Eleni Nikolakaki

Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation.

Glycogen synthase kinase‐3 (GSK‐3) is a protein serine kinase implicated in the cellular response to insulin. The enzyme is the mammalian homologue of the zeste‐white3 (shaggy) homeotic gene of Drosophila melanogaster and has been implicated in the regulation of the c‐Jun/AP‐1 transcription factor. In mammals this protein serine kinase is encoded by two related genes termed GSK‐3 alpha and beta. Here, we demonstrate that these two proteins and the fruit fly protein are phosphorylated on tyrosine in vivo. Moreover, GSK‐3 beta activity and function are shown to be dependent on tyrosine phosphorylation. The modified tyrosine residue is conserved in all members of the GSK‐3 family and is equivalent to that required for activity by mitogen‐activated protein (MAP) kinases. However, unlike MAP kinases, GSK‐3 is highly phosphorylated on tyrosine and thus active in resting cells.

Regulation of Alternative Splicing of Human Tau Exon 10 by Phosphorylation of Splicing Factors

Tau is a microtubule-associated protein whose transcript undergoes regulated splicing in the mammalian nervous system. Exon 10 of the gene is an alternatively spliced cassette that is adult-specific and encodes a microtubule-binding domain. Mutations increasing the inclusion of exon 10 result in the production of tau protein which predominantly contains four microtubule-binding repeats and were shown to cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we show that exon 10 usage is regulated by CDC2-like kinases CLK1, 2, 3, and 4 that phosphorylate serine-arginine-rich proteins, which in turn regulate pre-mRNA splicing. Cotransfection experiments suggest that CLKs achieve this effect by releasing specific proteins from nuclear storage sites. Our results show that changing pre-mRNA-processing pathways through phosphorylation could be a new therapeutic concept for tauopathies.

Mitotic Phosphorylation of the Lamin B Receptor by a Serine/Arginine Kinase and p34cdc2

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that is modified at interphase by a nuclear envelope-bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH2-terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, we performed phosphopeptide mapping of in vitro and in vivo 32P-labeled LBR and analyzed a series of recombinant proteins and synthetic peptides. Our results show that LBR undergoes two types of mitotic phosphorylation mediated by the RS and the p34cdc2 protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser76, Ser78, Ser80, Ser82, Ser84), whereas p34cdc2 mainly phosphorylates Ser71. These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsible for nuclear envelope assembly and disassembly.

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.

On the bioreactivity of triorganotin aminobenzoates. Investigation of trialkyl and triarylyltin(IV) esters of 3-amino and 4-aminobenzoic acids.

The synthesis and study of trimethyl-, tributyl- and triphenyltin esters of the 3- and 4-aminobenzoic acids are reported. The triorganotin derivatives are characterized by elemental analyses, FT-IR and solution (1)H and (13)C NMR spectra. The structure of the trimethyltin 4-aminobenzoate is solved by X-ray diffraction and proves to be polymeric in nature with bridging carboxylates and trigonal bipyramidal tin(IV) environment. However, all the compounds become monomeric in solution with a tetrahedral tin coordination environment in chloroform and trigonal bipyramidal in DMSO due to coordination of the solvent as the NMR spectra have revealed. The compounds exhibit variable cytotoxic activity when tested against Kappa562 myelogenous leukaemia, HeLa cervical cancer and HepG2 hepatocellular carcinoma cell lines, with the butyl derivatives being the more effective and the methyl ones the less. Interestingly, their antibacterial action was significantly lower when tested against Escherichia coli, while not appreciable direct interaction with DNA has been observed. The above observations account for a mode of action that may be related to their potential interaction with cell membranes and the subsequent inhibition of various signaling processes.

Relative expression of human telomerase catalytic subunit (hTERT) transcripts in astrocytic gliomas

Human telomerase catalytic subunit (hTERT) expression has been reported as a marker for malignancy in various tumor systems. The aim of the present study was to investigate the relative expression of hTERT (relhTERT) and its transcripts A+B+ (contained in the full-length product), Adel and Bdel in astrocytic gliomas (grades I–IV, n=38). relhTERT was assessed by duplex reverse transcription-PCR and the expression profile of Adel, Bdel and A+B+ transcripts by nested real time-PCR. relhTERT and A+B+ presence correlated well with each other (P<0.001) and with histological grading [grades I–II (low) vs III–IV (high), PrelhTERT=0.002 and PA+B+<0.001]. A+B+ was detected in one out of seven hTERT-positive low-grade tumors, while it was present in 96.3%, and predominantly expressed in 59.3% of high-grade tumors. Bdel predominance was observed only in three cases, irrespective of grading, while Bdel levels equal or close to those of A+B+ were found in 30.4% of grade IV tumors. In situ hybridization with specific Bplus and Bdel probes revealed positive signals for both mRNAs in association with relhTERT and respective variant profiles. In addition, this method was useful in assessing hTERT expression in cases where sampling errors for RT-PCR were unavoidable. Our findings show that except for differences in relhTERT, low- and high-grade astrocytic gliomas exhibit distinct hTERT variant profiles, most of which seem to be in line with the role attributed to hTERT regarding its contribution to the acquisition of malignant potential during astrocyte carcinogenesis. Low-grade tumors mainly express Adel and Bdel. High-grade tumors, especially grade IV, always express A+B+, mostly but not always in predominance over Adel and Bdel. In this same group, profiles with Bdel predominance or relatively equal A+B+/Bdel expression are also observed, and Adel is often missing. Whether these differences characterize tumors with different biological behavior remains to be elucidated.

The enzymatic activity of SR protein kinases 1 and 1a is negatively affected by interaction with scaffold attachment factors B1 and 2

SR protein kinases (SRPKs) phosphorylate Ser/Arg dipeptide‐containing proteins that play crucial roles in a broad spectrum of basic cellular processes. Phosphorylation by SRPKs constitutes a major way of regulating such cellular mechanisms. In the past, we have shown that SRPK1a interacts with the nuclear matrix protein scaffold attachment factor B1 (SAFB1) via its unique N‐terminal domain, which differentiates it from SRPK1. In this study, we show that SAFB1 inhibits the activity of both SRPK1a and SRPK1 in vitro and that its RE‐rich region is redundant for the observed inhibition. We demonstrate that kinase activity inhibition is caused by direct binding of SAFB1 to SRPK1a and SRPK1, and we also present evidence for the in vitro binding of SAFB2 to the two kinases, albeit with different affinity. Moreover, we show that both SR protein kinases can form complexes with both scaffold attachment factors B in living cells and that this interaction is capable of inhibiting their activity, depending on the tenacity of the complex formed. Finally, we present data demonstrating that SRPK/SAFB complexes are present in the nucleus of HeLa cells and that the enzymatic activity of the nuclear matrix‐localized SRPK1 is repressed. These results suggest a new role for SAFB proteins as regulators of SRPK activity and underline the importance of the assembly of transient intranuclear complexes in cellular regulation.

Identification of distinct SET/TAF-Iβ domains required for core histone binding and quantitative characterisation of the interaction

BackgroundThe assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Iβ belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Iβ, we designed several SET/TAF-Iβ truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays.ResultsWild type SET/TAF-Iβ binds to histones H2B and H3 with Kd values of 2.87 and 0.15 μM, respectively. The preferential binding of SET/TAF-Iβ to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Iβ, as well as the H3 amino-terminal tail, are dispensable for this interaction.ConclusionThis type of analysis allowed us to assess the relative affinities of SET/TAF-Iβ for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Iβ and can be valuable to understand the role of SET/TAF-Iβ in chromatin function.

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-β. 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 ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells

SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells.