The PSF·p54nrb Complex Is a Novel Mnk Substrate That Binds the mRNA for Tumor Necrosis Factor α

Abstract

To identify new potential substrates for the MAP kinase signal-integrating kinases (Mnks), we employed a proteomic approach. The Mnks are targeted to the translational machinery through their interaction with the cap-binding initiation factor complex. We tested whether proteins retained on cap resin were substrates for the Mnks in vitro, and identified one such protein as PSF (the PTB (polypyrimidine tract-binding protein)-associated splicing factor). Mnks phosphorylate PSF at two sites in vitro, and our data show that PSF is an Mnk substrate in vivo. We also demonstrate that PSF, together with its partner, p54 nrb , binds RNAs that contain AU-rich elements (AREs), such as those for proinflammatory cytokines (e.g. tumor necrosis factor ␣ (TNF␣)). Indeed, PSF associates specifically with the TNF␣ mRNA in living cells. PSF is phosphorylated at two sites by the Mnks. Our data show that Mnk-mediated phosphorylation increases the binding of PSF to the TNF␣ mRNA in living cells. These findings identify a novel Mnk substrate. They also suggest that the Mnk-catalyzed phosphorylation of PSF may regulate the fate of specific mRNAs by modulating their binding to PSF⅐p54 nrb .Polypyrimidine tract-binding protein (PTB) 3 -associated splicing factor (PSF) and p54 nrb are highly homologous DNA/ RNA-binding proteins that form a multifunctional heterodimer implicated in nuclear processes such as transcription, nuclear RNA processing, nuclear retention of edited RNA, DNA relaxation, and tumorigenesis (reviewed in Ref. 1). These proteins also cooperate in the inhibition of human immunodeficiency virus type 1 mRNA expression (2). Moreover, PSF is also been reported to repress gene expression through its association with nuclear hormone receptors (3) or through binding to insulin-like growth factor-1 response elements (4). PSF and p54 nrb are both phosphoproteins. Phosphorylation may be involved in the relocalization of PSF during apoptosis (5) and in regulating the binding properties of p54 nrb during mitosis (6). All eukaryotic cytoplasmic mRNAs have a 5Ј-terminal cap structure that contains 7-methyl-GTP (m 7 GTP) and promotes their efficient translation (7,8). The cap is bound by eukaryotic translation initiation factor eIF4E, which also binds to the scaffold eIF4G and through this with other translational factors to recruit the 40 S ribosomal subunit to the mRNA. eIF4G also binds the poly(A)-binding protein (PABP), which interacts with the 3Ј-end of the mRNA, thus circularizing it (reviewed in Ref. 9). eIF4E is phosphorylated in vitro and in vivo by the MAP kinase-signal integrating (or MAP kinase-interacting) kinases (Mnks) (10 -13). There are two Mnk genes in humans, each of which generates two different polypeptides as a consequence of alternative splicing (14, 15). The longer Mnk1 isoform, Mnk1a, is switched on by signaling through the ERK and p38 MAP kinase pathways, whereas Mnk2a (the longer Mnk2 isoform), in contrast, shows high basal activity (11,16,17). We recently showed that Mnks play an important role in the control ...

Mnk kinases in cytokine signaling and regulation of cytokine responses

“…As previous studies had shown that PSF along with its binding partner p54nrb can bind ARE containing mRNAs [37], this study established that Mnk1 mediated phosphorylation of PSF in response to T cell activation facilitates the interaction between the PSF/p54nrb and the ARE containing mRNAs such as TNFα and GM-CSF [36]. The authors also demonstrated that PSF/p54nrb binding to ARE does not affect the nuclear-cytoplasmic distribution or TNFα mRNA stability [36]. The importance of Mnk induced binding of PSF/p54nrb to ARE containing mRNAs and its effects on translation of the respective mRNAs remains to be determined.…”

“…A separate study systematically used a proteomic approach to identify novel Mnk1 substrates based on their abilities to interact with a 5’-7-methylguanylate cap bound resin [36]. These studies identified PSF [PTB (polypyrimidine tract binding protein) associated splicing factor] as a Mnk substrate that undergoes phosphorylation on Ser8 and Ser283 [36].…”

“…These studies identified PSF [PTB (polypyrimidine tract binding protein) associated splicing factor] as a Mnk substrate that undergoes phosphorylation on Ser8 and Ser283 [36]. A series of immunoprecipitation experiments indicated that the retention of PSF on the cap bound resin reflected its ability to interact with mRNA and not its ability to associate with cap binding proteins [36].…”

“…These studies identified PSF [PTB (polypyrimidine tract binding protein) associated splicing factor] as a Mnk substrate that undergoes phosphorylation on Ser8 and Ser283 [36]. A series of immunoprecipitation experiments indicated that the retention of PSF on the cap bound resin reflected its ability to interact with mRNA and not its ability to associate with cap binding proteins [36]. As previous studies had shown that PSF along with its binding partner p54nrb can bind ARE containing mRNAs [37], this study established that Mnk1 mediated phosphorylation of PSF in response to T cell activation facilitates the interaction between the PSF/p54nrb and the ARE containing mRNAs such as TNFα and GM-CSF [36].…”

“…A series of immunoprecipitation experiments indicated that the retention of PSF on the cap bound resin reflected its ability to interact with mRNA and not its ability to associate with cap binding proteins [36]. As previous studies had shown that PSF along with its binding partner p54nrb can bind ARE containing mRNAs [37], this study established that Mnk1 mediated phosphorylation of PSF in response to T cell activation facilitates the interaction between the PSF/p54nrb and the ARE containing mRNAs such as TNFα and GM-CSF [36]. The authors also demonstrated that PSF/p54nrb binding to ARE does not affect the nuclear-cytoplasmic distribution or TNFα mRNA stability [36].…”