Thomas Giannakouros

Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation‐dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub‐set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross‐link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB‐binding protein, revealing a new mechanism for anchoring domains of under‐acetylated chromatin to the inner nuclear membrane.

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.

Functional potential of P2P-R: a role in the cell cycle and cell differentiation related to its interactions with proteins that bind to matrix associated regions of DNA?

P2P‐R is the alternately spliced product of the P2P‐R/PACT gene in that P2P‐R lacks one exon encoding 34 amino acids. The 250 kDa P2P‐R protein is the predominate product expressed in multiple murine cell lines. It is a highly basic protein that contains multiple domains including an N‐terminal RING type zinc finger, a proline rich domain, an RS region, and a C‐terminal lysine‐rich domain. P2P‐R binds the p53 and the Rb1 tumor suppressors and is phosphorylated by the cdc2 and SRPK1a protein kinases. P2P‐R also interacts with scaffold attachment factor‐B (SAF‐B), a well characterized MARs (for matrix attachment regions) binding factor, and may interact with nucleolin, another MARs binding factor. In addition, P2P‐R binds single strand DNA (ssDNA). The expression of P2P‐R is regulated by differentiation and cell cycle events. P2P‐R mRNA is markedly repressed during differentiation, whereas immunoreactive P2P‐R protein levels are >10‐fold higher in mitotic than in G0 cells. The localization of P2P‐R also is modulated during the cell cycle. During interphase, P2P‐R is present primarily in nucleoli and nuclear speckles whereas during mitosis, P2P‐R associates with the periphery of chromosomes. Overexpression of near full length P2P‐R induces mitotic arrest in prometaphase and mitotic apoptosis, and overexpression of selected P2P‐R segments also can promote apoptosis. This compendium of data supports the possibility that P2P‐R may form complexes with the Rb1 and/or p53 tumor suppressors and MARs‐related factors, in a cell cycle and cell differentiation‐dependent manner, to influence gene transcription/expression and nuclear organization. J. Cell. Biochem. 90: 6–12, 2003.

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.

Concentration-dependent effects of natural polyamines on peptide chain initiation and elongation in a cell-free system of protein synthesis

Spermidine and spermine at submillimolar concentrations stimulate the rate of incorporation of amino acid into protein in a cell-free system, directed either by endogenous or exogenous mRNA (TMV, globin). The stimulatory effects of these polyamines are exerted at both the stages of initiation and elogation and are more pronounced in the case of TMV or globin mRNA, amounting to approximately 2.3-fold stimulation over the polyamine-free system. The number of polysomes and the polysome-associated radioactivity increase approximately 2-fold in the presence of spermine. Synthesis of large polypeptides is a characteristic feature of the stimulatory event. However, elevated concentrations of spermidine and spermine strongly inhibit amino acid incorporation into protein. Inhibition is manifest at the stage of peptide elongation. In the case of endogenous mRNA the addition of an excess of polyamines results in a non uniform inhibition of amino acid incorporation. A most interesting finding is that, with increasing concentrations of polyamines, the intensity of four bands with Mr values of 63000, 44000, 15500 and 12500 respectively, increases or leastwise remains constant while others fade, indicading differential translation of proteins in the presence of polyamines.

Protein kinase CK2 phosphorylates and activates the SR protein-specific kinase 1.

The serine/arginine subfamily of protein kinases has been conserved throughout evolution and its members are thought to play important roles in the regulation of multiple cellular processes. Mammalian SRPK1 has been considered as a constitutively active kinase that is predominantly expressed in testis. In the present study, recombinant GST-SRPK1 was used as substrate to identify potential protein kinase(s) in testis extracts, involved in phosphorylating and thereby regulating the activity of this enzyme. Using a panel of chromatography media, inhibition by heparin, immunoblot analysis, and phosphopeptide mapping, CK2 was determined to be the major kinase that phosphorylates SRPK1. Phosphorylation of SRPK1 by CK2 occurred mainly at Ser(51) and Ser(555) in vitro, and resulted in approximately 6-fold activation of the enzyme. These findings suggest that SRPK1 may be an important cellular target for CK2 action.

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.

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.