Bjorn Skalhegg - Academia.edu (original) (raw)

Papers by Bjorn Skalhegg

Journal of cell science, 2001

Protein kinase A regulatory subunit RIIalpha is tightly bound to centrosomal structures during in... more Protein kinase A regulatory subunit RIIalpha is tightly bound to centrosomal structures during interphase through interaction with the A-kinase anchoring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34(cdc2) kinase (CDK1) has been shown to phosphorylate RIIalpha on T54 and this has been proposed to alter the subcellular localization of RIIalpha. We have made stable transfectants from an RIIalpha-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RIIalpha (RIIalpha(T54E)). When expressed, RIIalpha detaches from centrosomes at mitosis and dissociates from its centrosomal location in purified nucleus-centrosome complexes by incubation with CDK1 in vitro. By contrast, centrosomal RIIalpha(T54E) is not redistributed at mitosis, remains mostly associated with centrosomes during all phases of the cell cycle and cannot be solubilized by CDK1 in vitro. Furthermore, RIIalpha is solubilized from particular cell fraction...

European Journal of Biochemistry, 2001

European Journal of Biochemistry, 1994

Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of CAMP-d... more Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of CAMP-dependent protein kinases (PKA) were transcribed and translated in a cell-free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all

The FASEB Journal, 2007

In this study, we provide novel insight into the mechanism of how ERK2 can be sorted to different... more In this study, we provide novel insight into the mechanism of how ERK2 can be sorted to different intracellular compartments and thereby mediate different responses. MEK1-activated ERK2 accumulated in the nucleus and induced proliferation. Conversely, MEK2-activated ERK2 was retained in the cytoplasm and allowed survival. Localization was a determinant for ERK2 functions since MEK1 switched from providing proliferation to be a mediator of survival when ERK2 was routed to the cytoplasm by the attachment of a nuclear export site. MEK1-mediated ERK2 nuclear translocation and proliferation were shown to depend on phosphorylation of S298 and T292 sites in the MEK1 proline-rich domain. These sites are phosphorylated on cellular adhesion in MEK1 but not MEK2. Whereas p21-activated kinase phosphorylates S298 and thus enhances the MEK1-ERK2 association, ERK2 phosphorylates T292, leading to release of active ERK2 from MEK1. On the basis of these results, we propose that the requirement of adhesion for cells to proliferate in response to growth factors, in part, may be explained by the MEK1 S298/T292 control of ERK2 nuclear translocation. In addition, we suggest that ERK2 intracellular localization determines whether growth factors mediate proliferation or survival and that the sorting occurs in an adhesion-dependent manner.-Skarpen, E., Flinder, L. I., Rosseland, C. M., Ørstavik, S., Wierød, L., Pedersen Oksvold, M., Skålhegg, B. S., Huitfeldt, H. S. MEK1 and MEK2 regulate distinct functions by sorting ERK2 to different intracellular compartments. FASEB J. 22, 466 -476 (2008)

Science, 1994

Selective activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pro... more Selective activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase type I (cAKI), but not type II, is sufficient to mediate inhibition of T cell replication induced through the antigen-specific T cell receptor-CD3 (TCR-CD3) complex. Immunocytochemistry and immunoprecipitation studies of the molecular mechanism by which cAKI inhibits TCR-CD3-dependent T cell replication demonstrated that regulatory subunit I alpha, along with its associated kinase activity, translocated to and interacted with the TCR-CD3 complex during T cell activation and capping. Regulatory subunit II alpha did not. When stimulated by cAMP, the cAKI localized to the TCR-CD3 complex may release kinase activity that, through phosphorylation, might uncouple the TCR-CD3 complex from intracellular signaling systems.

Scandinavian Journal of Immunology, 1994

We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cA... more We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cAKI, RI alpha 2-C beta 2) mediates the inhibitory effects of cAMP on T-cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3- and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti-CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replication. Combined stimulation with anti-CD3 and anti-CD28, which gives a synergistic response in T-cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anti-CD3 and anti-CD28, the inhibitory effect of cAMP on both T-cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T-cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP).

… of reproduction and …, 1993

This study was undertaken to examine the expression and cellular location of the various cAMP-dep... more This study was undertaken to examine the expression and cellular location of the various cAMP-dependent protein kinase (PKA) subunits in different testicular cell types, using cDNA probes, isoenzyme-specific antibodies and activity measurements. Amounts of mRNA and protein were examined in cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), Leydig cell tumours as well as whole testes from rats of various ages. In Sertoli cells, there was a good correlation between the amount of mRNA and the respective immunoreactive proteins. In other types of cell, such as germ cells and Leydig tumour cells, this was not always the case. Large amounts of RII beta mRNA were found in Leydig tumour cells, whereas the amount of immunoreactive protein was low. Furthermore, large amounts of small-sized, germ cell-specific mRNAs for RI alpha (1.7 kb) and RII alpha (2.2 kb) were also found in the developing rat testis after 30 to 40 days of age, but the large amounts of mRNA were only partially reflected at the protein level. Pachytene spermatocytes and round spermatids were practically devoid of both RII alpha and RII beta protein. During spermatid differentiation, there was a decrease in RI alpha and an increase in RII alpha protein. Cell specific distribution of the various PKA subunits in testicular cell types is described. In some types of cell, discrepancies between mRNA and protein were demonstrated, which clearly suggest cell specific differences in translational efficiencies for some of these mRNAs, particularly the small-sized mRNAs for RI alpha and RII alpha in meiotic and post-meiotic germ cells.

Proceedings of the National Academy of Sciences, 1996

Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and th... more Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Ca and C,B, and the Cf3 gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous

Molecular and Cellular Biology, 2002

HA95, a nuclear protein homologous to AKAP95, has been identified in immune precipitates of the E... more HA95, a nuclear protein homologous to AKAP95, has been identified in immune precipitates of the Epstein-Barr virus (EBV) coactivating nuclear protein EBNA-LP from EBV-transformed lymphoblastoid cells (LCLs). We now find that HA95 and EBNA-LP are highly associated in LCLs and in B-lymphoma cells where EBNA-LP is expressed by gene transfer. Binding was also evident in yeast two-hybrid assays. HA95 binds to the EBNA-LP repeat domain that is the principal coactivator of transcription. EBNA-LP localizes with HA95 and causes HA95 to partially relocalize with EBNA-LP in promyelocytic leukemia nuclear bodies. Protein kinase A catalytic subunit ␣ (PKAcs␣) is significantly associated with HA95 in the presence or absence of EBNA-LP.

Journal of Virology, 2001

EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with F... more EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with Flag epitope-tagged EBNA-LP (FLP) from lymphoblasts in which FLP was stably expressed. The association of EBNA-LP with Hsp70 (72/73) was confirmed, and sequences of DNA-PK catalytic subunit (DNA-PKcs), HA95, Hsp27, prolyl 4-hydroxylase ␣-1 subunit, ␣-tubulin, and ␤-tubulin were identified. The fraction of total cellular HA95 that associated with FLP was very high, while progressively lower fractions of the total DNA-PKcs, Hsp70, Hsp 27, ␣-tubulin, and ␤-tubulin specifically associated with EBNA-LP as determined by immunoblotting with antibodies to these proteins.

In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor- CD3 ... more In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor- CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 z chain after T cell activation, and

Journal of Biological Chemistry, 2009

Protein kinase A (PKA) is targeted to distinct subcellular localizations by specific protein kina... more Protein kinase A (PKA) is targeted to distinct subcellular localizations by specific protein kinase A anchoring proteins (AKAPs). AKAPs are divided into subclasses based on their ability to bind type I or type II PKA or both. Dual-specificity AKAPs were recently reported to have an additional PKA binding determinant called the RI specifier region. A bioinformatic search with the consensus RI specifier region identified a novel AKAP, the splicing factor arginine/serine-rich 17A (SFRS17A). Here, we show by a variety of protein interaction assays that SFRS17A binds both type I and type II PKA in vitro and inside cells, demonstrating that SFRS17A is a dual-specific AKAP. Moreover, immunofluorescence experiments show that SFRS17A colocalizes with the catalytic subunit of PKA as well as the splicing factor SC35 in splicing factor compartments. Using the E1A minigene splicing assay, we found that expression of wild type SFRS17A conferred regulation of E1A alternative splicing, whereas the mutant SFRS17A, which is unable to bind PKA, did not. Our data suggest that SFRS17A is an AKAP involved in regulation of pre-mRNA splicing possibly by docking a pool of PKA in splicing factor compartments.

Experimental cell …, 1993

European Journal of Endocrinology, 1996

Disciplines in medicine and the distinction between them develop and change with progress in the ... more Disciplines in medicine and the distinction between them develop and change with progress in the field, both scientifically and in terms of clinical practice.

European Journal of Clinical Investigation, 2004

Highly active antiretroviral therapy (HAART) often leads to a dramatic improvement in clinical, v... more Highly active antiretroviral therapy (HAART) often leads to a dramatic improvement in clinical, viral and immunologic parameters in HIV-infected individuals. However, the emergence of long-term side-effects of HAART and in particular dylipidaemia is increasingly reported. Based on the potential lipid-lowering and immunomodulatory properties of tetradecylthioacetic acid (TTA) we examined whether TTA in combination with dietary intervention could modify lipid levels in peripheral blood in HIV-infected patients on HAART. Ten HIV-infected patients on protease inhibitor-based HAART with hyperlipidaemia followed a cholesterol-lowering diet throughout the study period (8 weeks). During the last 4 weeks of the study all patients received TTA (1 g qd) in addition to the cholesterol-lowering diet. Our main and novel findings were: (i) TTA in combination with dietary intervention reduces total cholesterol, LDL cholesterol, triglycerides and LDL/HDL cholesterol in these patients, and a particularly suppressing effect was observed during the TTA phase regarding total cholesterol. (ii) During the TTA phase, the cholesterol-lowering effect was accompanied by a significant reduction in plasma levels of tumour necrosis factor alpha. (iii) Our studies in peripheral blood mononuclear cells from these patients and in the liver from wild-type mice receiving TTA suggest that the hypolipidaemic effects of TTA may involve up-regulation of scavenger and LDL-receptor expression. Although few patients were studied, the present pilot study suggests that TTA combined with dietary intervention could be an interesting therapeutic approach in HIV-infected patients on HAART, potentially resulting in both hypolipidaemic and anti-inflammatory effects.

Endocrinology, 1991

Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of c... more Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels.

Cytogenetic and Genome Research, 2000

... Paper. Molecular cloning, chromosomal localization, and expression of the murine SALL1 orthol... more ... Paper. Molecular cloning, chromosomal localization, and expression of the murine SALL1 ortholog Sall1 A. Buck, L. Archangelo, C. Dixkens, J. Kohlhase Institut für Humangenetik ... For first strand cDNA synthesis, primer MS3R3 (5′ GGAGGCTGGACTTTCATTG 3′) was used. ...

Current Drug Targets, 2005

ABSTRACT In several cases of immunodeficiency and autoimmunity, the dysfunctional immune system i... more ABSTRACT In several cases of immunodeficiency and autoimmunity, the dysfunctional immune system is associated with either hypo- or hyperactive T and B cells. In autoimmune conditions such as systemic lupus erythematosus (SLE) and immunodeficiencies such as acquired immunodeficiency syndrome (AIDS), it has been demonstrated that the regulatory effect of the signaling pathway of cyclic 3', 5' adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) is abrogated. PKA is well-known as a key regulator of immune responses in that it inhibits both early and late phases of antigen induced T and B cell activation. Here we will discuss a potential useful strategy for therapeutic interventions of dysfunctional T cells associated with SLE and HIV by modulation of the cAMP-PKA pathway. Therefore, we will describe the components and architecture of the cAMP-PKA signaling pathway in T cells in order to point out one or several steps which potentially may serve as targets for therapeutic intervention.

Anatomy and Embryology, 2001

To identify intracellular signalling pathways that transduce muscle electrical activity, we have ... more To identify intracellular signalling pathways that transduce muscle electrical activity, we have investigated the Protein Kinase A (PKA) pathway in fast and slow skeletal muscle. The slow soleus muscle (SOL) displayed approximately twice as much PKA catalytic activity and cAMP-binding compared to the fast Extensor Digitorum Longus (EDL) muscle. These results were confirmed by Western blot analysis using antibodies directed against the catalytic or regulatory subunits of PKA. PKA subunits were concentrated at the neuromuscular junction in innervated and denervated muscle fibers demonstrating that PKA is expressed post-synaptically. In addition, we also detected PKA subunits outside the junctional area, suggesting that PKA functions outside of the synaptic regions. Following denervation, levels of cyclic AMP, PKA C activity, R cAMP-binding and RI alpha protein levels increased significantly in the SOL, in contrast to the EDL where only elevated levels of RI alpha protein were observed. These observations demonstrate that PKA levels in skeletal muscle are subject to control at several levels and suggest that some of the differences may be in the pattern of electrical activity that motoneurons impose on the SOL and EDL.