Biomedical subjects

Thomas Küntziger

Publications and source records attributed to Thomas Küntziger.

6 recordsLinked to original sources

PubMed2006

The KH-Tudor domain of a-kinase anchoring protein 149 mediates RNA-dependent self-association.

A-Kinase anchoring proteins (AKAPs) control the subcellular localization and temporal specificity of protein phosphorylation mediated by cAMP-dependent protein kinase. AKAP149 (AKAP1) is found in mitochondria and in the endoplasmic reticulum-nuclear envelope network where it anchors protein kinases, phosphatases, and a phosphodiesterase. AKAP149 harbors in its COOH-terminal part one KH and one Tudor domain, both known to be involved in RNA binding. We investigated the properties of the COOH-terminal domain of AKAP149. We show here that AKAP149 is a self-associating protein with RNA binding features. The KH domain of AKAP149 is sufficient for self-association in a RNA-dependent manner. The Tudor domain is not necessary for self-association, but it is required together with the KH domain for targeting to well-defined nuclear foci. These foci are spatially closely related to nucleolar subcompartments. We also show that the KH-Tudor-containing domain of AKAP149 binds RNA in vitro and in RNA coprecipitation experiments. AKAP149 emerges as a scaffolding protein involved in the integration of intracellular signals and possibly in RNA metabolism.

A Kinase Anchor Proteins↗

PubMed2006

Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149.

Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1 remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking the RVXF PP1-binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing the RVXF motif and five flanking serines, either wild type (wt) or bearing S-->A or S-->D mutations, specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149. Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunoprecipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities. Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro; however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149. AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be involved in the integration of intracellular signals that converge at the nuclear envelope.

A Kinase Anchor Proteins↗

PubMed2006

In vitro reprogramming of nuclei and cells.

Directly turning a somatic cell type into another would be beneficial for producing replacement cells for therapeutic purposes. To this end, novel cell reprogramming strategies are being developed. We describe here methods for functionally reprogramming a somatic cell using an extract derived from another somatic cell type. The procedure involves reversible permeabilization of 293T fibroblasts, incubation of the permeabilized cells in a nuclear and cytoplasmic extract of T-cells, resealing of the "reprogrammed" cells, and culture for assessment of reprogramming. Reprogramming has been evidenced by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodeling complex, changes in chromatin composition, activation of lymphoid cell-specific genes, and expression of T-cell-specific surface molecules. The system is likely to constitute a powerful tool to examine the processes of nuclear reprogramming, at least as they occur in vitro.

Bacterial Proteins↗

PubMed2005

PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner.

PP1 (protein phosphatase-1) is a serine/threonine phosphatase involved in mitosis exit and chromosome decondensation. In the present study, we characterize the subcellular and subnuclear localization of PNUTS (PP1 nuclear targeting subunit), a nuclear regulatory subunit of PP1, and report a stimulatory role of PNUTS in the decondensation of prometaphase chromosomes in two in vitro systems. In interphase, PNUTS co-fractionates, together with a fraction of nuclear PP1, primarily with micrococcal nuclease-soluble chromatin. Immunofluorescence analysis shows that PNUTS is targeted to the reforming nuclei in telophase following the assembly of nuclear membranes and concomitantly with chromatin decondensation. In interphase cytosolic extract, ATP-dependent decondensation of prometaphase chromosomes is blocked by PP1-specific inhibitors. In contrast, a recombinant PNUTS(309-691) fragment accelerates chromosome decondensation. This decondensation-promoting activity requires the consensus RVXF PP1-binding motif of PNUTS, whereas a secondary, inhibitory PP1-binding site is dispensable. In a defined buffer system, PNUTS(309-691) also elicits decondensation in an exogenous PP1-dependent manner and, as in the cytosolic extract, a W401A (Thr401-->Ala) mutation that destroys PP1 binding abolishes this activity. The results illustrate an involvement of the PNUTS:PP1 holoenzyme in chromosome decondensation in vitro and argue that PNUTS functions as a PP1-targeting subunit in this process. We hypothesize that targeting of PNUTS to reforming nuclei in telophase may be a part of a signalling event promoting chromatin decondensation as cells re-enter interphase.

Cell Nucleus↗

PubMed2005

Long-term in vitro, cell-type-specific genome-wide reprogramming of gene expression.

We demonstrate a cell extract-based, genome-wide and heritable reprogramming of gene expression in vitro. Kidney epithelial 293T cells have previously been shown to take on T cell properties following a brief treatment with an extract of Jurkat T cells. We show here that 293T cells exposed for 1 h to a Jurkat cell extract undergo genome-wide, target cell-type-specific and long-lasting transcriptional changes. Microarray analyses indicate that on any given week after extract treatment, approximately 2500 genes are upregulated >3-fold, of which approximately 900 are also expressed in Jurkat cells. Concomitantly, approximately 1500 genes are downregulated or repressed, of which approximately 500 are also downregulated in Jurkat cells. Gene expression changes persist for over 30 passages ( approximately 80 population doublings) in culture. Target cell-type specificity of these changes is shown by the lack of activation or repression of Jurkat-specific genes by extracts of 293T cells or carcinoma cells. Quantitative RT-PCR analysis confirms the long-term transcriptional activation of genes involved in key T cell functions. Additionally, growth of cells in suspended aggregates, expression of CD3 and CD28 T cell surface markers, and interleukin-2 secretion by 293T cells treated with extract of adult peripheral blood T cells illustrate a functional nuclear reprogramming. Therefore, target cell-type-specific and heritable changes in gene expression, and alterations in cell function, can be promoted by extracts derived from transformed cells as well as from adult primary cells.

Adult↗

PubMed2002

Reprogrammed gene expression in a somatic cell-free extract.

We have developed a somatic cell-free system that remodels chromatin and activates gene expression in heterologous differentiated nuclei. Extracts of stimulated human T cells elicit chromatin binding of transcriptional activators of the interleukin-2 (IL-2) gene, anchoring and activity of a chromatin-remodeling complex and hyperacetylation of the IL-2 promoter in purified exogenous resting T-cell nuclei. The normally repressed IL-2 gene is transcribed in nuclei from quiescent human T cells and from various non-T-cell lines. This demonstrates that somatic cell extracts can be used to reprogram gene expression in differentiated nuclei. In vitro reprogramming may be useful for investigating regulation of gene expression and for producing replacement cells for the treatment of a wide variety of diseases.

Acetylation↗