The O-ß-linked N-acetylglucosaminylation of the Lamin B receptor and its impact on DNA binding and phosphorylation.

Lamin B Receptor (LBR) is an integral protein of the interphase inner nuclear membrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has been extensively studied, very little is known about other post-translational modifications of the protein. There is only one report on the O-ß-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites by using as substrates of O-ß-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA binding while it marginally affects RS domain phosphorylation mediated by SRPK1, Akt2 and cdk1 kinases. GENERAL SIGNIFICANCE: Our methodology providing a quantitative description of O-GlcNAc patterns based on a combination of mass spectrometry and high resolution NMR spectroscopy on short peptide substrates allows subsequent functional analyses. Hence, our approach is of general interest to a wide audience of biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation.

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.

Expression of SRPK1 in gliomas and its role in glioma cell lines viability.

Among factors regulating the splicing of major importance is serine/arginine protein kinase 1 (SRPK1) that phosphorylates SR splicing factors. SRPK1 is expressed in the mammalian central nervous system in a region- and neuron-specific manner. Based on previous observations that glial cells are practically devoid of SRPK1 and reports showing aberrant expression of SRPK1 in numerous tumors, but with conflicting roles, this study aims to investigate the expression of SRPK1 in glioma and its influence on tumor cell biological features. As shown by immunohistochemical analysis, malignant glioma cells express SRPK1 in glioblastomas with significant association between SRPK1 expression and patients' survival. SRPK1 expression was also significantly upregulated at the messenger RNA (mRNA) and protein level in glioma cell lines. Small interfering RNA-mediated downregulation of SRPK1 had little effect on cell viability, while it slightly enhanced the sensitivity of cells to killing by cisplatin. These results support the idea that at least in vitro, the effect of SRPK1 knockdown on the viability of glioma cell lines is rather limited, while the in vivo effects could be attributed to the modulation of angiogenesis by SRPK1.

Phosphorylation of the arginine/serine repeats of lamin B receptor by SRPK1-insights from molecular dynamics simulations.

BACKGROUND: Arginine/serine (RS) repeats are found in several proteins in metazoans with a wide variety of functions, many of which are regulated by SR protein kinase 1 (SRPK1)-mediated phosphorylation. Lamin B receptor (LBR) is such a protein implicated in chromatin anchorage to the nuclear envelope. METHODS: Molecular dynamics simulations were used to investigate the conformation of two LBR peptides containing four (human-) and five (turkey-orthologue) consecutive RS dipeptides, in their unphosphorylated and phosphorylated forms and of a conserved peptide, in isolation and in complex with SRPK1. GST pull-down assays were employed to study LBR interactions. RESULTS: Unphosphorylated RS repeats adopt short, transient helical conformations, whereas serine phosphorylation induces Arginine-claw-like structures. The SRSRSRSPGR peptide, overlapping with the LBR RS repeats, docks into the known, acidic docking groove of SRPK1, in an extended conformation. Phosphorylation by SRPK1 is necessary for the association of LBR with histone H3. CONCLUSIONS: The C-terminal region of the LBR RS domain constitutes a recognition platform for SRPK1, which uses the same recognition mechanism for LBR as for substrates with long RS domains. This docking may promote unfolding of the RS repeats destined to be phosphorylated. Phosphorylation induces Arginine-claw-like conformations, irrespective of the RS-repeat length, that may facilitate interactions with basic partners. GENERAL SIGNIFICANCE: Our results shed light on the conformational preferences of an important class of repeats before and after their phosphorylation and support the idea that even short RS domains may be constituents of recognition platforms for SRPK1, thus adding to knowledge towards a full understanding of their phosphorylation mechanism.

Synthesis and Anti-Angiogenic Activity of Novel c(RGDyK) Peptide-Based JH-VII-139-1 Conjugates.

Peptide-drug conjugates are delivery systems for selective delivery of cytotoxic agents to target cancer cells. In this work, the optimized synthesis of JH-VII-139-1 and its c(RGDyK) peptide conjugates is presented. The low nanomolar SRPK1 inhibitor, JH-VII-139-1, which is an analogue of Alectinib, was linked to the ανß3 targeting oligopeptide c(RGDyK) through amide, carbamate and urea linkers. The chemostability, cytotoxic and antiangiogenic properties of the synthesized hybrids were thoroughly studied. All conjugates retained mid nanomolar-level inhibitory activity against SRPK1 kinase and two out of four conjugates, geo75 and geo77 exhibited antiproliferative effects with low micromolar IC50 values against HeLa, K562, MDA-MB231 and MCF7 cancer cells. The activities were strongly related to the stability of the linkers and the release of JH-VII-139-1. In vivo zebrafish screening assays demonstrated the ability of the synthesized conjugates to inhibit the length or width of intersegmental vessels (ISVs). Flow cytometry experiments were used to test the cellular uptake of a fluorescein tagged hybrid in MCF7 and MDA-MB231 cells that revealed a receptor-mediated endocytosis process. In conclusion, most conjugates retained the inhibitory potency against SRPK1 as JH-VII-139-1 and demonstrated antiproliferative and antiangiogenic activities. Further animal model experiments are needed to uncover the full potential of such peptide conjugates in cancer therapy and angiogenesis-related diseases.

Good Cop, Bad Cop: The Different Roles of SRPKs.

SR Protein Kinases (SRPKs), discovered approximately 30 years ago, are widely known as splice factor kinases due to their decisive involvement in the regulation of various steps of mRNA splicing. However, they were also shown to regulate diverse cellular activities by phosphorylation of serine residues residing in serine-arginine/arginine-serine dipeptide motifs. Over the last decade, SRPK1 has been reported as both tumor suppressor and promoter, depending on the cellular context and has been implicated in both chemotherapy sensitivity and resistance. Moreover, SRPK2 has been reported to exhibit contradictory functions in different cell contexts promoting either apoptosis or tumor growth. The aim of the current review is to broaden and deepen our understanding of the SRPK function focusing on the subcellular localization of the kinases. There is ample evidence that the balance between cytoplasmic and nuclear SRPK levels is tightly regulated and determines cell response to external signals. Specific cell states coupled to kinase levels, spatial specific interactions with substrates but also changes in the extent of phosphorylation that allow SRPKs to exhibit a rheostat-like control on their substrates, could decide the proliferative or antiproliferative role of SRPKs.

SR Protein Kinase 1 Inhibition by TAF15.

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases' partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins.

Nuclear Translocation of SRPKs Is Associated with 5-FU and Cisplatin Sensitivity in HeLa and T24 Cells.

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 and anchors the kinases in the cytoplasm is a unique structural feature of SRPKs. Here, we report that exposure of HeLa and T24 cells to DNA damage inducers triggers the nuclear translocation of SRPK1 and SRPK2. Furthermore, we show that nuclear SRPKs did not protect from, but on the contrary, mediated the cytotoxic effects of genotoxic agents, such as 5-fluorouracil (5-FU) and cisplatin. Confirming previous data showing that the kinase activity is essential for the entry of SRPKs into the nucleus, SRPIN340, a selective SRPK1/2 inhibitor, blocked the nuclear accumulation of the kinases, thus diminishing the cytotoxic effects of the drugs. ATR/ATM-dependent phosphorylation of threonine 326 and serine 408 in the spacer domain of SRPK1 was essential for the redistribution of the kinase to the nucleus. Substitution of either of these two residues to alanine or inhibition of ATR/ATM kinase activity abolished nuclear localization of SRPK1 and conferred tolerance to 5-FU treatment. These findings suggest that SRPKs may play an important role in linking cellular signaling to DNA damage in eukaryotic cells.

Deletion of LBR N-terminal domains recapitulates Pelger-Huet anomaly phenotypes in mouse without disrupting X chromosome inactivation.

Mutations in the gene encoding Lamin B receptor (LBR), a nuclear-membrane protein with sterol reductase activity, have been linked to rare human disorders. Phenotypes range from a benign blood disorder, such as Pelger-Huet anomaly (PHA), affecting the morphology and chromatin organization of white blood cells, to embryonic lethality as for Greenberg dysplasia (GRBGD). Existing PHA mouse models do not fully recapitulate the human phenotypes, hindering efforts to understand the molecular etiology of this disorder. Here we show, using CRISPR/Cas-9 gene editing technology, that a 236bp N-terminal deletion in the mouse Lbr gene, generating a protein missing the N-terminal domains of LBR, presents a superior model of human PHA. Further, we address recent reports of a link between Lbr and defects in X chromosome inactivation (XCI) and show that our mouse mutant displays minor X chromosome inactivation defects that do not lead to any overt phenotypes in vivo. We suggest that our N-terminal deletion model provides a valuable pre-clinical tool to the research community and will aid in further understanding the etiology of PHA and the diverse functions of LBR.

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.

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.

Identification of protamine 1 as a novel cancer-testis antigen in early chronic lymphocytic leukaemia.

Early chronic lymphocytic leukaemia (CLL) is an ideal disease for immunotherapy. We previously showed that SEMG 1 is a cancer-testis (CT) antigen in CLL. In this study, SEMG 1 was applied as the bait in a yeast two-hybrid system of a testicular cDNA library. Seven clones were isolated and Protamine (Prm) 1 was identified as a novel CT antigen in early CLL. PRM1 transcripts were detected in 11/41 (26.8%) patients. Prm 1 protein was also expressed but heterogeneously within individual patients. Of the 11 patients expressing Prm 1, four expressed Zap 70 protein and seven did not. These results, therefore, indicate that Prm 1 could potentially be a suitable target for the design of tumour vaccine for patients with early CLL, including for those with poor risk CLL. High titres of Prm 1 IgG antibodies could be detected in 20 of these 41 CLL patients but not in any of the 20 healthy donors (P = 0.0001), suggesting the presence of Prm 1-reactive immune responses within the immune repertoire of patients with early CLL. Further work is warranted, especially in approaches to upregulate Prm 1 expression, and to determine the role of Prm 1 as an immunotherapeutic target for early CLL.

SEMG-1 expression in early stage chronic lymphocytic leukemia.

BACKGROUND AIMS: Chronic lymphocytic leukemia (CLL) is an indolent disease. It is currently recommended that patients with CLL stages 0 and I follow a watchful waiting strategy. These patients are, therefore, a suitable group for testing immunotherapeutic approaches to avoid problems of immunosuppression as a result of disease progression and chemotherapy. In this study, we investigated the expression of SEMG-1 in early CLL to determine the suitability of SEMG-1 as a target for further development of tumor vaccines for early CLL. METHODS: A combination of reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunocytochemistry was used to evaluate the expression of SEMG-1 in early CLL. The results were correlated with Zap 70 expression. Recombinant SEMG-1 protein was used in an enzyme-linked immunosorbent assay (ELISA) to determine the presence of SEMG-1 antibodies (Ab) in serum from these patients. RESULTS: The SEMG-1 gene was expressed in 19/41 (46%) patients with early CLL. Gene expression was associated with protein synthesis in CLL cells. Protein expression, however, was heterogeneous within individual patients. Only transcripts encoding the SEMG-1(50) variant and not SEMG-1(43) were detected. SEMG-1(50) was expressed irrespective of the Zap 70 status. High-titer SEMG-1 IgG but not IgM Ab were detected in some of these patients, suggesting that SEMG-1-reactive immune responses are intact within the immune repertoire of early CLL patients. CONCLUSIONS: SEMG-1 is expressed in nearly half of patients with early CLL and may be a target for further investigations into its use for immunotherapy of early CLL.

Data on the expression of SRPK1a in mammals.

SRPK1 is an evolutionary conserved protein kinase that specifically phosphorylates its substrates at serine residues located within arginine-serine-rich (RS) domains. We have previously reported the existence of a second less abundant isoform in humans, SRPK1a, which is formed from alternative splicing of the SRPK1 gene and contains an insertion of 171 amino acids at its N-terminal domain (Nikolakaki et al., 2001). In the NCBI database SRPK1a is annotated as a related to SRPK1-mRNA sequence coding for protein CAC39299.1. Here, we present data on the conservation of the extra sequence of SRPK1a in mammals. Furthermore, the retrieved sequences were comparatively analyzed and data on their evolutionary origin and relationships are also presented.

Lamin B Receptor: Interplay between Structure, Function and Localization.

Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane, containing a hydrophilic N-terminal end protruding into the nucleoplasm, eight hydrophobic segments that span the membrane and a short, nucleoplasmic C-terminal tail. Two seemingly unrelated functions have been attributed to LBR. Its N-terminal domain tethers heterochromatin to the nuclear periphery, thus contributing to the shape of interphase nuclear architecture, while its transmembrane domains exhibit sterol reductase activity. Mutations within the transmembrane segments result in defects in cholesterol synthesis and are associated with diseases such as the Pelger-Huët anomaly and Greenberg skeletal dysplasia, whereas no such harmful mutations related to the anchoring properties of LBR have been reported so far. Recent evidence suggests a dynamic regulation of LBR expression levels, structural organization, localization and function, in response to various signals. The molecular mechanisms underlying this dynamic behavior have not yet been fully unraveled. Here, we provide an overview of the current knowledge of the interplay between the structure, function and localization of LBR, and hint at the interconnection of the two distinct functions of LBR.

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.

SRPK1 and Akt Protein Kinases Phosphorylate the RS Domain of Lamin B Receptor with Distinct Specificity: A Combined Biochemical and In Silico Approach.

Activated Akt has been previously implicated in acting on RS domain-containing proteins. However, it has been questioned whether its action is direct or it is mediated by co-existing SR kinase activity. To address this issue we studied in detail the phosphorylation of Lamin B Receptor (LBR) by Akt. Using synthetic peptides and a set of recombinant proteins expressing mutants of the LBR RS domain we now demonstrate that while all serines of the RS domain represent more or less equal phosphoacceptor sites for SRPK1, Ser80 and Ser82 are mainly targeted by Akt. 3D-modeling combined with molecular dynamics (MD) simulations show that amongst short, overlapping LBR RS-containing peptides complying with the minimum Akt recognition consensus sequence, only those bearing phosphosites either at Ser80 or Ser82 are able to fit into the active site of Akt, at least as effectively as its known substrate, GSK3-ß. Combined our results provide evidence that Akt kinases directly phosphorylate an RS domain-containing protein and that both the residues N-terminal the phosphosite and at position +1 are essential for Akt specificity, with the latter substrate position being compatible with the arginine residue of RS-repeats.

The intracellular fate of zonula occludens 2 is regulated by the phosphorylation of SR repeats and the phosphorylation/O-GlcNAcylation of S257.

Zona occludens 2 (ZO-2) has a dual localization. In confluent epithelia, ZO-2 is present at tight junctions (TJs), whereas in sparse proliferating cells it is also found at the nucleus. Previously we demonstrated that in sparse cultures, newly synthesized ZO-2 travels to the nucleus before reaching the plasma membrane. Now we find that in confluent cultures newly synthesized ZO-2 goes directly to the plasma membrane. Epidermal growth factor induces through AKT activation the phosphorylation of the kinase for SR repeats, serine arginine protein kinase 1, which in turn phosphorylates ZO-2, which contains 16 SR repeats. This phosphorylation induces ZO-2 entry into the nucleus and accumulation in speckles. ZO-2 departure from the nucleus requires intact S257, and stabilizing the ß-O-linked N-acetylglucosylation (O-GlcNAc) of S257 with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, an inhibitor of O-GlcNAcase, triggers nuclear exportation and proteosomal degradation of ZO-2. At the plasma membrane ZO-2 is not O-GlcNAc, and instead, as TJs mature, it becomes phosphorylated at S257 by protein kinase Cζ. This late phosphorylation of S257 is required for the correct cytoarchitecture to develop, as cells transfected with ZO-2 mutant S257A or S257E form aberrant cysts with multiple lumens. These results reveal novel posttranslational modifications of ZO-2 that regulate the intracellular fate of this protein.

The deletion variant of α2b-adrenergic receptor is associated with decreased risk in Alzheimer's disease and mild cognitive impairment.

A common genetic polymorphism of the α2b-adrenergic receptor (ADRA2B) resulting in a deletion of three glutamic acids located on the third intracellular loop of the protein, has been associated with memory formation enhanced by emotional events. Additionally, there are several studies documenting the involvement of this polymorphism in other types of cognition, such as episodic memory. The aim of this study was to investigate the possible relationship of this genetic variance with a common memory affecting disease, Alzheimer's disease. Our study was carried out in a total number of 311 Greek subjects, including 119 sporadic AD patients, 95 MCI cases and 97 controls. Genomic DNA was extracted from whole blood and the fragments containing the polymorphism were amplified by PCR analysis. A genotypic analysis of the APOE polymorphism was also carried out. A significant difference in the frequency of the ADRA2B genetic variation among the three groups was observed. Specifically, the deletion variant is more prevalent in controls than in AD and MCI patients. Our data demonstrate for the first time an independent contribution of the ADRA2B genetic polymorphism to memory impairment and we further suggest a possible protective role of the deletion variant against the disease development.

Serine-arginine protein kinases: a small protein kinase family with a large cellular presence.

Serine-arginine protein kinases (SPRKs) constitute a relatively novel subfamily of serine-threonine kinases that specifically phosphorylate serine residues residing in serine-arginine/arginine-serine dipeptide motifs. Fifteen years of research subsequent to the purification and cloning of human SRPK1 as a SR splicing factor-phosphorylating protein have lead to the accumulation of information on the function and regulation of the different members of this family, as well as on the genomic organization of SRPK genes in several organisms. Originally considered to be devoted to constitutive and alternative mRNA splicing, SRPKs are now known to expand their influence to additional steps of mRNA maturation, as well as to other cellular activities, such as chromatin reorganization in somatic and sperm cells, cell cycle and p53 regulation, and metabolic signalling. Similarly, SRPKs were considered to be constitutively active kinases, although several modes of regulation of their function have been demonstrated, implying an elaborate cellular control of their activity. Finally, SRPK gene sequence information from bioinformatics data reveals that SRPK gene homologs exist either in single or multiple copies in every single eukaryotic organism tested, emphasizing the importance of SRPK protein function for cellular life.