Serine-arginine Protein Kinase Research Articles

Deep PIM kinase substrate profiling reveals new rational cotherapeutic strategies for acute myeloid leukemia

AbstractProvirus integration site for Moloney murine leukemia virus (PIM) family serine/threonine kinases perform protumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of PIM kinase functions is currently lacking. Multiple small-molecule PIM kinase inhibitors are currently being evaluated as cotherapeutics in patients with cancer. To further illuminate PIM kinase functions in cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative PIM substrates nominated RNA splicing and ribosomal RNA (rRNA) processing as PIM-regulated cellular processes. PIM inhibition elicited reproducible splicing changes in PIM-inhibitor–responsive acute myeloid leukemia (AML) cell lines. PIM inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. PIM inhibition also altered rRNA processing, and PIM inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic cotherapeutic strategies. This approach may expand the cotherapeutic armamentarium to overcome kinase inhibitor–resistant disease that limits durable responses in malignant disease.

SRPK2 Mediates HBV Core Protein Phosphorylation and Capsid Assembly via Docking Interaction.

Members of the serine-arginine protein kinase (SRPK) family, SRPK1 and SRPK2, phosphorylate the hepatitis B core protein (Cp) and are crucial for pregenomic RNA encapsidation during viral nucleocapsid assembly. Among them, SRPK2 exhibits higher kinase activity toward Cp. In this study, we identified Cp sites that are phosphorylated by SRPK2 and demonstrated that the kinase utilizes an SRPK-specific docking groove to interact with and regulate the phosphorylation of the C-terminal arginine rich domain of Cp. We determined that direct interaction between the docking groove of SRPK2 and unphosphorylated Cp inhibited premature viral capsid assembly in vitro, whereas the phosphorylation of the viral protein reactivated the process. Pull-down assays together with the new cryo-electron microscopy structure of the HBV capsid in complex with SRPK2 revealed that the kinases decorate the surface of the viral capsid by interacting with the C-terminal domain of Cp, underscoring the importance of the docking interaction in regulating capsid assembly and pregenome packaging. Moreover, SRPK2-knockout in HepG2 cells suppressed Cp phosphorylation, indicating that SRPK2 is an important cellular kinase for HBV life cycle.

Role of Serine arginine protein kinase 1 and Minichromosome maintenance protein 2 in predicting epithelial ovarian cancer response to treatment and prognosis.

Serine-Arginine (SR) proteins are a conserved family of proteins involved in RNA splicing and are reported to be over-expressed in multiple cancers. The aim of the study is evaluation of the expression of Serine arginine protein kinase 1 (SRPK1) and Minichromosome maintenance protein 2 (MCM2) in epithelial ovarian cancer (EOC) and their correlation with clinicopathological features, response to therapy, progression-free survival (PFS), and cancer-specific survival (CSS). This study was carried out on surgical specimens of 65 patients diagnosed with EOC which were submitted to immunohistochemical staining by SRPK1 and MCM2 antibodies. About 89.2% of cases showed SRPK1 expression and its high expression was significantly associated with type II tumors and advanced stage. All cases showed nuclear immunoreaction for MCM2 with high expression in 49.2% of cases. There was a significant relationship between high values of SRPK1 H-score and percentage of MCM2. Postmenopause, type II pathology, advanced stage, absence of complete response to the treatment, resistance to platinum-based chemotherapy, and surgery done by a general surgeon were the factors affecting PFS. Response to treatment and platinum sensitivity were the most independent factors affecting patients' PFS. The factors associated with shorter CSS were suboptimal debulking, advanced stage, absence of complete response to the treatment, platinum resistance, and high SRPK1. High SRPK1 expression and platinum sensitivity were the independent factors affecting patients' CSS. SRPK1 is an unfavorable biomarker in EOC patients because of its association with aggressive histologic type, advanced International Federation of Gynecology and Obstetrics (FIGO) stage, and worse survival. SRPK1 could promote the proliferation of EOC by up-regulation of MCM2.

Targeting hepatic serine-arginine protein kinase 2 ameliorates alcohol-associated liver disease by alternative splicing control of lipogenesis.

Lipid accumulation induced by alcohol consumption is not only an early pathophysiological response but also a prerequisite for the progression of alcohol-associated liver disease (ALD). Alternative splicing regulates gene expression and protein diversity; dysregulation of this process is implicated in human liver diseases. However, how the alternative splicing regulation of lipid metabolism contributes to the pathogenesis of ALD remains undefined. Serine-arginine-rich protein kinase 2 (SRPK2), a key kinase controlling alternative splicing, is activated in hepatocytes in response to alcohol, in mice with chronic-plus-binge alcohol feeding, and in patients with ALD. Such induction activates sterol regulatory element-binding protein 1 and promotes lipogenesis in ALD. Overexpression of FGF21 in transgenic mice abolishes alcohol-mediated induction of SRPK2 and its associated steatosis, lipotoxicity, and inflammation; these alcohol-induced pathologies are exacerbated in FGF21 knockout mice. Mechanistically, SRPK2 is required for alcohol-mediated impairment of serine-arginine splicing factor 10, which generates exon 7 inclusion in lipin 1 and triggers concurrent induction of lipogenic regulators-lipin 1β and sterol regulatory element-binding protein 1. FGF21 suppresses alcohol-induced SRPK2 accumulation through mammalian target of rapamycin complex 1 inhibition-dependent degradation of SRPK2. Silencing SRPK2 rescues alcohol-induced splicing dysregulation and liver injury in FGF21 knockout mice. These studies reveal that (1) the regulation of alternative splicing by SRPK2 is implicated in lipogenesis in humans with ALD; (2) FGF21 is a key hepatokine that ameliorates ALD pathologies largely by inhibiting SRPK2; and (3) targeting SRPK2 signaling by FGF21 may offer potential therapeutic approaches to combat ALD.

AB054. SOH23ABS_054. Under-expression of serine-arginine protein kinase 1 (SRPK1) in mucinous colorectal cancer, mediates resistance to oxaliplatin

Background: Approximately 10% of all colorectal cancers (CRCs) are mucinous. Mucinous CRCs are resistant to multiple chemotherapy agents, including oxaliplatin. Serine-arginine protein kinase 1 (SRPK1) is an enzyme, which modulates the activity of multiple splicing factors. The objectives of this study were to evaluate SRPK1 expression in mucinous CRC and investigate its potential role in oxaliplatin resistance.

Serine-arginine protein kinase 1 (SRPK1) promotes EGFR-TKI resistance by enhancing GSK3β Ser9 autophosphorylation independent of its kinase activity in non-small-cell lung cancer

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a major challenge for clinicians and patients with non-small cell lung cancer (NSCLC). Serine-arginine protein kinase 1 (SRPK1) is a key oncoprotein in the EGFR/AKT pathway that participates in tumorigenesis. We found that high SRPK1 expression was significantly associated with poor progression-free survival (PFS) in patients with advanced NSCLC undergoing gefitinib treatment. Both in vitro and in vivo assays suggested that SRPK1 reduced the ability of gefitinib to induce apoptosis in sensitive NSCLC cells independently of its kinase activity. Moreover, SRPK1 facilitated binding between LEF1, β-catenin and the EGFR promoter region to increase EGFR expression and promote the accumulation and phosphorylation of membrane EGFR. Furthermore, we verified that the SRPK1 spacer domain bound to GSK3β and enhanced its autophosphorylation at Ser9 to activate the Wnt pathway, thereby promoting the expression of Wnt target genes such as Bcl-X. The correlation between SRPK1 and EGFR expression was confirmed in patients. In brief, our research suggested that the SRPK1/GSK3β axis promotes gefitinib resistance by activating the Wnt pathway and may serve as a potential therapeutic target for overcoming gefitinib resistance in NSCLC.

First evidence of a serine arginine protein kinase (SRPK) in leishmania braziliensis and its potential as therapeutic target

Leishmaniasis is a parasitic disease found in tropical and subtropical regions around the world, caused by parasites of the genus Leishmania. The disease is a public health concern and presents clinical manifestations that can cause death, disability, and mutilation. The parasite has promastigote (vector) and amastigote (vertebrate host) forms and kinase enzymes are involved in this differentiation process. In the present investigation, we show, for the first time, evidence of a serine/arginine protein kinase in Leshmania braziliensis (LbSRPK). Our results show that amastigotes express more LbSRPK than promastigotes. Analogues of SRPIN340 (a known inhibitor of SRPK) were evaluated for their leishmanicidal activity and two of them, namely SRVIC22 and SRVIC32 showed important leishmanicidal activity in vitro. SRVIC22 and SRVIC32 were able to reduce the infection rate in macrophages and the number of intracellular amastigotes by 55 and 60%, respectively. Bioinformatics analysis revealed the existence of two different amino acid residues in the active site of LbSRPK compared to their human homologue (Tyr/Leu-and Ser/Tyr), which could explain the absence of leishmanicidal activity of SRPIN340 on infected macrophages. In order to enhance leishmanicidal activity of the analogues, optimizations were proposed in the structures of the ligands, suggesting strong interactions with the catalytic site of LbSRPK. Although the evidence on the action of inhibitors upon LbSRPK is only indirect, our studies not only reveal, for the first time, evidence of a SRPK in Leishmania, but also shed light on a new therapeutic target for drug development.

SRPK1 promotes sepsis-induced acute lung injury via regulating PI3K/AKT/FOXO3 signaling

Objective Sepsis is the most common cause of death in intensive care unit. Moreover, sepsis is the leading cause of acute lung injury (ALI). Serine-arginine protein kinase 1 (SRPK1) was demonstrated to promote the development of ALI. However, the potentials of SRPK1 in sepsis-induced ALI are still unknown. This study aimed to investigate the potentials of SRPK1 in sepsis-induced ALI and the underlying mechanisms. Methods Cecal ligation and puncture (CLP) was performed to establish sepsis-induced ALI model in vivo. Primary human pulmonary microvascular endothelial cells (HPMECs) were exposed to lipopolysaccharide (LPS) to construct sepsis-induced ALI model in vitro. Gene expression was detected using western blot and qRT-PCR. The interaction between forkhead box O3 (FOXO3) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) was detected using luciferase and Chromatin immunoprecipitation (ChIP) assay. Cellular functions were CCK-8, colony formation, PI staining, and flow cytometry assay. Results SRPK1 was downregulated in patients with sepsis-induced ALI. Overexpression of SRPK1 suppressed the pyroptosis of HPMECs as well as promoted cell proliferation. Additionally, SRPK1 overexpression alleviated sepsis-induced ALI in vivo. SRPK1 activated phosphatidylinositol3-kinase (PI3K) signaling pathways. Blocking the activation of PI3K degraded the cellular functions of HPMECs. Moreover, FOXO3 transcriptionally inactivated NLRP3 and suppressed its mRNA and protein expression. Conclusion Taken together, SRPK1 suppressed sepsis-induced ALI via regulating PI3K/AKT/FOXO3/NLRP3 signaling. SRPK1 may be the potential biomarker for sepsis-induced ALI.

Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.

S7.4d Candida albicans SR-like protein kinases regulate different cellular processes: Sky1 is involved in thecontrol of ion homeostasis, while Sky2 is important for dipeptide utilization

S7.4 Pathogenesis and host defense, September 23, 2022, 10:30 AM - 12:00 PM Protein kinases play a crucial role in regulating cellular processes such as growth, proliferation, environmental adaptation, and stress responses. Candida albicans genome comprises of 108 predicted protein kinases, of which the exact role of nearly half of those kinases remains unknown. One family of protein kinases is serine-arginine (SR) protein kinases, which are highly conserved in eukaryotes and regulate fundamental processes such as constitutive and alternative splicing, mRNA processing, and ion homeostasis. The C. albicans genome encodes two (Sky1, Sky2) and the C. glabrata genome has one homolog (Sky1) of the human SR protein kinase 1, but their functions have not yet been investigated. We used deletion strains of the corresponding genes in both fungi to study their cellular functions. C. glabrata and C. albicans strains lacking SKY1 exhibited higher resistance to osmotic stress and toxic polyamine concentrations, similar to their ortholog Sky1 in Saccharomyces cerevisiae. Deletion of SKY2 in C. albicans resulted in impaired utilization of various dipeptides as the sole nitrogen source which was shown by utilizing a high-throughput phenotypic screen. Subsequent phosphoproteomic analysis identified the di- and tri-peptide transporter Ptr22 as a potential Sky2 substrate. Our data suggest that Sky2 seems to be involved in Ptr22 regulation since overexpression of PTR22 in the sky2∆ mutant restored the ability to grow on dipeptides and made the cells more susceptible to the dipeptide antifungals Polyoxin D and Nikkomycin Z. Altogether, our results demonstrate that C. albicans and C. glabrata Sky1 protein kinases are functionally similar to Sky1 in S. cerevisiae, whereas C. albicans Sky2, a unique kinase of the CTG clade, likely regulates dipeptide uptake via Ptr22.

MiR-659-3p inhibits osteosarcoma progression and metastasis by inhibiting cell proliferation and invasion via targeting SRPK1

ObjectiveOsteosarcoma is the most common primary bone cancer that affects mostly children and young adults. Despite the advances in osteosarcoma treatment, the long-term survival rate of metastatic patients has not significantly improved in the past few decades, thus demonstrating the need for novel therapeutic targets or methods to improve metastatic osteosarcoma treatment. In this study we aimed to elucidate the role of miR-659-3p and SRPK1 in osteosarcoma.MethodsWe evaluated miR-659-3p and SRPK1 function in osteosarcoma cell proliferation, migration, and cell cycle progression in vitro by using gain- and loss-of-function strategies. The effect of miR-659-3p in tumor progression and metastasis was determined by in vivo mouse model.ResultsWe revealed that expression of miR-659-3p was significantly downregulated in osteosarcoma compared with normal bone cells and was inversely correlated with serine-arginine protein kinase 1 (SRPK1) expression. We proved that miR-659-3p targets 3’ UTR of SRPK1 and negatively regulates SRPK1 expression in osteosarcoma cells via luciferase assay. In vitro studies revealed that gain of miR-659-3p function inhibited osteosarcoma cells growth, migration, and invasion by down-regulating SRPK1 expression. Inversely, inhibiting miR-659-3p in osteosarcoma cells promoted cell growth, migration, and invasion. Cell cycle profile analysis revealed that miR-659-3p inhibited osteosarcoma cells’ G1/G0 phase exit by down-regulating SRPK1 expression. By using an in vivo mouse model, we demonstrated that miR-659-3p inhibits osteosarcoma tumor progression and lung metastasis by inhibiting SRPK1 expression and potentially downstream cell proliferation, and epithelial-to-mesenchymal transition genes.ConclusionsThis study demonstrated that miR-659-3p is a potential therapeutic method and SRPK1 is a potential therapeutic target for osteosarcoma treatment.

SRPK1 regulates RNA binding in a pre-spliceosomal complex using a catalytic bypass mechanism.

Serine-arginine protein kinase 1 (SRPK1) phosphorylates serine-arginine (SR) proteins in the cytoplasm, directing them to the nucleus for splicing function. SRPK1 has also been detected in the nucleus but its function here is still not fully understood. We now demonstrate that nuclear SRPK1 can regulate U1-70K, a protein component of the uridine-rich 1 small nuclear ribonucleoprotein (U1 snRNP) that binds SR proteins and facilitates 5' splice-site selection in precursor mRNA. We found that SRPK1 uses a large, disordered domain to bind U1-70K, regulating the interaction of an exonic splicing enhancer (ESE) to the associated SR protein. Surprisingly, the catalytic activity of SRPK1 is not required for this phenomenon. Instead, SRPK1 associates directly with the N-terminus of U1-70K and alters the regulatory function of the distal C-terminus, modifying interactions between the U1-70K:SR protein complex and the ESE. Disruption of SRPK1 binding to this complex affects the alternative splicing of genes modulated by the C-terminus of U1-70K. Such findings suggest that, in addition to operating as a traditional serine-modifying catalyst, SRPK1 can also bypass this intrinsic activity to regulate RNA contacts in an early pre-spliceosomal complex.

Icariside Ⅱ Attenuates Palmitic Acid-Induced Endothelial Dysfunction Through SRPK1-Akt-eNOS Signaling Pathway.

Background: Endothelial dysfunction is commonly accompanied by a reduced capacity for nitric oxide (NO) production and decreased NO sensitivity, playing a central role in numerous vascular diseases. Saturated free fatty acids are known to reduce NO production and then induce endothelial dysfunction. Alternative splicing participates in the regulation of cellular and tissular homeostasis and is highly regulated by serine-arginine protein kinase (SRPK1). The role of SRPK1 in the biology of endothelial cells remains elusive. Icariside Ⅱ (ICA Ⅱ) has been reported to have protective effects on endothelial function. However, the specific molecular mechanisms are still unknown. The purpose of this study is to explore the role of SRPK1 in the biology of endothelial cells and the underlying mechanism of ICA Ⅱ on palmitic acid (PA) induced endothelial dysfunction. Methods: Endothelial dysfunction was induced using PA in human umbilical vein endothelial cells (HUVECs). The expression and phosphorylation of related proteins in the SRPK1-Akt-eNOS signaling pathway were detected by Western Blot. Cell Counting Kit-8 assay and Ki-67 immunofluorescence were used to estimate cell viability. Endothelial cell function was assessed by detecting NO production using DAF-FM DA. Interaction between ICA Ⅱ and SRPK1 was demonstrated by a biotinylated protein interaction pull-down assay. Results: The expressions of eNOS, Akt, and SRPK1 were down-regulated in the endothelial dysfunction stimulated by PA. SRPK1 inhibitor SPHINX31 restrained endothelial cell viability in a dose-dependent manner. Moreover, inhibition of SRPK1 using SPHINX31 and knockdown of SRPK1 by shRNA also showed a down-regulation of the proteins associated with the SRPK1-Akt-eNOS signaling pathway. Biotinylated protein interaction pull-down assay revealed that ICA Ⅱ could be directly bound with SRPK1. On the other hand, ICA Ⅱ could attenuate the PA-induced endothelial dysfunction and restore cell viability through the SRPK1-Akt-eNOS pathway. Conclusions: ICA Ⅱ, bound with SRPK1, could attenuate the endothelial dysfunction induced by the PA in HUVECs via the SRPK1-Akt-eNOS signaling pathway.

Stress Granules Determine the Development of Obesity-Associated Pancreatic Cancer.

: We show that stress adaptation via the phase-separation organelles SGs mediates PDAC development. Moreover, preexisting stress conditions such as obesity are a driving force behind tumor SG dependence, and enhanced SG levels are key determinants and a chemopreventive target for obesity-associated PDAC. This article is highlighted in the In This Issue feature, p. 1825.

Candida albicans SR-Like Protein Kinases Regulate Different Cellular Processes: Sky1 Is Involved in Control of Ion Homeostasis, While Sky2 Is Important for Dipeptide Utilization.

Protein kinases play a crucial role in regulating cellular processes such as growth, proliferation, environmental adaptation and stress responses. Serine-arginine (SR) protein kinases are highly conserved in eukaryotes and regulate fundamental processes such as constitutive and alternative splicing, mRNA processing and ion homeostasis. The Candida albicans genome encodes two (Sky1, Sky2) and the Candida glabrata genome has one homolog (Sky1) of the human SR protein kinase 1, but their functions have not yet been investigated. We used deletion strains of the corresponding genes in both fungi to study their cellular functions. C. glabrata and C. albicans strains lacking SKY1 exhibited higher resistance to osmotic stress and toxic polyamine concentrations, similar to Saccharomyces cerevisiae sky1Δ mutants. Deletion of SKY2 in C. albicans resulted in impaired utilization of various dipeptides as the sole nitrogen source. Subsequent phosphoproteomic analysis identified the di- and tripeptide transporter Ptr22 as a potential Sky2 substrate. Sky2 seems to be involved in Ptr22 regulation since overexpression of PTR22 in the sky2Δ mutant restored the ability to grow on dipeptides and made the cells more susceptible to the dipeptide antifungals Polyoxin D and Nikkomycin Z. Altogether, our results demonstrate that C. albicans and C. glabrata Sky1 protein kinases are functionally similar to Sky1 in S. cerevisiae, whereas C. albicans Sky2, a unique kinase of the CTG clade, likely regulates dipeptide uptake via Ptr22.

A presynaptic phosphosignaling hub for lasting homeostatic plasticity

Stable function of networks requires that synapses adapt their strength to levels of neuronal activity, and failure to do so results in cognitive disorders. How such homeostatic regulation may be implemented in mammalian synapses remains poorly understood. Here we show that the phosphorylation status of several positions of the active-zone (AZ) protein RIM1 are relevant for synaptic glutamate release. Position RIMS1045 is necessary and sufficient for expression of silencing-induced homeostatic plasticity and is kept phosphorylated by serine arginine protein kinase 2 (SRPK2). SRPK2-induced upscaling of synaptic release leads to additional RIM1 nanoclusters and docked vesicles at the AZ and is not observed in the absence of RIM1 and occluded by RIMS1045E. Our data suggest that SRPK2 and RIM1 represent a presynaptic phosphosignaling hub that is involved in the homeostatic balance of synaptic coupling of neuronal networks.

Phosphorylation Regulates CIRBP Arginine Methylation, Transportin-1 Binding and Liquid-Liquid Phase Separation.

Arginine-glycine(-glycine) (RG/RGG) regions are highly abundant in RNA-binding proteins and involved in numerous physiological processes. Aberrant liquid-liquid phase separation (LLPS) and stress granule (SGs) association of RG/RGG regions in the cytoplasm have been implicated in several neurodegenerative disorders. LLPS and SG association of these proteins is regulated by the interaction with nuclear import receptors, such as transportin-1 (TNPO1), and by post-translational arginine methylation. Strikingly, many RG/RGG proteins harbour potential phosphorylation sites within or close to their arginine methylated regions, indicating a regulatory role. Here, we studied the role of phosphorylation within RG/RGG regions on arginine methylation, TNPO1-binding and LLPS using the cold-inducible RNA-binding protein (CIRBP) as a paradigm. We show that the RG/RGG region of CIRBP is in vitro phosphorylated by serine-arginine protein kinase 1 (SRPK1), and discovered two novel phosphorylation sites in CIRBP. SRPK1-mediated phosphorylation of the CIRBP RG/RGG region impairs LLPS and binding to TNPO1 in vitro and interferes with SG association in cells. Furthermore, we uncovered that arginine methylation of the CIRBP RG/RGG region regulates in vitro phosphorylation by SRPK1. In conclusion, our findings indicate that LLPS and TNPO1-mediated chaperoning of RG/RGG proteins is regulated through an intricate interplay of post-translational modifications.

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

Serine-arginine protein kinase-like protein, SrpkF, stimulates both cellobiose-responsive and D-xylose-responsive signaling pathways in Aspergillus aculeatus.

Aspergillus aculeatus produces cellulolytic enzymes in the presence of their substrates. We screened a library of 12,000 A. aculeatus T-DNA-inserted mutants to identify a regulatory factor involved in the expression of their enzyme genes in response to inducers. We found one mutant that reduced the expression of FIII-avicelase (chbI) in response to cellulose. T-DNA was inserted into a putative protein kinase gene similar to AN10082 in A. nidulans, serine-arginine protein kinase F, SrpkF. Fold increases in srpkF gene expression in response to various carbon sources were 2.3 (D-xylose), 44 (Avicel®), 59 (Bacto™ Tryptone), and 98 (no carbon) compared with D-glucose. Deletion of srpkF in A. aculeatus resulted in a significant reduction in cellulose-responsive expression of chbI, hydrocellulase (cel7b), and FIb-xylanase (xynIb) genes at an early induction phase. Further, the srpkF-overexpressing strain showed upregulation of the srpkF gene from four- to nine-fold higher than in the control strain. srpkF overexpression upregulated cbhI and cel7b in response to cellobiose and the FI-carboxymethyl cellulase gene (cmc1) and xynIb in response to D-xylose. However, the srpkF deletion did not affect the expression of xynIb in response to D-xylose due to the less expression of srpkF under the D-xylose condition. Our data demonstrate that SrpkF is primarily involved in cellulose-responsive expression, though it has a potential to stimulate gene expression in response to both cellobiose and D-xylose in A. aculeatus.

Oncogenic dysregulation of pre-mRNA processing by protein kinases: challenges and therapeutic opportunities.

Alternative splicing and polyadenylation represent two major steps in pre‐mRNA‐processing, which ensure proper gene expression and diversification of human transcriptomes. Deregulation of these processes contributes to oncogenic programmes involved in the onset, progression and evolution of human cancers, which often result in the acquisition of resistance to existing therapies. On the other hand, cancer cells frequently increase their transcriptional rate and develop a transcriptional addiction, which imposes a high stress on the pre‐mRNA‐processing machinery and establishes a therapeutically exploitable vulnerability. A prominent role in fine‐tuning pre‐mRNA‐processing mechanisms is played by three main families of protein kinases: serine arginine protein kinase (SRPK), CDC‐like kinase (CLK) and cyclin‐dependent kinase (CDK). These kinases phosphorylate the RNA polymerase, splicing factors and regulatory proteins involved in cleavage and polyadenylation of the nascent transcripts. The activity of SRPKs, CLKs and CDKs can be altered in cancer cells, and their inhibition was shown to exert anticancer effects. In this review, we describe key findings that have been reported on these topics and discuss challenges and opportunities of developing therapeutic approaches targeting splicing factor kinases.