with heterogeneous ribonucleoproteins in the nucleus, and the recruitment of mTORC1 to this complex induces the activation of S6K2 upon serum stimulation. 30 This finding leads us to speculate that mTORC1 might regulate the recruitment of S6K2 to the NCoR1/PPARa complex in response to nutrient levels.…”

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“…On the other hand, the importance of S6K2 in cell proliferation is illustrated in study demonstrating S6K2 was responsible for the interleukin-3 (IL-3)-driven cell proliferation since S6K2 was activated in lymphocytes and primary mouse bone marrow-derived mast cells upon IL-3 induced proliferation; and cell cycle progression was accelerated by overexpression of constitutively active S6K2 in lymphocytes (Cruz et al, 2005). Furthermore, the association of heterogeneous ribonucleoprotein (hnRNP) F with mTOR and S6K2, but not S6K1, is essential for driving cell proliferation (Goh et al, 2010). Taking collectively, both S6K1 and S6K2 are involved in mTORC1-mediated cell cycle progression.…”

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“…HnRNPs are chromatin-associated nuclear proteins that bind specifically to different RNA sequences and are involved in several steps of RNA processing, including splicing, polyadenylation, mRNA stability and translational regulation [167]. Upon serum stimulation, mTOR associates with the hnRNP-S6K2 complex and activates S6K2 which promotes cell proliferation through unknown mechanisms [120]. Recently, it was also shown that S6K2, but not S6K1, can bind to the transcription factor YY1 in serum stimulated cells [116] (Figure 10).…”

“…However, SH3-binding motifs, which can be found in S6K2, are shown to facilitate interaction of cofactors with ERα [138], allowing to speculate that S6K2 also has the potential to act as a cofactor of the receptor. Nuclear S6K2, but not S6K1 has been shown to associate with several RNA-binding proteins, including the hnRNPs [120]. HnRNPs are chromatin-associated nuclear proteins that bind specifically to different RNA sequences and are involved in several steps of RNA processing, including splicing, polyadenylation, mRNA stability and translational regulation [167].…”