A structural and functional study delineates how the interaction between NF-κB subunit RelA and co-activator CBP/p300 helps drive transcription of NF-κB target genes.
NF-κB plays a vital role in cellular immune and inflammatory response, survival, and proliferation by regulating the transcription of various genes involved in these processes. To activate transcription, RelA (a prominent NF-κB family member) interacts with transcriptional co-activators like CREB-binding protein (CBP) and its paralog p300 in addition to its cognate κB sites on the promoter/enhancer regions of DNA. The RelA:CBP/p300 complex is comprised of two components—first, DNA binding domain of RelA interacts with the KIX domain of CBP/p300, and second, the transcriptional activation domain (TAD) of RelA binds to the TAZ1 domain of CBP/p300. A phosphorylation event of a well-conserved RelA(Ser276) is prerequisite for the former interaction to occur and is considered a decisive factor for the overall RelA:CBP/p300 interaction. The role of the latter interaction in the transcription of RelA-activated genes remains unclear. Here we provide the solution structure of the latter component of the RelA:CBP complex by NMR spectroscopy. The structure reveals the folding of RelA–TA2 (a section of TAD) upon binding to TAZ1 through its well-conserved hydrophobic sites in a series of grooves on the TAZ1 surface. The structural analysis coupled with the mechanistic studies by mutational and isothermal calorimetric analyses allowed the design of RelA-mutants that selectively abrogated the two distinct components of the RelA:CBP/p300 interaction. Detailed studies of these RelA mutants using cell-based techniques, mathematical modeling, and genome-wide gene expression analysis showed that a major set of the RelA-activated genes, larger than previously believed, is affected by this interaction. We further show how the RelA:CBP/p300 interaction controls the nuclear response of NF-κB through the negative feedback loop of NF-κB pathway. Additionally, chromatin analyses of RelA target gene promoters showed constitutive recruitment of CBP/p300, thus indicating a possible role of CBP/p300 in recruitment of RelA to its target promoter sites.
The NF-κB family of transcription factors regulate the expression of numerous genes involved in the immune response, cell survival, differentiation, and proliferation. The interaction of the RelA subunit of NF-κB with the general co-activator protein CBP/p300 is vital for RelA-dependent gene transcription. Although the recruitment of RelA to its cognate genomic κB sites for target gene activation is well-established, the involvement of CBP/p300 in this process remains unclear. Through our structure/function-based approach we provide the molecular and functional details of the RelA:CBP/p300 interaction and its contribution to the regulation of distinct subsets of target genes. We also show that disruption of this interaction deregulates the NF-κB pathway by interfering with its negative feedback loop. Furthermore, our study indicates a possible reciprocal role for CBP/p300 in the recruitment of RelA to its target gene promoters.
Besides activating NFκB by phosphorylating IκBs, IKKα/IKKβ kinases are also involved in regulating metabolic insulin signaling, the mTOR pathway, Wnt signaling, and autophagy. How IKKβ enzymatic activity is targeted to stimulus-specific substrates has remained unclear. We show here that NEMO, known to be essential for IKKβ activation by inflammatory stimuli, is also a specificity factor that directs IKKβ activity towards IκBα. Physical interaction and functional competition studies with mutant NEMO and IκB proteins indicate that NEMO functions as a scaffold to recruit IκBα to IKKβ. Interestingly, expression of NEMO mutants that allow for IKKβ activation by the cytokine IL-1 but fail to recruit IκBs, results in hyperphosphorylation of alternative IKKβ substrates. Furthermore IKK's function in autophagy, which is independent of NFκB is significantly enhanced without NEMO as IκB scaffold. Our work establishes a role for scaffolds such as NEMO in determining stimulus-specific signal transduction via the pleiotropic signaling hub IKK.
Conformational change in human IKK2 permits dimers to form higher-order oligomers that support interaction between kinase domains and promote activation through trans auto-phosphorylation.
Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.
IκB kinase (IKK) is an enzyme that quickly becomes active in response to diverse stresses on a cell. Once activated, IKK promotes an array of cellular defense processes by phosphorylating IκB, thereby promoting its degradation and liberating its partner, the pro-survival transcription factor NF-κB; NF-κB is then free to relocate to the nucleus where it can modulate gene expression. Our X-ray crystallographic studies on an active version of the human IKK2 isoform reveal that the enzyme adopts a unique open conformation that permits pairs of IKK2 enzymes to form higher order assemblies in which their catalytic domains are in close proximity. Disruption of IKK2's ability to form these assemblies, by introducing changes that interfere with the surfaces that mediate oligomerization, results in IKK2 enzymes that are greatly impaired in their ability to become activated in cells. We propose that after oligomerization the neighboring catalytic domains then phosphorylate each other as part of the activation process. Our findings also suggest that targeted small molecules might disrupt cell survival by blocking IKK2 assembly in cells.
ASF/SF2, a member of the serine-arginine (SR) protein family, has two RRM domains (RRM1 and RRM2) and a C-terminus domain rich in RS dipeptides. The SR protein kinase 1 (SRPK1) phosphorylates approximately 12 of these serines using a semi-processive mechanism. The x-ray structure of the ASF/SF2:SRPK1 complex revealed several features of the complex that raised intriguing questions of how the substrate is phosphorylated by the kinase: The part of the RS domain destined to be phosphorylated at later stages of the reaction docks to a kinase groove distal to the active site while the neighboring RRM2 binds near the active site (1). In this study we investigated the interplay between the RS domain and RRM2 for stable association and phosphorylation of ASF/SF2. Despite several contacts in the enzyme-substrate complex, free RRM2 does not bind efficiently to SRPK1 unless the docking groove is occupied by the RS domain. This domain cross-talk enhances the processive phosphorylation of the RS domain. The RRM:SRPK1 contact residues control the folding of a critical beta strand in RRM2. Unfolding of this structural element may force the N-terminal serines of the RS domain into the active site for sequential phosphorylation. Thus, ASF/SF2 represents a new class of substrates that use unique primary sequence to induce allosteric binding, processive phosphorylation, and product release.
The splicing of mRNA requires a group of essential factors known as SR proteins that participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition domains (RRMs) and a C-terminal domain rich in Arg-Ser repeats (RS domain). SR proteins are phosphorylated at numerous serines in the RS domain by the SRPK family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus and may also play important roles in alternative splicing, mRNA export and other processing events. Although SR proteins are polyphosphorylated in vivo, the mechanism underlying this complex reaction has only been recently elucidated. SRSF1, a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a posttranslational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity and rapidly modifies about 10–12 serines in the N-terminal portion of the RS domain (RS1) using a mechanism that incorporates sequential, C-to-N phosphorylation and several processive steps. SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound to a docking groove in the large lobe of the kinase domain. Upon subsequent rounds of phosphorylation, this N-terminal segment translocates into the active site and a β strand in RRM2 unfolds and occupies the docking groove. These studies indicate that efficient regiospecific phosphorylation of SRSF1 is the result of a contoured binding cavity in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain and a highly directional processing mechanism.
Mechanism; Protein Kinase; Splicing; SR protein; Structure
SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, region-specific mapping of phosphorylation sites in ASF/SF2 was monitored as a function of the protein phosphatase PP1. We showed that ten phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs (RRMs) in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RRMs protects against dephosphorylation suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.
protein kinase; protein phosphatase; phosphorylation; splicing; SR protein
Mutations that impair activity of the ER stress response kinase Ire1 inhibit resolution of the unfolded protein response (see also a related paper by Rubio et al. in this issue).
The unfolded protein response (UPR) activates Ire1, an endoplasmic reticulum (ER) resident transmembrane kinase and ribonuclease (RNase), in response to ER stress. We used an in vivo assay, in which disappearance of the UPR-induced spliced HAC1 messenger ribonucleic acid (mRNA) correlates with the recovery of the ER protein-folding capacity, to investigate the attenuation of the UPR in yeast. We find that, once activated, spliced HAC1 mRNA is sustained in cells expressing Ire1 carrying phosphomimetic mutations within the kinase activation loop, suggesting that dephosphorylation of Ire1 is an important step in RNase deactivation. Additionally, spliced HAC1 mRNA is also sustained after UPR induction in cells expressing Ire1 with mutations in the conserved DFG kinase motif (D828A) or a conserved residue (F842) within the activation loop. The importance of proper Ire1 RNase attenuation is demonstrated by the inability of cells expressing Ire1-D828A to grow under ER stress. We propose that the activity of the Ire1 kinase domain plays a role in attenuating its RNase activity when ER function is recovered.
SR proteins promote spliceosome formation by recognizing exonic splicing enhancers (ESEs) during pre-mRNA splicing. Each SR protein binds diverse ESEs using strategies that are yet to be elucidated. Here, we show that the RNA-binding domain (RBD) of SRSF1 optimally binds to decameric purine rich ESE sequences although locations of purines are not stringently specified. The presence of uracils either within or outside of the recognition site is detrimental for binding with SRSF1. The entire RBD, comprised of two RRMs and a glycine-rich linker, is essential for ESE binding. Mutation within each segment reduced or nearly abolished binding, suggesting that these segments mediate cooperative binding. The linker plays a decisive role in organizing ESE binding. The flanking basic regions of the linker appear to communicate with each other in bringing the two RRMs close together to form the complex with RNA. Our study thus suggests semi-conservative adaptable interaction between ESE and SRSF1, and such binding mode is not only essential for the recognition of plethora of physiological ESE sequences but may also be essential for the interaction with various factors during the spliceosome assembly.
The specific binding of transcription factors to cognate sequence elements is thought to be critical for the generation of specific gene expression programs. Members of the nuclear factor κB (NF-κB) and interferon (IFN) regulatory factor (IRF) transcription factor families bind to the κB site and the IFN response element (IRE), respectively, of target genes, and they are activated in macrophages after exposure to pathogens. However, how these factors produce pathogen-specific inflammatory and immune responses remains poorly understood. Combining top-down and bottom-up systems biology approaches, we have identified the NF-κB p50 homodimer as a regulator of IRF responses. Unbiased genome-wide expression and biochemical and structural analyses revealed that the p50 homodimer repressed a subset of IFN-inducible genes through a previously uncharacterized subclass of guanine-rich IRE (G-IRE) sequences. Mathematical modeling predicted that the p50 homodimer might enforce the stimulus specificity of composite promoters. Indeed, the production of the antiviral regulator IFN-β was rendered stimulus-specific by the binding of the p50 homodimer to the G-IRE–containing IFNβ enhancer to suppress cytotoxic IFN signaling. Specifically, a deficiency in p50 resulted in the inappropriate production of IFN-β in response to bacterial DNA sensed by Toll-like receptor 9. This role for the NF-κB p50 homodimer in enforcing the specificity of the cellular response to pathogens by binding to a subset of IRE sequences alters our understanding of how the NF-κB and IRF signaling systems cooperate to regulate antimicrobial immunity.
The pro-survival activity of NF-κB in response to a variety of stimuli has been extensively characterized. Although there have been a few reports addressing the pro-cell death role of NF-κB, the precise mechanism of NF-κB's pro-cell death function still remains elusive.
In the present study, we investigated the role of NF-κB in cell death induced by chronic insult with hydrogen peroxide (H2O2). Here, we show that NF-κB promotes H2O2 induced caspase independent but PARP dependent fibroblast cell death. The pro-death activity of NF-κB is due to the DNA binding activity of RelA, which is induced through IKK- mediated IκBα degradation. NF-κB dependent pro-survival genes, Bcl-2 and XIAP, were significantly repressed, while NF-κB dependent pro-death genes, TNFα and Fas Ligand, were induced in response to H2O2.
We discovered an unexpected function of NF-κB, in that it potentiates chronic H2O2 exposure induced cell death, and suggest that NF-κB mediates cell death through the repression of pro-survival genes and induction of pro-death genes. Since unremitting exposure of tissues to H2O2 and other reactive oxygen species can lead to several degenerative disorders and diseases, our results have important implications for the use of NF-κB inhibitors in therapeutic drug design.
The SR proteins are essential factors that control the splicing of precursor mRNA by regulating multiple steps in spliceosome development. The prototypical SR protein ASF/SF2 contains two N-terminal RRMs (RRM1 and RRM2) and a 50-residue C-terminal RS (arginine-serine rich) domain that can be phosphorylated at numerous serines by the protein kinase SRPK1. The RS domain is further divided into N-terminal (RS1) and C-terminal (RS2) segments whose modification guides the nuclear localization of ASF/SF2. While previous studies revealed that SRPK1 phosphorylates RS1, regio- and temporal-specific control within the largely redundant RS domain is not well understood. To address this issue, engineered footprinting and single turnover experiments were performed to determine where and how SRPK1 initiates phosphorylation within the RS domain. The data show that local sequence elements in the RS domain control the strong kinetic preference for RS1 phosphorylation. SRPK1 initiates phosphorylation in a small region of serines (initiation box) in the middle of the RS domain at the C-terminal end of RS1 and then proceeds in an N-terminal direction. This initiation process requires both a viable docking groove in the large lobe of SRPK1 and one RRM (RRM2) on the N-terminal flank of the RS domain. Thus, while local RS/SR content steers regional preferences in the RS domain, distal contacts with SRPK1 guide initiation and directional phosphorylation within these regions.
protein kinase; regiospecificity; phosphorylation; splicing; SR protein
The 2.9 Å crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a β strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.
The SR protein ASF/SF2, an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain). The serine protein kinase SRPK1 phosphorylates the RS domain at multiple serines using a directional (C-to-N-terminal) and processive mechanism, a process that directs the SR protein to the nucleus and influences protein-protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme-substrate complex was analyzed using single and multi-turnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1-RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible, protein-protein interactions to facilitate both early and late phases of processive phosphorylation of SR proteins.
kinase; kinetics; phosphorylation; splicing; SR protein
The prosurvival transcription factor NF-κB specifically binds promoter DNA to activate target gene expression. NF-κB is regulated through interactions with IκB inhibitor proteins. Active proteolysis of these IκB proteins is, in turn, under the control of the IκB kinase complex (IKK). Together, these three molecules form the NF-κB signaling module. Studies aimed at characterizing the molecular mechanisms of NF-κB, IκB, and IKK in terms of their three-dimensional structures have lead to a greater understanding of this vital transcription factor system.
Structural studies of the NF-κB transcription factor and its regulators I-κB and IKK provide insights into NF-κB dimerization, activation, and DNA binding.
Transcription complex components frequently show coupled folding and binding but the functional significance of this mode of molecular recognition is unclear. IκBα binds to and inhibits the transcriptional activity of NF-κB via its ankyrin repeat (AR) domain. The β-hairpins in ARs 5–6 in IκBα are weakly folded in the free protein, and their folding is coupled to NF-κB binding. Here we show that introduction of two stabilizing mutations in IκBα AR 6 causes ARs 5–6 to cooperatively fold to a conformation similar to that in NF-κB-bound IκBα. Free IκBα is degraded by a proteasome-dependent but ubiquitin-independent mechanism, and this process is slower for the pre-folded mutants both in vitro and in cells. Interestingly, the pre-folded mutants bind NF-κB more weakly as shown by both SPR and ITC in vitro and immunoprecipitation experiments from cells. One consequence of the weaker binding is that resting cells containing these mutants show incomplete inhibition of NF-κB activation; they have significant amounts of nuclear NF-κB. Additionally, the weaker binding combined with the slower degradation rate of the free protein results in reduced levels of nuclear NF-κB upon stimulation. These data clearly demonstrate that the coupled folding and binding of IκBα is critical for its precise control of NF-κB transcriptional activity.
coupled folding and binding; transcription factor regulation; protein-protein interactions; ankyrin repeat; ubiquitin-independent proteasome degradation
IκBα is an ankyrin repeat protein that inhibits NF-κB transcriptional activity by sequestering NF-κB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IκBα ankyrin repeat domain to NF-κB(p50/p65) using surface plasmon (SPR) resonance and isothermal titration calorimetry (ITC). SPR data showed that the IκBα and NF-κB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a KD.obs of approximately 40 pM at 37 °C. As reported previously, the amino-terminal/DNA binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high affinity binding. This was surprising given the small size of the binding interface formed by this part of the p65. The NF-κB(p50/p65) heterodimer and p65 homodimer bound IκBα with almost indistinguishable thermodynamics except that the NF-κB p65 homodimer was characterized by a more favorable ΔHobs relative to the NF-κB(p50p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (ΔCP,obs), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be readily accounted for by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.
Ankyrin repeat; NF-κB; rel family; IκBα; surface plasmon resonance; isothermal titration calorimetry; transcription factor
Splicing requires reversible phosphorylation of serine/arginine-rich (SR) proteins, which direct splice site selection in eukaryotic mRNA. These phosphorylation events are dependent on SR protein (SRPK) and cdc2-like kinase (CLK) families. SRPK1 phosphorylation of splicing factors is restricted by a specific docking interaction whereas CLK activity is less constrained. To understand functional differences between splicing factor targeting kinases, we determined crystal structures of CLK1 and CLK3. Intriguingly, in CLKs the SRPK1 docking site is blocked by insertion of a previously unseen helix αH. In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific β7/8-hairpin insert. Thus, the unconstrained substrate interaction together with the determined active-site mediated substrate specificity allows CLKs to complete the functionally important hyperphosphorylation of splicing factors like ASF/SF2. In addition, despite high sequence conservation, we identified inhibitors with surprising isoform specificity for CLK1 over CLK3.
Distal interactions between discrete elements of an enzyme are critical for communication and ultimately for regulation. However, identifying the components of such interactions has remained elusive due to the delicate nature of these contacts. Protein kinases are a prime example of an enzyme with multiple regulatory sites that are spatially separate, yet communicate extensively for tight regulation of activity. Kinase misregulation has been directly linked to a variety of cancers, underscoring the necessity for understanding intramolecular kinase regulation.
A genetic screen was developed to identify suppressor mutations that restored catalytic activity in vivo from two kinase-dead Protein Kinase A mutants in S. cerevisiae. The residues defined by the suppressors provide new insights into kinase regulation. Many of the acquired mutations were distal to the nucleotide binding pocket, highlighting the relationship of spatially dispersed residues in regulation.
The suppressor residues provide new insights into kinase regulation, including allosteric effects on catalytic elements and altered protein-protein interactions. The suppressor mutations identified in this study also share overlap with mutations identified from an identical screen in the yeast PKA homolog Tpk2, demonstrating functional conservation for some residues. Some mutations were independently isolated several times at the same sites. These sites are in agreement with sites previously identified from multiple cancer data sets as areas where acquired somatic mutations led to cancer progression and drug resistance. This method provides a valuable tool for identifying residues involved in kinase activity and for studying kinase misregulation in disease states.
Inflammatory NF-κB/RelA activation is mediated by the three canonical inhibitors, IκBα, -β, and -ε. We report here that nfκb2/p100 forms two distinct inhibitory complexes with RelA, one of which mediates developmental NF-κB activation. Our genetic evidence confirms that p100 is required and sufficient as a fourth IκB protein for non-canonical NF-κB signaling downstream of NIK and IKK1. A mathematical model of the four-IκB-containing NF-κB signaling module accounts for NF-κB/RelA:p50 activation in response to inflammatory and developmental stimuli. By exploring signaling crosstalk between them, we find that the gene expression program induced by lymphotoxin β receptor (LTβR)- signaling is determined by the cellular history of exposure to inflammatory stimuli. Further integrated computational and experimental studies reveal that mutant cells with altered balances between canonical and non-canonical IκB proteins may exhibit inappropriate inflammatory gene expression in response to developmental signals. Our results have important implications for physiological and pathological scenarios in which inflammatory and developmental signals converge.
Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.
Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.
Extracellular nucleotides play many biological roles, including intercellular communication and modulation of nucleotide receptor signaling, and are dependent on the phosphorylation state of the nucleotide. Regulation of nucleotide phosphorylation is necessary, and a specialized class of enzymes, nucleotide pyrophosphatases/phosphodiesterases (E-NPPs), has been identified in mammals to perform this function. Although the E-NPP class is conserved among complex eukaryotes, this system has not yet been identified in Saccharomyces cerevisiae. Using genetic and biochemical experiments, we show that two orthologs of the E-NPP family, referred to as Npp1p and Npp2p, exist in budding yeast and can perform nucleotide phosphate hydrolysis. This activity is enhanced during phosphate starvation, where hydrolyzed phosphates can be imported from extracellular sources and utilized to overcome phosphate starvation through the activity of the Pho5p acid phosphatase. The added compensatory effect by Pho5p is also a newly established role for Pho5p. This study demonstrates that extracellular nucleotide phosphate metabolism appears to be controlled by at least two independent regulatory mechanisms, uniting phosphate starvation with extracellular nucleotide regulation.
IκBβ, one of the major IκB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IκBβ exists in at least two different forms: one that is bound to the NF-κB dimer and the other bound to both NF-κB and κB-Ras, a Ras-like small G protein. Removal of cellular κB-Ras enhances whereas excess κB-Ras blocks induced IκBβ degradation. Remarkably, κB-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of κB-Ras abrogate its ability to block degradation of IκBβ. κB-Ras also directly blocks the in vitro phosphorylation of IκBβ by IKKβ. These observations suggest that IκBβ in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IκBβ.