SRPK1 and LBR protein kinases show identical substrate specificities

Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs. SR splicing factors that are essential for pre-mRNA splicing and the lamin B receptor (LBR), an integral protein of the inner nuclear membrane, are among the best characterized proteins that contain RS domains. Two SR Protein-specific Kinases, SRPK1 and SRPK2, have been shown to phosphorylate specifically the RS motifs of the SR family of splicing factors and play an important role in regulating both the spliceosome assembly and their intranuclear distribution, whereas an LBR-associated kinase, that specifically phosphorylates a stretch of RS repeats located at the NH2-terminal region of LBR, has been recently purified and characterized from turkey erythrocyte nuclear envelopes. Using synthetic peptides representing different regions of LBR and recombinant proteins produced in bacteria we now demonstrate that SRPK1 modifies LBR with similar kinetics and on the same sites as the LBR kinase, that are also phosphorylated in vivo. These data provide significant evidence for a new role of SRPK1 in addition to that of pre-mRNA splicing.     

Acetylation and phosphorylation of SRSF2 control cell fate decision in response to cisplatin

SRSF2 is a serine/arginine-rich protein belonging to the family of SR proteins that are crucial regulators of constitutive and alternative pre-mRNA splicing. Although it is well known that phosphorylation inside RS domain controls activity of SR proteins, other post-translational modifications regulating SRSF2 functions have not been described to date. In this study, we provide the first evidence that the acetyltransferase Tip60 acetylates SRSF2 on its lysine 52 residue inside the RNA recognition motif, and promotes its proteasomal degradation. We also demonstrate that the deacetylase HDAC6 counters this acetylation and acts as a positive regulator of SRSF2 protein level. In addition, we show that Tip60 downregulates SRSF2 phosphorylation by inhibiting the nuclear translocation of both SRPK1 and SRPK2 kinases. Finally, we demonstrate that this acetylation/phosphorylation signalling network controls SRSF2 accumulation as well as caspase-8 pre-mRNA splicing in response to cisplatin and determines whether cells undergo apoptosis or G(2)/M cell cycle arrest. Taken together, these results unravel lysine acetylation as a crucial post-translational modification regulating SRSF2 protein level and activity in response to genotoxic stress.     

Phosphorylation mechanism and structure of serine-arginine protein kinases

The splicing of mRNA requires a group of essential factors known as SR proteins, which participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition motifs 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 SR-specific protein kinase (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. Human alternative splicing factor [serine/arginine-rich splicing factor 1 (SRSF1)], a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a post-translational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10-12 serines in the N-terminal region of the RS domain (RS1), using a mechanism that incorporates sequential, C-terminal to N-terminal 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 RNA recognition motif 2 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.     

SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells

Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH₂ terminus, and a recent study showed that this NH₂-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.     

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340)

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.     

Arginine/serine repeats are sufficient to constitute a splicing activation domain

SR proteins are essential pre-mRNA splicing factors that have been shown to bind a number of exonic splicing enhancers where they function to stimulate the splicing of adjacent introns. Members of the SR protein family contain one or two N-terminal RNA binding domains, as well as a C-terminal arginine–serine (RS) rich domain. The RS domains mediate protein–protein interactions with other RS domain containing proteins and are essential for many, but not all, SR protein functions. Hybrid proteins containing an RS domain fused to the bacteriophage MS2 coat protein are sufficient to activate enhancer-dependent splicing in HeLa cell nuclear extract when bound to the pre-mRNA. Here we report progress towards determining the protein sequence requirements for RS domain function. We show that the RS domains from non-SR proteins can also function as splicing activation domains when tethered to the pre-mRNA. Truncation experiments with the RS domain of the human SR protein 9G8 identified a 29 amino acid segment, containing 26 arginine or serine residues, that is sufficient to activate splicing when fused to MS2. We also show that synthetic domains composed solely of RS dipeptides are capable of activating splicing, although their potency is proportional to their size.     

Mass spectrometric and kinetic analysis of ASF/SF2 phosphorylation by SRPK1 and Clk/Sty

Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.     

The N-terminal fragment from caspase-cleaved serine/arginine protein-specific kinase2 (SRPK2) translocates into the nucleus and promotes apoptosis

SRPK2 belongs to a family of serine/arginine (SR) protein-specific kinases (SRPKs), which phosphorylate SR domain-containing proteins in the nuclear speckles and mediate the pre-mRNA splicing. Previous studies have shown that SRPK2 plays a pivotal role in cell proliferation and apoptosis. However, how SRPK2 is regulated during the apoptosis is unclear. Here, we show that SRPK2 is cleaved by caspases at Asp-139 and -403 residues. Its N terminus cleaved product translocates into the nucleus and promotes VP16-induced apoptosis. Akt phosphorylation of SRPK2 prevents its apoptotic cleavage by caspases. 14-3-3β, the binding partner of Akt-phosphorylated SRPK2, further protects it from degradation. Hence, our results suggest that the N-terminal domain of SRPK2 cleaved by caspases translocates into the nucleus, where it promotes chromatin condensation and apoptotic cell death.     

ICP27 interacts with SRPK1 to mediate HSV splicing inhibition by altering SR protein phosphorylation

Infection with some viruses can alter cellular mRNA processing to favor viral gene expression. We present evidence that herpes simplex virus 1 (HSV-1) protein ICP27, which contributes to host shut-off by inhibiting pre-mRNA splicing, interacts with essential splicing factors termed SR proteins and affects their phosphorylation. During HSV-1 infection, phosphorylation of several SR proteins was reduced and this correlated with a subnuclear redistribution. Exogenous SR proteins restored splicing in ICP27-inhibited nuclear extracts and SR proteins isolated from HSV-1-infected cells activated splicing in uninfected S100 extracts, indicating that inhibition occurs by a reversible mechanism. Spliceosome assembly was blocked at the pre-spliceosomal complex A stage. Furthermore, we show that ICP27 interacts with SRPK1 and relocalizes it to the nucleus; moreover, SRPK1 activity was altered in the presence of ICP27 in vitro. We propose that ICP27 modifies SRPK1 activity resulting in hypophosphorylation of SR proteins impairing their ability to function in spliceosome assembly.     

RNA association or phosphorylation of the RS domain prevents aggregation of RS domain-containing proteins

Domains rich in alternating arginine and serine residues (RS domains) are found in a large number of eukaryotic proteins involved in several cellular processes. According to the prevailing view RS domains function as protein interaction domains, thereby promoting the assembly of higher-order cellular structures. Furthermore, recent data demonstrated that the RS regions of several SR splicing factors directly contact the pre-mRNA in a nonsequence specific but functionally important fashion. Using a variety of biochemical approaches, we now demonstrate that the RS domains of three proteins, not directly associated with the splicing reaction, such as lamin b receptor, acinus and peroxisome proliferator-activated receptor gamma coactivator-1 alpha, associate mainly with nuclear RNA and that this association is conducive in retaining the proteins in a soluble form. Phosphorylation by SRPK1 prevents RNA association, yet it greatly increases the fraction of the proteins recovered in soluble form, thereby mimicking the RNA effect. Based on these results we propose that the tendency to self-associate and form aggregates is a general property of RS domain-containing proteins and could be attributed to their disordered structure. RNA binding or SRPK1-mediated phosphorylation prevents aggregation and may serve to modulate the RS domain interaction modes.     

Interacting factors and cellular localization of SR protein-specific kinase Dsk1

Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A)(+) RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G(2) phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.     

Identification and characterization of srp1, a gene of fission yeast encoding a RNA binding domain and a RS domain typical of SR splicing factors.

The SR protein family is involved in constitutive and regulated pre-mRNA splicing and has been found to be evolutionarily conserved in metazoan organisms. In contrast, the genome of the unicellular yeast Saccharomyces cerevisiae does not contain genes encoding typical SR proteins. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD respectively, and a RS (arginine/serine-rich) domain which gave the family its name. We have now cloned from the fission yeast Schizosaccharomyces pombe the gene srp1. This gene is the first yeast gene encoding a protein with typical features of mammalian SR protein family members. The gene is not essential for growth. We show that overexpression of the RNA binding domain inhibits pre-mRNA splicing and that the highly conserved sequence RDAEDA in the RBD is involved. Overexpression of Srp1 containing mutations in the RS domain also inhibits pre-mRNA splicing activity. Furthermore, we show that overexpression of Srp1 and overexpression of the mammalian SR splicing factor ASF/SF2 suppress the pre-mRNA splicing defect of the temperature-sensitive prp4-73 allele. prp4 encodes a protein kinase involved in pre-mRNA splicing. These findings are consistent with the notion that Srp1 plays a role in the splicing process.     

Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

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, we monitored region-specific mapping of phosphorylation sites in ASF/SF2 as a function of the protein phosphatase PP1. We showed that 10 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 in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RNA recognition motifs 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.     

Adaptable molecular interactions guide phosphorylation of the SR protein ASF/SF2 by SRPK1

The SR (arginine-serine rich) protein ASF/SF2 (also called human alternative splicing factor), an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRMs; RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain, a domain rich in arginine-serine repeats). The SR-specific protein kinase (SRPK) 1 phosphorylates the RS domain at multiple serines using a directional (C-terminal-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-turnover and multiturnover 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 the processive phosphorylation of SR proteins.     

Paraquat Modulates Alternative Pre-mRNA Splicing by Modifying the Intracellular Distribution of SRPK2

Paraquat (PQ) is a neurotoxic herbicide that induces superoxide formation. Although it is known that its toxic properties are linked to ROS production, the cellular response to PQ is still poorly understood. We reported previously that treatment with PQ induced genome-wide changes in pre-mRNA splicing. Here, we investigated the molecular mechanism underlying PQ-induced pre-mRNA splicing alterations. We show that PQ treatment leads to the phosphorylation and nuclear accumulation of SRPK2, a member of the family of serine/arginine (SR) protein-specific kinases. Concomitantly, we observed increased phosphorylation of SR proteins. Site-specific mutagenesis identified a single serine residue that is necessary and sufficient for nuclear localization of SRPK2. Transfection of a phosphomimetic mutant modified splice site selection of the E1A minigene splicing reporter similar to PQ-treatment. Finally, we found that PQ induces DNA damage and vice versa that genotoxic treatments are also able to promote SRPK2 phosphorylation and nuclear localization. Consistent with these observations, treatment with PQ, cisplatin or γ-radiation promote changes in the splicing pattern of genes involved in DNA repair, cell cycle control, and apoptosis. Altogether, our findings reveal a novel regulatory mechanism that connects PQ to the DNA damage response and to the modulation of alternative splicing via SRPK2 phosphorylation.     

Localization of serine kinases, SRPK1 (SFRSK1) and SRPK2 (SFRSK2), specific for the SR family of splicing factors in mouse and human chromosomes

The serine- and arginine-rich (SR) splicing factors play an important role in both constitutive and alternative pre-mRNA splicing, and the functions of these splicing factors are regulated by phosphorylation. We have previously characterized SRPK1 (SFRSK1) and SRPK2 (SFRSK2), which are highly specific protein kinases for the SR family of splicing factors. Here we report the chromosomal localization of the mouse and human genes for both kinases. SRPK1 probes detected two loci that were mapped to mouse Chromosomes 17 and X using The Jackson Laboratory interspecific backcross DNA panel, and SRPK2 probes identified a single locus on mouse Chromosome 5. Using a somatic cell hybrid mapping panel and by fluorescence in situ hybridization, SRPK1 and SRPK2 were respectively mapped to human chromosomes 6p21.2-p21.3 (a region of conserved synteny to mouse Chromosome 17) and 7q22-q31.1 (a region of conserved synteny to mouse Chromosome 5). In addition, we also found multiple SRPK-related sequences on other human chromosomes, one of which appears to correspond to a SRPK2 pseudogene on human chromosome 8.     

Phosphorylation of serine-arginine rich proteins--pleiotropic effect of one type posttranslational modification

SR proteins are components of the spliceosome, a dynamic complex catalysing removal of pre-mRNA introns during splicing reaction. Recent studies have shown additional functions for SR proteins in mRNA transport and translation. Several protein kinases have been identified that can phosphorylate SR proteins. Phosphorylation is a common posttranslational modification among proteins. However in the case of SR proteins it has got a special meaning. Phosphorylation of serine residues clustered in a narrow area of RS domains affects all aspects of SR proteins functions. It influences subcellular localization and determines process in which the proteins take part.     

Deletion of the N-terminus of SF2/ASF permits RS-domain-independent pre-mRNA splicing

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.