Proteomic identification of PSF and p54(nrb) as TopBP1-interacting proteins

TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by co-immunoprecipitation assays and LC-ESI-MS-analyses. As TopBP1 binding partners we identified p54(nrb) and PSF, and confirmed the physical interactions by GST pull-down assays, co-immunoprecipitations and by yeast two-hybrid experiments. Recent evidence shows an involvement of p54(nrb) and PSF in DNA double-strand break repair (DSB) and radioresistance. To get a first picture of the physiological significance of the interaction of TopBP1 with p54(nrb) and PSF we investigated in real time the spatiotemporal behaviour of the three proteins after laser microirradiation of living cells. Localisation of TopBP1 at damage sites was noticed as early as 5 s following damage induction, whereas p54(nrb) and PSF localised there after 20 s. Both p54(nrb) and PSF disappeared after 20 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair.     

Mnk kinase pathway: Cellular functions and biological outcomes

The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.     

Sequences in PSF/SFPQ mediate radioresistance and recruitment of PSF/SFPQ-containing complexes to DNA damage sites in human cells

PSF (gene name SFPQ) is a member of a small family of proteins with dual functions in RNA biogenesis and DNA repair. PSF and PSF-containing complexes stimulate double-strand break repair in cell free systems, most likely via direct interaction with the repair substrate. Prior in vitro studies are, however, insufficient to demonstrate whether PSF contributes to DNA repair in living cells. Here, we investigate the effect of miRNA-mediated PSF knockdown in human (HeLa) cells. We find that PSF is essential for reproductive viability. To circumvent this and investigate the DNA damage sensitivity phenotype, we established a genetic rescue assay based on co-transfection of PSF miRNA and mutant PSF expression constructs. Mutational analysis suggests that sequences required for viability and radioresistance are partially separable, and that the latter requires a unique N-terminal PSF domain. As an independent means to investigate PSF sequences involved in DNA repair, we established an assay based on real-time relocalization of PSF-containing complexes to sites of dense, laser-induced DNA damage in living cells. We show that relocalization is driven by sequences in PSF, rather than its dimerization partner, p54(nrb)/NONO, and that sequences required for relocalization reside in the same N-terminal domain that contributes to radioresistance. Further evidence for the importance of PSF sequences in mediating relocalization is provided by observations that PSF promotes relocalization of a third protein, PSPC1, under conditions where p54(nrb) is limiting. Together, these observations support the model derived from prior biochemical studies that PSF influences repair via direct, local, interaction with the DNA substrate.     

PSF/p54(nrb) stimulates "jumping" of DNA topoisomerase I between separate DNA helices

We have previously shown [Straub et al. (1998) J. Biol. Chem. 273, 26261] that the pyrimidine tract binding protein associated splicing factor PSF/p54(nrb) binds and stimulates DNA topoisomerase I. Here we show that cleavage and religation half-reactions of topoisomerase I are unaffected by PSF/p54(nrb), whereas the propensity of the enzyme to jump between separate DNA helices is stimulated. To demonstrate such an effect, topoisomerase I was first captured in suicidal cleavage of an oligonucleotide substrate. Subsequently, a cleavage/ligation equilibrium was established by adding a ligation donor under conditions allowing recleavage of the ligated substrate. Finally, a second oligonucleotide was added to the mixture, which also allowed suicidal cleavage by topoisomerase I, but did not accommodate the ligation donor of the first oligonucleotide. Thus, topoisomerase I was given the choice to engage in repeated cleavage/ligation cycles of the first oligonucleotide or to jump to the second suicide substrate and get trapped. PSF/p54(nrb) enhanced the cleavage rate of the second oligonucleotide (11-fold), suggesting that it stimulates the dissociation of topoisomerase I after ligation. Thus, stimulation of topoisomerase I catalysis by PSF/p54(nrb) seems to be affected by mobilization of the enzyme.     

Features of the catalytic domains and C termini of the MAPK signal-integrating kinases Mnk1 and Mnk2 determine their differing activities and regulatory properties

The MAPK signal-integrating kinases Mnk1 and Mnk2 are closely related but show marked differences in their basal activities and regulation. Both possess, within their C termini, motifs for binding to MAPKs, although these differ between Mnk1 and Mnk2. Mnk2 shows much higher activity in unstimulated cells than Mnk1, whose activity is greatly increased, e.g. by stimulation of the MEK/ERK pathway. Such increases are sensitive to blockade of that pathway, whereas the activation state of Mnk2 is relatively insensitive to inhibition of upstream signaling. Here we have studied the roles of features in their catalytic domains and C termini in determining their regulatory properties and basal activities. Mnk2 can bind to phosphorylated, active ERK, whereas Mnk1 cannot. Such binding apparently protects ERK against dephosphorylation and inactivation. The high basal activity of Mnk2 and its binding to (phospho)ERK requires features both of the catalytic domain and of the C terminus. For example, within the catalytic region an aspartate in Mnk2 plays a key role. Mutation to alanine inactivates Mnk2. In the C terminus, features within the MAPK-binding motif and to either side of it, including potential phosphorylation sites, affect MAPK binding and activity. The association of Mnks with the scaffold protein eukaryotic initiation factor 4G is negatively modulated by Mnk activity. These data indicate that multiple features determine the activities of the Mnks and thus impact on their ability to phosphorylate physiological substrates such as eukaryotic initiation factor 4E.     

Identification of the polypyrimidine tract binding protein-associated splicing factor.p54(nrb) complex as a candidate DNA double-strand break rejoining factor

The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF.p54(nrb) complex cooperates with Ku protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF.p54(nrb) complex cooperate to align separate DNA molecules.     

Purification and cDNA cloning of HeLa cell p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6.

While searching for a human homolog of the S.cerevisiae splicing factor PRP18, we found a polypeptide that reacted strongly with antibodies against PRP18. We purified this polypeptide from HeLa cells using a Western blot assay, and named it p54nrb (for nuclear RNA-binding protein, 54 kDa). cDNAs encoding p54nrb were cloned with probes derived from partial sequence of the purified protein. These cDNAs have identical coding sequences but differ as a result of alternative splicing in the 5' untranslated region. The cDNAs encode a 471 aa polypeptide that contains two RNA recognition motifs (RRMs). Human p54nrb has no homology to yeast PRP18, except for a common epitope, but is instead 71% identical to human splicing factor PSF within a 320 aa region that includes both RRMs. In addition, both p54nrb and PSF are rich in Pro and Gln residues outside the main homology region. The Drosophila puff-specific protein BJ6, one of three products encoded by the alternatively spliced no-on-transient A gene (nonA), which is required for normal vision and courtship song, is 42% identical to p54nrb in the same 320 aa region. The striking homology between p54nrb, PSF, and NONA/BJ6 defines a novel phylogenetically conserved protein segment, termed DBHS domain (for Drosophila behavior, human splicing), which may be involved in regulating diverse pathways at the level of pre-mRNA splicing.     

PSF and p54nrb bind a conserved stem in U5 snRNA

PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.     

The mitogen-activated protein kinase signal-integrating kinase Mnk2 is a eukaryotic initiation factor 4E kinase with high levels of basal activity in mammalian cells

The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKalpha, and p38MAPKbeta. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.     

Involvement of p54(nrb), a PSF partner protein,in DNA double-strand break repair and radioresistance

Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a γ-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.     

Regulation of arsenic trioxide-induced cellular responses by Mnk1 and Mnk2

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As(2)O(3) induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38alpha MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1(-/-) or Mnk2(-/-), or double Mnk1(-/-)Mnk2(-/-) knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.     

Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo

Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.