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

<p>Abstract</p> <p>Background</p> <p>The 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.</p> <p>Results</p> <p>Wild type SET/TAF-Iβ binds to histones H2B and H3 with K_dvalues 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.</p> <p>Conclusion</p> <p>This 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.</p

The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells

AbstractSRPK1, 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

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

PROTEIN PHOSPHORYLATION IN A CELL FREE SYSTEM OF PROTEIN SYNTHESIS FROM MOUSE LIVER. PURIFICATION AND CHARACTERIZATION OF A NOVEL PROTEIN KINASE REGULATED BY CYCLIC NUCLEOTIDES

IN THE FIRST PART OF THE THESIS AN INVESTIGATION ON THE PHOSPHORYLATION OF THE PROTEIN COMPONENTS OF A CELL FREE SYSTEM OF PROTEIN SYNTHESIS OF MOUSE LIVER (POLYSOMES,"PH5" FRACTION) AS WELL AS THE EFFECT OF VARIOUS AGENTS ON PROTEIN PHOSPHORYLATION IS REPORTED. THE ENZYME IS A SERINE- THREONINE KINASE WITH A MOLECULAR MASS OF 90 KDA. IT CONTAINS A 47 KDA AUTOPHOSPHORYLATABLE PEPTIDE AND CONSEQUENTLY IN ALL PROBABILITY THE NATIVE ENZYME IS A HOMODIMER. IT ACTS OPTIMALLYAT PH 7.5 AND PHOSPHORYLATES HISTONES AND POLYSOMAL PROTEINS BUT NOT PROTAMINE, CASEIN OR ANY OF THE PROTEINS OF THE "PH5" FRACTION. AT HIGH (10- 5 M) ATP CONCENTRATIONS MG2+ IS THE MOST EFFICIENT ACTIVATING METAL WHILE AT LOW (10-9 M) ATP CONCENTRATIONS MN2+ IS. THE ACTIVITY OF THE PURIFIED KINASE IS AFFECTED BY THE PRESENCE OF ANY OF THE FOUR 3', 5' CYCLIC NUCLEOTIDES. THIS EFFECT WHICH MAY BE EITHER STIMULATORY OR INHIBITORY, DEPENDS ON THE CONCENTRATION OF ATP, ON THE TYPE OF ACTIVATING METAL EMPLOYED AS WELL AS ON THE SUBSTRATE USED. ON THE BASIS OF THE AFOREMENTIONED EFFECTS OF THE 3', 5' CYCLIC NUCLEOTIDES AND THE FACT THAT THE NATIVE ENZYME IS NOT DISSOCIATED INTO REGULATORY AND CATALYTIC SUBUNITS, THIS NOVEL KINASE NAMED CYCLIC NUCLEOTIDE- REGULATED PROTEIN KINASE (NRPK).ΣΤΟ ΠΡΩΤΟ ΜΕΡΟΣ ΤΗΣ ΔΙΑΤΡΙΒΗΣ ΔΙΕΡΕΥΝΗΘΗΚΕ Η ΦΩΣΦΟΡΥΛΙΩΣΗ ΤΩΝ ΕΠΙΜΕΡΟΥΣ ΠΡΩΤΕΙΝΙΚΩΝ ΣΥΣΤΑΤΙΚΩΝ ΕΝΟΣ ΣΥΣΤΗΜΑΤΟΣ ΠΡΩΤΕΙΝΟΣΥΝΘΕΣΗΣ ΕΛΕΥΘΕΡΟΥ ΚΥΤΤΑΡΩΝ (ΠΟΛΥΣΩΜΑΤΑ, ΚΛΑΣΜΑ "PH5") ΑΠΟ ΣΥΚΩΤΙ ΠΟΝΤΙΚΟΥ ΚΑΘΩΣ ΚΑΙ Η ΕΠΙΔΡΑΣΗ ΔΙΑΦΟΡΩΝ ΠΑΡΑΓΟΝΤΩΝ ΠΟΥ ΤΗΝ ΕΠΗΡΕΑΖΟΥΝ. ΣΤΟ ΔΕΥΤΕΡΟ ΜΕΡΟΣ ΤΗΣ ΔΙΑΤΡΙΒΗΣ ΑΠΟΜΟΝΩΘΗΚΕ ΚΑΙ ΜΕΛΕΤΗΘΗΚΕ ΜΙΑ ΝΕΑ ΚΙΝΑΣΗ ΠΡΩΤΕΙΝΩΝ ΑΠΟ ΤΟ ΚΥΤΤΑΡΟΠΛΑΣΜΑ ΚΑΙ ΣΥΓΚΕΚΡΙΜΕΝΑ ΑΠΟ ΤΟ ΚΛΑΣΜΑ "PH5". Η ΚΙΝΑΣΗ ΑΥΤΗ ΤΩΝ ΠΡΩΤΕΙΝΩΝ ΕΙΝΑΙ ΚΙΝΑΣΗ ΣΕΡΙΝΗΣ- ΘΡΕΟΝΙΝΗΣ ΚΑΙ ΕΧΕΙ ΜΟΡΙΑΚΟΒΑΡΟΣ 90 KDA. ΠΕΡΙΕΧΕΙ ΜΙΑ ΑΥΤΟΦΩΣΦΟΡΥΛΙΟΥΜΕΝΗ ΥΠΟΜΟΝΑΔΑ ΤΩΝ 47 KDA ΚΑΙ ΓΙΑ ΤΟΛΟΓΟ ΑΥΤΟ ΚΑΤΑ ΠΑΣΑ ΠΙΘΑΝΟΤΗΤΑ ΤΟ ΔΡΑΣΤΙΚΟ ΕΝΖΥΜΟ ΕΙΝΑΙ ΕΝΑ ΟΜΟΔΙΜΕΡΕΣ. ΕΧΕΙ ΒΕΛΤΙΣΤΟ PH ΔΡΑΣΗΣ 7.5 ΚΑΙ ΦΩΣΦΟΡΥΛΙΩΝΕΙ ΙΣΤΟΝΕΣ ΚΑΙ ΡΙΒΟΣΗΜΙΚΕΣ ΠΡΩΤΕΙΝΕΣ ΑΛΛΑ ΟΧΙ ΚΑΖΕΙΝΗ, ΠΡΟΤΑΜΙΝΗ ΚΑΙ ΠΡΩΤΕΙΝΕΣ ΤΟΥ ΚΛΑΣΜΑΤΟΣ "PH5". ΣΕ ΨΗΛΕΣ ΣΥΓΚΕΝΤΡΩΣΕΙΣ ATP (10-5 Μ)ΔΡΑ ΚΑΛΥΤΕΡΑ ΠΑΡΟΥΣΙΑ MG2+, ΕΝΩ ΑΝΤΙΘΕΤΑ ΣΕ ΧΑΜΗΛΕΣ ΣΥΓΚΕΝΤΡΩΣΕΙΣATP (10-9 Μ) ΠΑΡΟΥΣΙΑ MN2+. Η ΔΡΑΣΗ ΤΗΣ ΚΙΝΑΣΗΣ ΕΠΗΡΕΑΖΕΤΑΙ ΑΠΟ ΤΗΝ ΠΑΡΟΥΣΙΑ ΤΩΝ 3', 5' ΚΥΚΛΙΚΩΝ ΝΟΥΚΛΕΟΤΙΔΙΩΝ ΚΑΙ ΤΟ ΑΠΟΤΕΛΕΣΜΑ ΕΞΑΡΤΑΤΑΙ ΤΟΣΟ ΑΠΟ ΤΗ ΣΥΓΚΕΝΤΡΩΣΗ ΤΟΥ ATP, ΟΣΟ ΚΑΙ ΑΠΟ ΤΟ ΜΕΤΑΛΛΟ ΠΟΥ ΧΡΗΣΙΜΟΠΟΙΕΙΤΑΙ ΩΣ ΕΝΕΡΓΟΠΟΙΗΤΗΣ ΚΑΘΩΣ ΚΑΙ ΑΠΟ ΤΗ ΦΥΣΗ ΤΟΥ ΥΠΟΣΤΡΩΜΑΤΟΣ. ΜΕ ΒΑΣΗ ΤΙΣ ΠΙΟ ΠΑΝΩ ΕΠΙΠΤΩΣΕΙΣ ΤΩΝ 3', 5' ΚΥΚΛΙΚΩΝ ΝΟΥΚΛΕΟΤΙΔΙΩΝ ΣΤΗ ΔΡΑΣΗ ΤΟΥ ΕΝΖΥΜΟΥ ΚΑΘΩΣ ΚΑΙ ΑΠΟ ΤΟ ΓΕΓΟΝΟΣ ΟΤΙ ΔΕΝ ΔΙΑΣΠΑΤΑΙ ΣΕ ΚΑΤΑΛΥΤΙΚΗ ΚΑΙ ΡΥΘΜΙΣΤΙΚΗ ΥΠΟΜΟΝΑΔΑ Η ΚΙΝΑΣΗ ΑΥΤΗ ΟΝΟΜΑΣΤΗΚΕ : ΚΙΝΑΣΗ ΠΟΥ Η ΔΡΑΣΗ ΤΗΣ ΡΥΘΜΙΖΕΤΑΙ ΑΠΟ ΚΥΚΛΙΚΑ ΝΟΥΚΛΕΟΤΙΔΙΑ (DNRPK, CYCLIC NUCLETIDE-REGULATED PROTEIN KINASE)

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

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&rsquo; 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

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

Temporal Association of Protamine 1 with the Inner Nuclear Membrane Protein Lamin B Receptor during Spermiogenesis

International audienceDuring mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH(2)-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32

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

International audienc