SR Protein Kinase Research Articles

SR/RS Motifs as Critical Determinants of Coronavirus Life Cycle.

SR/RS domains are found in almost all eukaryotic genomes from C. elegans to human. These domains are thought to mediate interactions between proteins but also between proteins and RNA in complex networks associated with mRNA splicing, chromatin structure, transcription, cell cycle and cell structure. A precise and tight regulation of their function is achieved through phosphorylation of a number of serine residues within the SR/RS motifs by the Serine-Arginine protein kinases (SRPKs) that lead to delicate structural alterations. Given that coronavirus N proteins also contain SR/RS domains, we formulate the hypothesis that the viruses exploit the properties of these motifs to promote unpacking of viral RNA and virion assembly.

A conserved sequence motif bridges two protein kinases for enhanced phosphorylation and nuclear function of a splicing factor.

The assembly and activation of the spliceosome rely upon the phosphorylation of an essential family of splicing factors known as the serine-arginine (SR) proteins. Although it has been demonstrated recently that two enzyme families, the SR protein kinases (SRPKs) and the Cdc2-like kinases (CLKs), can function as a complex to efficiently phosphorylate these SR proteins in the nucleus, the molecular features involved in such a connection are unknown. In this study, we identified a group of conserved residues in the large lobe of SRPK1 that interact with the N terminus of CLK1 stabilizing the SRPK1-CLK1 complex. Mutations in this motif not only disrupt formation of the kinase-kinase complex but also impair SRPK1-dependent release of the phospho-SR protein from CLK1. The binding motif potently up-regulates CLK1-specific phosphorylation sites, enhances SR protein diffusion from nuclear speckles, and impacts the alternative splicing of several target genes. These results indicate that CLK1 binds a conserved, electronegative surface on SRPK1, thereby controlling SR protein phosphorylation levels for enhanced subnuclear trafficking and alternative splicing regulation.

Phosphorylation of SR Proteins and the Effect of Hydrogen Peroxide Stress on the Splicing Cycle in S. pombe Fission Yeast

The spliceosome, a large and dynamic RNA‐protein complex, is responsible for effective pre‐mRNA splicing. Correct spliceosome assembly, function, and regulation have often been shown to involve SR proteins, which are dynamically phosphorylated. Alterations in the splicing process can lead to a variety of human diseases and the fission yeast, Schizosaccharomyces pombe, is one model organism for this research. It was previously determined that the conserved SR protein kinase in fission yeast, Dsk1 (Srpk1 in humans), is required for efficient splicing of introns. Previous identification of Dsk1 substrates and alanine mutation to prevent phosphorylation showed a minor splicing defect in the two fission yeast SR proteins (Srp1 and Srp2) under optimal growth conditions. Our current research takes a chemical‐genetic approach to exploring phosphorylation’s physical effects on specifically Srp1 and Srp2 protein substrates, and whether environmental stress, simulated by hydrogen peroxide exposure, can cause a higher retention rate of introns in a regulated way. Through the use of RT‐qPCR we can determine whether there is a splicing defect under the stress condition, and how phosphorylation of SR proteins affects the level of pre‐mRNA splicing defect.Support or Funding InformationPrevious work in Christine Guthrie’s lab in collaboration with Kevan Shokat and postdoctoral fellow Jesse Lipp, as well as previous work by Alex Piening of Rockhurst class 2018 and Clayton Carroll of Rockhurst class 2019. Thanks to Jeff Pleiss for microarray design as well as Nick Hertz & Alma Burlingame for mass spectrometry analysis. In addition, NIH NRSA postdoctoral fellowship for funding. Current funding and support provided by Rockhurst University.Rpl25a intron splicing in WT SR protein yeast strains and alanine mutated strains under optimal conditions.Figure 1S. pombe cell responses due to increasing concentrations of hydrogen peroxide (Veal, E. 2014).Figure 2

Human papillomavirus type 16 infection activates the host serine arginine protein kinase 1 (SRPK1) - splicing factor axis.

The infectious life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Evidence suggests a sophisticated interplay between host gene regulation and virus replication. Alternative splicing is an essential process for host and viral gene expression, and is generally upregulated by serine arginine-rich splicing factors (SRSFs). SRSF activity can be positively or negatively controlled by cycles of phosphorylation/dephosphorylation. Here we show that HPV16 infection leads to accumulation of the paradigm SRSF protein, SRSF1, in the cytoplasm in a keratinocyte differentiation-specific manner. Moreover, HPV16 infection leads to increased levels of cytoplasmic and nuclear phosphorylated SRSF1. SR protein kinase 1 (SRPK1) phosphorylates SRSF1. Similar to HPV upregulation of SRSF1, we demonstrate HPV upregulation of SRPK1 via the viral E2 protein. SRPK1 depletion or drug inhibition of SRPK1 kinase activity resulted in reduced levels of SRSF1, suggesting that phosphorylation stabilizes the protein in differentiated HPV-infected keratinocytes. Together, these data indicate HPV infection stimulates the SRPK1–SRSF axis in keratinocytes.

Phosphoproteomics Reveals Novel Targets and Phosphoprotein Networks in Cell Cycle Mediated by Dsk1 Kinase.

As the ortholog of human SR protein kinase 1 in fission yeast Schizosaccharomyces pombe, Dsk1 specifically phosphorylates SR proteins (serine/arginine-rich proteins) and promotes splicing of nonconsensus introns. The SRPK (SR protein-specific kinase) family performs highly conserved functions in eukaryotic cells including cell proliferation, differentiation, development, and apoptosis. Although Dsk1 was originally identified as a mitotic regulator, its specific targets involved in cell cycle have yet been unexplored. In this study, using a phosphoproteomics approach, we examined differential protein phosphorylation between wild-type cells and dsk1-deletion mutants. We found reduced phosphorylation of 149 peptides corresponding to 133 proteins in the dsk1-null cells. These proteins are involved in various cellular processes, including cytoskeleton organization and signal transduction, and specifically enriched in multiple steps of cell cycle control. Further, targeted MS analyses and in vitro biochemical assays established Cdr2 protein kinase and kinesin motor Klp9 as novel substrates of Dsk1, which function in cell size control for mitotic entry and in chromosome segregation for mitotic exit, respectively. The phosphoprotein networks mediated by Dsk1 reveal, for the first time, the molecular links connecting Dsk1 to mitotic phase transition, sister-chromatid segregation, and cytokinesis, providing further evidence of Dsk1's diverse influence on cell cycle progression and regulation.

CRKL regulates alternative splicing of cancer-related genes in cervical cancer samples and HeLa cell

BackgroundAberrant spliced isoforms are specifically associated with cancer progression and metastasis. The cytoplasmic adaptor CRKL (v-crk avian sarcoma virus CT10 oncogene homolog-like) is a CRK like proto-oncogene, which encodes a SH2 and SH3 (src homology) domain-containing adaptor protein. CRKL is tightly linked to leukemia via its binding partners BCR-ABL and TEL-ABL, upregulated in multiple types of human cancers, and induce cancer cell proliferation and invasion. However, it remains unclear whether signaling adaptors such as CRKL could regulate alternative splicing.MethodsWe analyzed the expression level of CRKL in 305 cervical cancer tissue samples available in TCGA database, and then selected two groups of cancer samples with CRKL differentially expressed to analyzed potential CRKL-regulated alternative splicing events (ASEs). CRKL was knocked down by shRNA to further study CRKL-regulated alternative splicing and the activity of SR protein kinases in HeLa cells using RNA-Seq and Western blot techniques. We validated 43 CRKL-regulated ASEs detected by RNA-seq in HeLa cells, using RT-qPCR analysis of HeLa cell samples and using RNA-seq data of the two group of clinical cervical samples.ResultsThe expression of CRKL was mostly up-regulated in stage I cervical cancer samples. Knock-down of CRKL led to a reduced cell proliferation. CRKL-regulated alternative splicing of a large number of genes were enriched in cancer-related functional pathways, among which DNA repair and G2/M mitotic cell cycle, GnRH signaling were shared among the top 10 enriched GO terms and KEGG pathways by results from clinical samples and HeLa cell model. We showed that CRKL-regulated ASEs revealed by computational analysis using ABLas software in HeLa cell were highly validated by RT-qPCR, and also validated by cervical cancer clinical samples.ConclusionsThis is the first report of CRKL-regulation of the alternative splicing of a number of genes critical in tumorigenesis and cancer progression, which is consistent with CRKL reported role as a signaling adaptor and a kinase. Our results underline that the signaling adaptor CRKL might integrate the external and intrinsic cellular signals and coordinate the dynamic activation of cellular signaling pathways including alternative splicing regulation.

Caspase-mediated cleavage of X-ray repair cross-complementing group 4 promotes apoptosis by enhancing nuclear translocation of caspase-activated DNase

X-ray repair cross-complementing group 4 (XRCC4), a repair protein for DNA double-strand breaks, is cleaved by caspases during apoptosis. In this study, we examined the role of XRCC4 in apoptosis. Cell lines, derived from XRCC4-deficient M10 mouse lymphoma cells and stably expressing wild-type XRCC4 or caspase-resistant XRCC4, were established and treated with staurosporine (STS) to induce apoptosis. In STS-induced apoptosis, expression of wild-type, but not caspase-resistant, XRCC4 in XRCC4-deficient cells enhanced oligonucleosomal DNA fragmentation and the appearance of TUNEL-positive cells by promoting nuclear translocation of caspase-activated DNase (CAD), a major nuclease for oligonucleosomal DNA fragmentation. CAD activity is reportedly regulated by the ratio of two inhibitor of CAD (ICAD) splice variants, ICAD-L and ICAD-S mRNA, which, respectively, produce proteins with and without the ability to transport CAD into the nucleus. The XRCC4-dependent promotion of nuclear import of CAD in STS-treated cells was associated with reduction of ICAD-S mRNA and protein, and enhancement of phosphorylation and nuclear import of serine/arginine-rich splicing factor (SRSF) 1. These XRCC4-dependent, apoptosis-enhancing effects were canceled by depletion of SRSF1 or SR protein kinase (SRPK) 1. In addition, overexpression of SRSF1 in XRCC4-deficient cells restored the normal level of apoptosis, suggesting that SRSF1 functions downstream of XRCC4 in activating CAD. This XRCC4-dependent, SRPK1/SRSF1-mediated regulatory mechanism was conserved in apoptosis in Jurkat human leukemia cells triggered by STS, and by two widely used anti-cancer agents, Paclitaxel and Vincristine. These data imply that the level of XRCC4 expression could be used to predict the effects of apoptosis-inducing drugs in cancer treatment.

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function.

A Possible Conserved Role for SR Protein Kinase in the Regulation of Cell Death

Apoptosis, programmed cell death, is a process that mammalian cells frequently undergo to suppress tumors and regulate cell growth. It is unclear whether or not apoptosis is conserved across both multicellular and unicellular eukaryotes. In baker's yeast, Saccharomyces cerevisiae, specific stress conditions can cause nuclear DNA fragmentation and cytoplasmic release of reactive oxygen species (ROS), the molecular markers of apoptosis in mammalian cells. However, it is unclear whether these markers are part of a fungal specific stress response pathway or indicate that the machinery controlling cell death is conserved. One complication to resolving this question has been that the probes used to quantify DNA fragmentation and ROS levels are usually used to stain fixed cells. This means we only see cells at a fixed point in time and are unable to track the fate of a cell following appearance of apoptotic markers. We cannot confirm yeast cell death without observing the cells after the markers of cell death appear, because we do not know if the presence of ROS is indicative of a stress response or cell death pathway. To address this, we developed a novel technique using live cell confocal microscopy to quantify the levels of ROS in yeast grown in different stress conditions. Our preliminary data show that once ROS appear in a cell it never buds again over a 10‐hour time course. This correlation between expression of the mammalian apoptotic markers and loss of cell viability in yeast suggest that this is a cell death pathway. Here, we focus on a potentially conserved role for SR protein kinases in the regulation of apoptosis in yeast and mammals, using this system. Previous work has demonstrated a role for the mammalian SRPK2 in apoptosis. Specifically, SRPK2 appears to be cleaved in a caspase dependent system; the N‐terminus then translocates to the nucleus where it may be involved in nuclear apoptotic pathway. Yeast encode for a single SR protein, Sky1. In response to cation toxicity, LiCl, stress we see induction of apoptosis markers in yeast. Interestingly, the deletion of SKY1 reduces the presence of the molecular markers of apoptosis compared to wild‐type in both standard (1% and 2% respectively) and stress conditions (3% and 14% respectively). Consistent with this, cells that carry a sky1Δ grow better than WT under stress conditions; moreover, cells that have constructs that overexpress SKY1 grow much worse under stress conditions. Using our live cell microscopy assay, we quantify and compare release of ROS in WT to sky1Δ constructs and WT to sky1‐overexpression constructs. Furthermore, we are investigating whether the cleavage of Sky1 is conserved by using yeast with N‐ and C‐terminally fluorescent tags on Sky1. Determining that these results are representative of an apoptotic cell death pathway in yeast opens the door to using this genetically facile model organism to further probe the molecular mechanics of apoptosis.Support or Funding InformationHoward Hughes Medical Institute

Abstract IA03: mTORC1/S6K1: Regulation of RNA biogenesis, protein synthesis, and cell metabolism

Abstract The mTOR Complex 1 (mTORC1) signaling pathway has evolved to sense and respond to cellular energy status, nutrient availability and surrounding oxygen concentrations. In addition, mTORC1 signaling can be further activated by mitogen- and hormone-stimulated kinases including Akt, ERK and RSK, and suppressed by mTORC1 and its effector S6K1, via a variety of negative feedback loops. This incredible integration of extracellular and intracellular cues translates into carefully regulated outputs that control cell growth, motility and survival. A major mechanism through which mTOR regulates downstream processes is via the regulation of gene expression, mRNA processing, protein synthesis and protein degradation. In an mTOR phosphoproteomic screen, we and others identified SR Protein Kinase 2 (SRPK2) as a potential downstream effector of mTORC1. I will discuss a novel mechanism by which SRPK2 is regulated and contributes to mTORC1-dependent regulation of lipid metabolism via the regulation of mRNA processing. Citation Format: John Blenis, Gina Lee, Jamie Dempsey, Christina England. mTORC1/S6K1: Regulation of RNA biogenesis, protein synthesis, and cell metabolism. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA03.

AEE788 Inhibits Basal Body Assembly and Blocks DNA Replication in the African Trypanosome.

Trypanosoma brucei causes human African trypanosomiasis (HAT). The pyrrolopyrimidine AEE788 (a hit for anti-HAT drug discovery) associates with three trypanosome protein kinases. Herein we delineate the effects of AEE788 on T. brucei using chemical biology strategies. AEE788 treatment inhibits DNA replication in the kinetoplast (mitochondrial nucleoid) and nucleus. In addition, AEE788 blocks duplication of the basal body and the bilobe without affecting mitosis. Thus, AEE788 prevents entry into the S-phase of the cell division cycle. To study the kinetics of early events in trypanosome division, we employed an "AEE788 block and release" protocol to stage entry into the S-phase. A time-course of DNA synthesis (nuclear and kinetoplast DNA), duplication of organelles (basal body, bilobe, kinetoplast, nucleus), and cytokinesis was obtained. Unexpected findings include the following: 1) basal body and bilobe duplication are concurrent; 2) maturation of probasal bodies, marked by TbRP2 recruitment, is coupled with nascent basal body assembly, monitored by localization of TbSAS6 at newly forming basal bodies; and 3) kinetoplast division is observed in G2 after completion of nuclear DNA synthesis. Prolonged exposure of trypanosomes to AEE788 inhibited transferrin endocytosis, altered cell morphology, and decreased cell viability. To discover putative effectors for the pleiotropic effects of AEE788, proteome-wide changes in protein phosphorylation induced by the drug were determined. Putative effectors include an SR protein kinase, bilobe proteins, TbSAS4, TbRP2, and BILBO-1. Loss of function of one or more of these effectors can, from published literature, explain the polypharmacology of AEE788 on trypanosome biology.

Evidence for disulfide bonds in SR Protein Kinase 1 (SRPK1) that are required for activity and nuclear localization.

Serine/arginine protein kinases (SRPKs) phosphorylate Arg/Ser dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. The existence of a large internal spacer sequence that separates the bipartite kinase catalytic core is a unique structural feature of SRPKs. Previous structural studies on a catalytically active fragment of SRPK1, which lacks the main part of the spacer domain, revealed that SRPK1 remains in an active state without any post-translational modifications or specific intra-protein interactions, while the spacer domain is depicted as a loop structure, outside the kinase core. Using systematic mutagenesis we now provide evidence that replacement of any individual cysteine residue in the spacer, apart from Cys414, or in its proximal flaking ends of the two kinase catalytic domains has an impact on kinase activity. Furthermore, the cysteine residues are critical for nuclear translocation of SRPK1 in response to genotoxic stress and SRPK1-dependent splicing of a reporter gene. While replacement of Cys207, Cys502 and Cys539 of the catalytic domains is predicted to distort the kinase active structure, our findings suggest that Cys356, Cys386, Cys427 and Cys455 of the spacer domain and Cys188 of the first catalytic domain are engaged in disulfide bridging. We propose that such a network of intramolecular disulfide bonds mediates the bending of the spacer region thus allowing the proximal positioning of the two catalytic subunits which is a prerequisite for SRPK1 activity.

Lynamicin D an antimicrobial natural product affects splicing by inducing the expression of SR protein kinase 1

The first total synthesis of the antimicrobial natural product lynamicin D has been developed using a Suzuki coupling to construct the bisindole pyrrole skeleton. An evaluation of the biological activity of lynamicin D reveals that it has a minor effect on cell viability but it can modulate splicing of pre-mRNAs. We provide evidence that this effect is mainly due to the ability of lynamicin D to alter the levels of SRPK1, the key kinase involved in both constitutive and alternative splicing.

SPSB1-mediated HnRNP A1 ubiquitylation regulates alternative splicing and cell migration in EGF signaling.

Extracellular signals have been shown to impact on alternative pre-mRNA splicing; however, the molecular mechanisms and biological significance of signal-induced splicing regulation remain largely unknown. Here, we report that epidermal growth factor (EGF) induces splicing changes through ubiquitylation of a well-known splicing regulator, hnRNP A1. EGF signaling upregulates an E3 ubiquitin (Ub) ligase adaptor, SPRY domain-containing SOCS box protein 1 (SPSB1), which recruits Elongin B/C-Cullin complexes to conjugate lysine 29-linked polyUb chains onto hnRNP A1. Importantly, SPSB1 and ubiquitylation of hnRNP A1 have a critical role in EGF-driven cell migration. Mechanistically, EGF-induced ubiquitylation of hnRNP A1 together with the activation of SR protein kinases (SRPKs) results in the upregulation of a Rac1 splicing isoform, Rac1b, to promote cell motility. These findings unravel a novel crosstalk between protein ubiquitylation and alternative splicing in EGF/EGF receptor signaling, and identify a new EGF/SPSB1/hnRNP A1/Rac1 axis in modulating cell migration, which may have important implications for cancer treatment.

Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing

Phosphorylation has been generally thought to activate the SR family of splicing factors for efficient splice-site recognition, but this idea is incompatible with an early observation that overexpression of an SR protein kinase, such as the CDC2-like kinase 1 (CLK1), weakens splice-site selection. Here, we report that CLK1 binds SR proteins but lacks the mechanism to release phosphorylated SR proteins, thus functionally inactivating the splicing factors. Interestingly, CLK1 overcomes this dilemma through a symbiotic relationship with the serine-arginine protein kinase 1 (SRPK1). We show that SRPK1 interacts with an RS-like domain in the N terminus of CLK1 to facilitate the release of phosphorylated SR proteins, which then promotes efficient splice-site recognition and subsequent spliceosome assembly. These findings reveal an unprecedented signaling mechanism by which two protein kinases fulfill separate catalytic features that are normally encoded in single kinases to institute phosphorylation control of pre-mRNA splicing in the nucleus.

Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif

Multisite phosphorylation is required for the biological function of serine–arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine–arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine–serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2–RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function.

An extensive program of periodic alternative splicing linked to cell cycle progression.

Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control.

SR protein kinases promote splicing of nonconsensus introns.

Phosphorylation of the spliceosome is essential for RNA splicing, yet how and to what extent kinase signaling affects splicing have not been defined on a genome-wide basis. Using a chemical genetic approach, we show in Schizosaccharomyces pombe that the SR protein kinase Dsk1 is required for efficient splicing of introns with suboptimal splice sites. Systematic substrate mapping in fission yeast and human cells revealed that SRPKs target evolutionarily conserved spliceosomal proteins, including the branchpoint-binding protein Bpb1 (SF1 in humans), by using an RXXSP consensus motif for substrate recognition. Phosphorylation of SF1 increases SF1 binding to introns with nonconsensus splice sites in vitro, and mutation of such sites to consensus relieves the requirement for Dsk1 and phosphorylated Bpb1 in vivo. Modulation of splicing efficiency through kinase signaling pathways may allow tuning of gene expression in response to environmental and developmental cues.

Increased Serine-Arginine (SR) Protein Phosphorylation Changes Pre-mRNA Splicing in Hypoxia

The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Inhibitory PAS domain protein (IPAS), a dominant negative regulator of hypoxia-inducible gene expression, is generated from hypoxia inducible transcription factor-3α (HIF-3α) pre-mRNA by an alternative splicing mechanism. Inactivation of the IPAS transcript in mice leads to the neo-vascularization of the cornea, suggesting that IPAS is an important regulator of anti-angiogenesis in this tissue. For the first time we demonstrate that serine-arginine (SR) proteins are involved in oxygen tension-dependent changes in pre-mRNA splicing. SR proteins isolated from hypoxic cells differentially interact with RNA (compared with proteins isolated from cells cultured under normoxic conditions). They possess the differential ability to activate hypoxia-dependent splice sites, and they are more phosphorylated than those isolated from normoxic HeLa cells. We also show that expression of SR protein kinases (CLK1, SRPK1, SRPK2) in hypoxic cells is elevated at mRNA and protein levels. Increased expression of CLK1 kinase is regulated by HIFs. Reduction of CLK1 cellular expression levels reduces hypoxia-dependent full-length carbonic anhydrase IX (CAIX) mRNA and CAIX protein formation and changes hypoxia-dependent cysteine-rich angiogenic inducer 61 (Cyr61) mRNA isoform formation profiles.

The influence of SRPK1 on glioma apoptosis, metastasis, and angiogenesis through the PI3K/Akt signaling pathway under normoxia.

Gliomas, the most common primary brain tumors, have low survival rates and poorly defined molecular mechanisms to target for treatment. Serine/arginine SR protein kinases 1 (SRPK1) can highly and specifically phosphorylate the SR protein found in many tumors, which can influence cell proliferation and angiogenesis. However, the roles and regulatory mechanisms of SRPK1 in gliomas are not understood. The aim of this study was to determine the functions and regulation of SRPK1 in gliomas. We found that SRPK1 inhibition induces early apoptosis and significantly inhibits xenograft tumor growth. Our results indicate that SRPK1 affects Akt and eIF4E phosphorylation, Bax and Bcl-2 activation, and HIF-1 and VEGF production in glioma cells. Moreover, transfection of SRPK1 siRNA strongly reduced cell invasion and migration by regulating the expression of MMP2 and MMP9 and significantly decreased the volume of tumors and angiogenesis. We show here that a strong link exists among SRPK1, Akt, eIF4E, HIF-1, and VEGF activity that is functionally involved in apoptosis, metastasis, and angiogenesis of gliomas under normoxic conditions. Thus, SRPK1 may be a potential anticancer target to inhibit glioma progression.