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  • 1.
    Antonsson, Åsa
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hughes, Kate
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Edin, Sofia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Regulation of c-Rel Nuclear Localization by Binding of Ca2+/Calmodulin2003Ingår i: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, nr 4, s. 1418-1427Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The NF-κB/Rel family of transcription factors participates in the control of a wide array of genes, including genes involved in embryonic development and regulation of immune, inflammation, and stress responses. In most cells, inhibitory IκB proteins sequester NF-κB/Rel in the cytoplasm. Cellular stimulation results in the degradation of IκB and modification of NF-κB/Rel proteins, allowing NF-κB/Rel to translocate to the nucleus and act on its target genes. Calmodulin (CaM) is a highly conserved, ubiquitously expressed Ca2+ binding protein that serves as a key mediator of intracellular Ca2+ signals. Here we report that two members of the NF-κB/Rel family, c-Rel and RelA, interact directly with Ca2+-loaded CaM. The interaction with CaM is greatly enhanced by cell stimulation, and this enhancement is blocked by addition of IκB. c-Rel and RelA interact with CaM through a similar sequence near the nuclear localization signal. Compared to the wild-type protein, CaM binding-deficient mutants of c-Rel exhibit increases in both nuclear accumulation and transcriptional activity on the interleukin 2 and granulocyte macrophage colony-stimulating factor promoters in the presence of a Ca2+ signal. Conversely, for RelA neither nuclear accumulation nor transcriptional activity on these promoters is increased by mutation of the sequence interacting with CaM. Our results suggest that CaM binds c-Rel and RelA after their release from IκB and can inhibit nuclear import of c-Rel while letting RelA translocate to the nucleus and act on its target genes. CaM can therefore differentially regulate the activation of NF-κB/Rel proteins following stimulation.

  • 2.
    Bergqvist, Ingela
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Eriksson, Maria
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Saarikettu, Juha
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Eriksson, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Corneliussen, Brit
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development2000Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 30, nr 10, s. 2857-2863Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    E2A, HEB and E2-2 genes encode a group of basic helix-loop-helix (bHLH) transcription factors that are structurally and functionally similar. Deletion of the genes encoding either of these proteins leads to early lethality and a block in B lymphocyte development. Evidence for a function in T lymphocyte development has, however, only been reported for E2A and HEB. To further elucidate the role of E2-2 at developmental stages that have proven difficult to study due to the early lethality phenotype of mice defective in E2-2, we generated and analyzed mice conditionally mutated in the E2-2 gene. These mice are mosaic with respect to E2-2 expression, consisting of cells with either one functional and one null mutated E2-2 allele or two null mutated alleles. Using this experimental model, we find that cells with a homozygous null mutated E2-2 gene are under-represented in B lymphocyte as well as T lymphocyte cell lineages as compared to other hematopoietic or non-hematopoietic cell lineages. Our data suggests that E2-2 deficiency leads to a partial block in both B and T lymphocyte development. The block in T cell development appears to occur at an early stage in differentiation, since skewing in the mosaicism is observed already in CD4+8+ double-positive thymocytes.

  • 3.
    Bäckström, Stefan
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Huang, Shenghua
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Magnus
    Kemi.
    Xie, X Q
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Härd, Torleif
    Grundström, Thomas
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Crystallization and preliminary studies of the DNA-binding runt domain of AML1.2001Ingår i: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 57, nr Pt 2, s. 269-71Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

  • 4.
    Bäckström, Stefan
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Magnus
    Kemi.
    Grundström, Christine
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Härd, Torleif
    Grundström, Thomas
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding.2002Ingår i: J Mol Biol, ISSN 0022-2836, Vol. 322, nr 2, s. 259-72Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

  • 5.
    Edin, Sofia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Oruganti, Sreenivasa Rao
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Interaction of calmodulin with Bcl10 modulates NF-kappaB activation.2010Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 47, nr 11-12, s. 2057-2064Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Calcium signals resulting from antigen receptor activation are important in determining the responses of a T or B lymphocyte to an antigen. Calmodulin (CaM), a multi-functional sensor of intracellular calcium (Ca(2+)) signals in cells, is required in the pathway from the T cell receptor (TCR) to activation of the key transcription factor NF-kappaB. Here we searched for a partner in direct interaction with CaM in the pathway, and found that CaM interacts specifically with the signaling adaptor Bcl10. The binding is Ca(2+) dependent and of high affinity, with a K(d) of approximately 160 nM. Proximity of CaM and Bcl10 in vivo is induced by increases in the intracellular Ca(2+) level. The interaction is localized to the CARD domain of Bcl10, which interacts with the CARD domain of the upstream signaling partner Carma1. Binding of CaM to Bcl10 is shown to inhibit the ability of Bcl10 to interact with Carma1, an interaction that is required for signaling from the TCR to NF-kappaB. Furthermore, a mutant of Bcl10 with reduced binding to CaM shows increased activation of an NF-kappaB reporter, which is further enhanced by activating stimuli. We propose a novel mechanism whereby the Ca(2+) sensor CaM regulates T cell responses to antigens by binding to Bcl10, thereby modulating its interaction with Carma1 and subsequent activation of NF-kappaB.

  • 6.
    Flodbring Larsson, Per
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Karlsson, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Division of Experimental Cancer Research, Department of Translational Medicine, Clinical Research Centre, Lund University, Malmö, Sweden.
    Sarwar, Martuza
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Miftakhova, Regina R.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wang, Tianyan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Khaja, Azharuddin Sajid Syed
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Semenas, Julius
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Chen, Sa
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hedblom, Andreas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Division of Experimental Cancer Research, Department of Translational Medicine, Clinical Research Centre, Lund University, Malmö, Sweden.
    Amjad, Ali
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ekström-Holka, Kristina
    Simoulis, Athanasios
    Kumar, Anjani
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gjörloff Wingren, Anette
    Robinson, Brian
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Mongan, Nigel P.
    Heery, David M.
    Öhlund, Daniel
    Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper.
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ødum, Niels
    Persson, Jenny L.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Division of Experimental Cancer Research, Department of Translational Medicine, Clinical Research Centre, Lund University, Malmö, Sweden; Department of Biomedical Sciences, Malmö University, Malmö, Sweden.
    FcγRIIIa receptor interacts with androgen receptor and PIP5K1α to promote growth and metastasis of prostate cancer2022Ingår i: Molecular Oncology, ISSN 1574-7891, E-ISSN 1878-0261Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Low-affinity immunoglobulin gamma Fc region receptor III-A (FcγRIIIa) is a cell surface protein that belongs to a family of Fc receptors that facilitate the protective function of the immune system against pathogens. However, the role of FcγRIIIa in prostate cancer (PCa) progression remained unknown. In this study, we found that FcγRIIIa expression was present in PCa cells and its level was significantly higher in metastatic lesions than in primary tumors from the PCa cohort (P = 0.006). PCa patients with an elevated level of FcγRIIIa expression had poorer biochemical recurrence (BCR)-free survival compared with those with lower FcγRIIIa expression, suggesting that FcγRIIIa is of clinical importance in PCa. We demonstrated that overexpression of FcγRIIIa increased the proliferative ability of PCa cell line C4-2 cells, which was accompanied by the upregulation of androgen receptor (AR) and phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), which are the key players in controlling PCa progression. Conversely, targeted inhibition of FcγRIIIa via siRNA-mediated knockdown or using its inhibitory antibody suppressed growth of xenograft PC-3 and PC-3M prostate tumors and reduced distant metastasis in xenograft mouse models. We further showed that elevated expression of AR enhanced FcγRIIIa expression, whereas inhibition of AR activity using enzalutamide led to a significant downregulation of FcγRIIIa protein expression. Similarly, inhibition of PIP5K1α decreased FcγRIIIa expression in PCa cells. FcγRIIIa physically interacted with PIP5K1α and AR via formation of protein-protein complexes, suggesting that FcγRIIIa is functionally associated with AR and PIP5K1α in PCa cells. Our study identified FcγRIIIa as an important factor in promoting PCa growth and invasion. Further, the elevated activation of FcγRIII and AR and PIP5K1α pathways may cooperatively promote PCa growth and invasion. Thus, FcγRIIIa may serve as a potential new target for improved treatment of metastatic and castration-resistant PCa.

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  • 7.
    Grundström, Christine
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The transcription factor E2A can bind to and cleave single-stranded immunoglobulin heavy chain locus DNA2023Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 153, s. 51-59Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Class switch recombination (CSR) changes the constant region of the immunoglobulin heavy chain (IgH), and somatic hypermutation (SH) introduces point mutations in the variable regions of the antibody genes. Both these processes that optimize antibody responses of B lymphocytes are initiated by the enzyme Activation Induced cytidine Deaminase (AID). Here we have searched for CSR or SH coupled activities of the transcription factor E2A, since E2A is in a complex with AID and the transcription factors PAX5, ETS1 and IRF4 on key sequences of the Igh locus in B lymphocytes activated to CSR and SH. We report that E2A in contrast to other described transcription factors binds sequence specifically also to single-stranded DNA. The binding of E2A to single-stranded DNA has a strong sequence preference for one strand of a site in the intronic enhancer of the Igh locus. Furthermore, E2A was also found to cleave single-stranded DNA. The sequence profile of substrates cleaved by E2A is coupled to the sequences of substrates and products of AID, suggesting that E2A has a role not only in targeting of AID to switch regions and SH parts of antibody genes but also in cleavage of DNA at these sites.

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  • 8.
    Grundström, Christine
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kumar, Anjani
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Priya, Anshu
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Negi, Neema
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    ETS1 and PAX5 transcription factors recruit AID to Igh DNA2018Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 48, nr 10, s. 1687-1697Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    B lymphocytes optimize antibody responses by class switch recombination (CSR), which changes the expressed constant region exon of the immunoglobulin heavy chain (IgH), and by somatic hypermutation (SH) that introduces point mutations in the variable regions of the antibody genes. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates both these antibody diversification processes by deaminating cytosine to uracil. Here we asked the question if transcription factors can mediate the specific targeting of the antibody diversification by recruiting AID. We have recently reported that AID is together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus. Here we report that also ETS1 is together with AID in this complex on key sequences of the Igh locus in splenic B cells of mice. Furthermore, we show that both ETS1 and PAX5 can directly recruit AID to DNA sequences from the Igh locus with the specific binding site for the transcription factor. Taken together, our findings support the notion of a targeting mechanism for the selective diversification of antibody genes with limited genome wide mutagenesis by recruitment of AID by PAX5 and ETS1 in a transcription factor complex.

  • 9.
    Grundström, Thomas
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hauser, Jannek
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kumar, Anjani
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Priya, Anshu
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kumar, Ramesh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Regulation of diversification and affinity maturation of antibodies2016Ingår i: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 38, s. S43-S43Artikel i tidskrift (Övrigt vetenskapligt)
  • 10.
    Grundström, Thomas
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hauser, Jannek
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kumar, Ramesh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ahmed, Tanzeel
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Mechanisms controlling diversification and affinity maturation of antibodies2015Ingår i: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 36, s. S43-S43Artikel i tidskrift (Övrigt vetenskapligt)
  • 11.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Allelic Exclusion of IgH through Inhibition of E2A in a VDJ Recombination Complex2014Ingår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 192, nr 5, s. 2460-2470Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A key feature of the immune system is the paradigm that one lymphocyte has only one Ag specificity that can be selected for or against. This requires that only one of the alleles of genes for AgR chains is made functional. However, the molecular mechanism of this allelic exclusion has been an enigma. In this study, we show that B lymphocytes with E2A that cannot be inhibited by calmodulin are dramatically defective in allelic exclusion of the IgH locus. Furthermore, we provide data supporting that E2A, PAX5, and the RAGs are in a VDJ recombination complex bound to key sequences on the Igh gene. We show that pre-BCR activation releases the VDJ recombination complex through calmodulin binding to E2A. We also show that pre-BCR signaling downregulates several components of the recombination machinery, including RAG1, RAG2, and PAX5, through calmodulin inhibition of E2A.

  • 12.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kumar, Ramesh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Regulated localization of an AID complex with E2A, PAX5 and IRF4 at the Igh locus2016Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 80, s. 78-90Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates somatic hypermutation (SH) and class switch recombination (CSR) by deaminating cytosine to uracil. The targeting of AID and therefore SH and CSR to Ig genes is a central process of the immune system, but the trans-acting factors mediating the specific targeting have remained elusive. Here we show that defective calmodulin inhibition of the transcription factor E2A after activation of the B cell receptor (BCR) leads to reduced BCR, IL4 plus CD40 ligand stimulated CSR to IgE and instead CSR to other Ig classes. AID that initiates CSR is shown to be in a complex with the transcription factors E2A, PAX5 and IRF4 on key sequences of the Igh locus. Calmodulin shows proximity with each of them after BCR stimulation. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks these reductions. AID binds directly to the bHLH domain of E2A and to the PD domain of PAX5. E2A, AID, PAX5 and IRF4 are components of a CSR complex that is redistributed on the Igh locus by BCR signaling through calmodulin binding.

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  • 13.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kumar, Ramesh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Signal regulated localisation of a mutagenic protein complex at the Igh locus2015Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, s. 13-13Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Our system to produce antibodies is critical for our survival against numerous infections, but it causes also many tumors. B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates these processes by deaminating cytosine to uracil. How somatic hypermutation (SH) and class switch recombination (CSR) are targeted is key to understanding the defect DNA integrity in lymphomas and also in other tumors where inflammatory signals aberrantly induces AID. The trans-acting factors mediating specific targeting of AID and thereby SH and CSR have remained elusive. Here we show that mutant E2A with defect inhibition by the Ca2+sensor protein calmodulin results in reduced B cell receptor- (BCR-), IL4-plus CD40 ligand-stimulated CSR to IgE and instead aberrant CSR. AID is shown to be together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus in activated mouse splenic B cells. Calmodulin shows proximity with each of them after BCR stimulation. Direct protein-protein interactions enable formation of the complexes. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks reduction of binding to these target sites and increases binding to other target sites. Thus, E2A, AID, PAX5 and IRF4 are components of a CSR and SH complex that is redistributed on the IgH gene by BCR signaling through calmodulin binding.

  • 14.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Regulation of allelic exclusion of IgH by calmodulin inhibition of E2AManuskript (preprint) (Övrigt vetenskapligt)
  • 15.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kumar, Ramesh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wallenius, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ahmed, Tanzeel
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Regulation of diversification and affinity maturation of antibodies2014Ingår i: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 34, s. S50-S50Artikel i tidskrift (Övrigt vetenskapligt)
  • 16.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Saarikettu, Juha
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Calcium regulation of myogenesis by differential calmodulin inhibition of basic helix-loop-helix transcription factors2008Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 19, nr 6, s. 2509-2519Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors are critical regulators of skeletal muscle differentiation that function as heterodimers with ubiquitously expressed E-protein bHLH transcription factors. These heterodimers must compete successfully with homodimers of E12 and other E-proteins to enable myogenesis. Here, we show that E12 mutants resistant to Ca(2+)-loaded calmodulin (CaM) inhibit MyoD-initiated myogenic conversion of transfected fibroblasts. Ca(2+) channel blockers reduce, and Ca(2+) stimulation increases, transcription by coexpressed MyoD and wild-type E12 but not CaM-resistant mutant E12. Furthermore, CaM-resistant E12 gives lower MyoD binding and higher E12 binding to a MyoD-responsive promoter in vivo and cannot rescue myogenic differentiation that has been inhibited by siRNA against E12 and E47. Our data support the concept that Ca(2+)-loaded CaM enables myogenesis by inhibiting DNA binding of E-protein homodimers, thereby promoting occupancy of myogenic bHLH protein/E-protein heterodimers on promoters of myogenic target genes.

  • 17.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sveshnikova, Natalia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wallenius, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Baradaran, Sanna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Saarikettu, Juha
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins2008Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, nr 4, s. 1267-1272Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Upon encountering antigens, B-lymphocytes can adapt to produce a highly specific and potent antibody response. Somatic hypermutation, which introduces point mutations in the variable regions of antibody genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both somatic hypermutation and CSR. The mutagenic AID enzyme has to be tightly controlled. Here, we show that engagement of the membrane-bound antibodies of the B-cell receptor (BCR), which signals that good antibody affinity has been reached, inhibits AID gene expression and that calcium (Ca(2+)) signaling is essential for this inhibition. Moreover, we show that overexpression of the Ca(2+) sensor protein calmodulin inhibits AID gene expression, and that the transcription factor E2A is required for regulation of the AID gene by the BCR. E2A mutated in the binding site for calmodulin, and thus showing calmodulin-resistant DNA binding, makes AID expression resistant to the inhibition through BCR activation. Thus, BCR activation inhibits AID gene expression through Ca(2+)/calmodulin inhibition of E2A.

  • 18.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Verma-Gaur, Jiyoti
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Broad feedback inhibition of pre-B-cell receptor signaling components2013Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 54, nr 3-4, s. 247-253Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During B lymphocyte development, first immunoglobulin heavy chain gene segments and then immunoglobulin light chain gene segments are rearranged to create antibody diversity. Early in the development, expression of a pre-B-cell receptor (pre-BCR) that has membrane-bound Ig heavy chain protein associated with surrogate light chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces survival and clonal expansion to select cells with good heavy chains, but it also down-regulates transcription of the genes for the SLC proteins and CD19 and limits its own proliferative signaling. Here we have analyzed whether the down-regulation is limited to the SLC proteins and CD19, and we show that the pre-BCR of primary mouse pre-B-cells instead is subject to a broad feedback inhibition of pre-BCR signaling components. Activation of signaling leads to down-regulation of the receptor proteins, many co-receptors and proteins participating in signal pathways from the receptor. Thus the down-regulation of the pre-BCR is much broader than previously assumed. We also show that Ca2+/calmodulin inhibition of the transcription factor E2A is required for the feedback inhibition of the pre-BCR signaling proteins. (C) 2012 Elsevier Ltd. All rights reserved.

  • 19.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Verma-Gaur, Jiyoti
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wallenius, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Mechanisms controlling diversification and affinity maturation of antibodies2013Ingår i: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 32, s. S45-S45Artikel i tidskrift (Övrigt vetenskapligt)
  • 20.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Verma-Gaur, Jiyoti
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wallenius, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Initiation of antigen receptor-dependent differentiation into plasma cells by calmodulin inhibition of E2A2009Ingår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 183, nr 2, s. 1179-1187Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Differentiation of B lymphocytes into Ab-secreting plasmablasts and plasma cells is Ag driven. The interaction of Ag with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. However, not much is known about the coupling between BCR activation and the shift in transcription factor network from that of a B cell to that of ASC differentiation. Our genome-wide analysis shows that Ab-secreting cell differentiation of mouse B cells is induced by BCR activation through very fast regulatory events from the BCR. We identify activation of IFN regulatory factor-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression as immediate early events. Furthermore, the transcription factor E2A is required for the rapid key down-regulations after BCR activation, and the Ca(2+) sensor protein calmodulin has the corresponding regulatory effect as BCR activation. Moreover, mutants in the calmodulin binding site of E2A show that Ca(2+) signaling through calmodulin inhibition of E2A is essential for the rapid down-regulation of immediate early genes after BCR activation in initiation of plasma cell differentiation.

  • 21.
    Hauser, Jannek
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Verma-Gaur, Jiyoti
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wallenius, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Grundström, Thomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Mechanisms regulating diversification and affinity maturation of antibodies2012Ingår i: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 30, nr Suppl 1, s. S42-S42Artikel i tidskrift (Övrigt vetenskapligt)
  • 22.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Verma-Gaur, Jiyoti
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wallenius, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Regulatory mechanisms controlling diversification and affinity maturation of antibodies2011Konferensbidrag (Refereegranskat)
  • 23.
    Hauser, Jannek
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wallenius, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sveshnikova, Natalia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Saarikettu, Juha
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Calmodulin inhibition of E2A stops expression of surrogate light chains of the pre-B-cell receptor and CD192010Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 47, nr 5, s. 1031-1038Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To create antibody diversity, B lymphocyte development is characterized by the ordered rearrangement of first immunoglobulin (Ig) heavy chain gene segments and then Ig light-chain gene segments. Early in B-cell development, expression of a pre-B-cell receptor (pre-BCR) composed of membrane-bound Ig heavy chain protein associated with surrogate light-chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces clonal expansion, but it also turns off transcription of the genes for the SLC proteins lambda5 and VpreB, which limits this proliferation. Here we show that signaling from the pre-BCR rapidly down-regulates lambda5 and VpreB and also the co-receptor CD19 in primary pre-B-cells. We show that calcium (Ca(2+)) signaling is essential for this silencing of the SLC and CD19 genes. The SLC genes are activated by the E2A transcription factor, and we show that E2A is required for pre-BCR-mediated regulation of the genes. E2A mutated in its binding site for the Ca(2+) sensor protein calmodulin, and thus with calmodulin-resistant DNA binding, makes lambda5, VpreB and CD19 expression resistant to the inhibition following pre-BCR activation. Thus, Ca(2+) down-regulates SLC and CD19 gene expression upon pre-BCR activation through inhibition of E2A by Ca(2+)/calmodulin.

  • 24.
    Hermann, Stefan
    et al.
    Department of Biosciences, Karolinska institute, Novum, Huddinge, Sweden.
    Saarikettu, Juha
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Onions, Jacqueline
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hughes, Kate
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Calcium regulation of basic helix-loop-helix transcription factors1998Ingår i: Cell Calcium, ISSN 0143-4160, E-ISSN 1532-1991, Vol. 23, nr 2-3, s. 135-142Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The basic helix-loop-helix (bHLH) family of transcription factors is essential for numerous developmental and growth control processes. The regulation of bHLH proteins occurs at many levels, including tissue specific expression, differential oligomerization and DNA binding specificities, interaction with negatively acting HLH proteins and post-translational modifications. This review focuses on what is emerging as another level of bHLH protein regulation, calcium regulation through interaction with Ca2+ loaded calmodulin and S-100 proteins. The mechanism and implications of these Ca2+ regulated interactions are discussed.

  • 25.
    Hughes, Kate
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Antonsson, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Calmodulin dependence of NFκB activation1998Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 441, nr 1, s. 132-136Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The NF kappa B family of transcription factors is regulated by inhibitory I kappa B proteins. A diversity of stimuli leads to the phosphorylation and subsequent degradation of I kappa B, releasing NF kappa B to act on its target genes. Calmodulin (CaM) is a key regulator of numerous cellular processes and is the predominant intracellular receptor for Ca2+ signals. Here me report that several CaM antagonists inhibit the activation of NF kappa B, and that this is due to the prevention of inducible I kappa B phosphorylation. Our results suggest that CaM is involved in the phosphorylation of I kappa B, a finding that may help in elucidating the mechanism of this critical step of NF kappa B activation.

  • 26.
    Hughes, Kate
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Edin, Sofia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Antonsson, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Calmodulin-dependent Kinase II Mediates T Cell Receptor/CD3- and Phorbol Ester-induced Activation of IκB Kinase2001Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 38, s. 36008-36013Artikel i tidskrift (Refereegranskat)
  • 27.
    Hughes, Kate
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Saarikettu, Juha
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gene expression in transfected cells2002Ingår i: Calcium-Binding Protein Protocols: Volume 2: Methods and Techniques / [ed] Hans J. Vogel, Totowa, NJ: Humana Press, 2002, Vol. 173, s. 355-363Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    A general approach to address the biological function of a calcium-binding protein, or another protein, in living cells is to increase or decrease the activity of the protein in the cell and analyze the effects on cell functions. In many cases, it is desirable to determine the effects of overexpressing the protein or a constitutively active or dominantly negative derivative, or to express the protein in a cell that normally lacks it. This is achieved by introducing its gene exogenously. The cDNA for the protein is cloned downstream of an active promoter in a plasmid designed for expression in mammalian cells. This expression plasmid is then transfected into the cell.

  • 28.
    Karlsson, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Division of Experimental Cancer Research, Department of Translational Medicine, Lund University, Clinical Research Centre, Malmö, Sweden.
    Larsson, Per
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Miftakhova, Regina R.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Genetics, Kazan Federal University, Kazan, Russia.
    Khaja, Azharuddin Sajid Syed
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sarwar, Martuza
    Semenas, Julius
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Division of Experimental Cancer Research, Department of Translational Medicine, Lund University, Clinical Research Centre, Malmö, Sweden.
    Chen, Sa
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hedblom, Andreas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Division of Experimental Cancer Research, Department of Translational Medicine, Lund University, Clinical Research Centre, Malmö, Sweden.
    Wang, Tianyan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ekström-Holka, Kristina
    Simoulis, Athanasios
    Kumar, Anjani
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ødum, Nils
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Persson, Jenny L.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Division of Experimental Cancer Research, Department of Translational Medicine, Lund University, Clinical Research Centre, Malmö, Sweden; Department of Biomedical Sciences, Malmö University, Malmö, Sweden.
    Establishment of Prostate Tumor Growth and Metastasis Is Supported by Bone Marrow Cells and Is Mediated by PIP5K1α Lipid Kinase2020Ingår i: Cancers, ISSN 2072-6694, Vol. 12, nr 9, artikel-id 2719Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cancer cells facilitate growth and metastasis by using multiple signals from the cancer-associated microenvironment. However, it remains poorly understood whether prostate cancer (PCa) cells may recruit and utilize bone marrow cells for their growth and survival. Furthermore, the regulatory mechanisms underlying interactions between PCa cells and bone marrow cells are obscure. In this study, we isolated bone marrow cells that mainly constituted populations that were positive for CD11b and Gr1 antigens from xenograft PC-3 tumor tissues from athymic nu/nu mice. We found that the tumor-infiltrated cells alone were unable to form tumor spheroids, even with increased amounts and time. By contrast, the tumor-infiltrated cells together with PCa cells formed large numbers of tumor spheroids compared with PCa cells alone. We further utilized xenograft athymic nu/nu mice bearing bone metastatic lesions. We demonstrated that PCa cells were unable to survive and give rise to colony-forming units (CFUs) in media that were used for hematopoietic cell colony-formation unit (CFU) assays. By contrast, PC-3M cells survived when bone marrow cells were present and gave rise to CFUs. Our results showed that PCa cells required bone marrow cells to support their growth and survival and establish bone metastasis in the host environment. We showed that PCa cells that were treated with either siRNA for PIP5K1α or its specific inhibitor, ISA-2011B, were unable to survive and produce tumor spheroids, together with bone marrow cells. Given that the elevated expression of PIP5K1α was specific for PCa cells and was associated with the induced expression of VEGF receptor 2 in PCa cells, our findings suggest that cancer cells may utilize PIP5K1α-mediated receptor signaling to recruit growth factors and ligands from the bone marrow-derived cells. Taken together, our study suggests a new mechanism that enables PCa cells to gain proliferative and invasive advantages within their associated host microenvironment. Therapeutic interventions using PIP5K1α inhibitors may not only inhibit tumor invasion and metastasis but also enhance the host immune system.

    Ladda ner fulltext (pdf)
    fulltext
  • 29.
    Kumar, Anjani
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Priya, Anshu
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ahmed, Tanzeel
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Negi, Neema
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Regulation of the DNA Repair Complex during Somatic Hypermutation and Class-Switch Recombination2018Ingår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 200, nr 12, s. 4146-4156Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    B lymphocytes optimize Ab responses by somatic hypermutation (SH), which introduces pointmutations in the variable regions of the Ab genes and by class-switch recombination (CSR), which changes the expressed C region exon of the IgH. These Ab diversification processes are initiated by the deaminating enzyme activation-induced cytidine deaminase followed by many DNA repair enzymes, ultimately leading to deletions and a high mutation rate in the Ab genes, whereas DNA lesions made by activation-induced cytidine deaminase are repaired with low error rate on most other genes. This indicates an advanced regulation of DNA repair. In this study, we show that initiation of Ab diversification in B lymphocytes of mouse spleen leads to formation of a complex between many proteins in DNA repair. We show also thatBCR activation, which signals the end of successful SH, reduces interactions between some proteins in the complex and increases other interactions in the complex with varying kinetics. Furthermore, we show increased localization of SH-and CSR-coupled proteins on switch regions of the Igh locus upon initiation of SH/CSR and differential changes in the localization upon BCR signaling, which terminates SH. These findings provide early evidence for a DNA repair complex or complexes that may be of functional significance for carrying out essential roles in SH and/or CSR in B cells.

  • 30. Lai, Dazhi
    et al.
    Wan, Mimi
    Wu, Jie
    Preston-Hurlburt, Paula
    Kushwaha, Ritu
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Imbalzano, Anthony N
    Chi, Tian
    Induction of TLR4-target genes entails calcium/calmodulin-dependent regulation of chromatin remodeling.2009Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, nr 4, s. 1169-1174Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Upon toll-like receptor 4 (TLR4) signaling in macrophages, the mammalian Swi/Snf-like BAF chromatin remodeling complex is recruited to many TLR4 target genes where it remodels their chromatin to promote transcription. Here, we show that, surprisingly, recruitment is not sufficient for chromatin remodeling; a second event, dependent on calcium/calmodulin (CaM), is additionally required. Calcium/CaM directly binds the HMG domain of the BAF57 subunit within the BAF complex. Calcium/CaM antagonists, including a CaM-binding peptide derived from BAF57, abolish BAF-dependent remodeling and gene expression without compromising BAF recruitment. BAF57 RNAi and BAF57 dominant negative mutants defective in CaM binding similarly impair the induction of BAF target genes. Our data implicate calcium/CaM in TLR4 signaling, and reveal a previously undescribed, recruitment-independent mode of regulation of the BAF complex that is probably achieved through a direct CaM-BAF interaction.

  • 31.
    Larsson, Göran
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Schleucher, Jürgen
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Onions, Jacqueline
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hermann, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wijmenga, Sybren S
    Backbone dynamics of a symmetric calmodulin dimer in complex with the calmodulin-binding domain of the basic-helix-loop-helix transcription factor SEF2-1/E2-2: a highly dynamic complex.2005Ingår i: Biophys J, ISSN 0006-3495, Vol. 89, nr 2, s. 1214-26Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Calmodulin (CaM) interacts specifically as a dimer with some dimeric basic-Helix-Loop-Helix (bHLH) transcription factors via a novel high affinity binding mode. Here we report a study of the backbone dynamics by (15)N-spin relaxation on the CaM dimer in complex with a dimeric peptide that mimics the CaM binding region of the bHLH transcription factor SEF2-1. The relaxation data were measured at multiple magnetic fields, and analyzed in a model-free manner using in-house written software designed to detect nanosecond internal motion. Besides picosecond motions, all residues also experience internal motion with an effective correlation time of approximately 2.5 ns with squared order parameter (S(2)) of approximately 0.75. Hydrodynamic calculations suggest that this can be attributed to motions of the N- and C-terminal domains of the CaM dimer in the complex. Moreover, residues with significant exchange broadening are found. They are clustered in the CaM:SEF2-1mp binding interface, the CaM:CaM dimer interface, and in the flexible helix connecting the CaM N- and C-terminal domains, and have similar exchange times (approximately 50 micros), suggesting a cooperative mechanism probably caused by protein:protein interactions. The dynamic features presented here support the conclusion that the conformationally heterogeneous bHLH mimicking peptide trapped inside the CaM dimer exchanges between different binding sites on both nanosecond and microsecond timescales. Nature has thus found a way to specifically recognize a relatively ill-fitting target. This novel mode of target-specific binding, which neither belongs to lock-and-key nor induced-fit binding, is characterized by dimerization and continuous exchange between multiple flexible binding alternatives.

  • 32.
    Liu, Hebin
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Carlsson, Leif
    Umeå centrum för molekylär medicin (UCMM).
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Identification of an N-terminal transactivation domain of Runx1 that separates molecular function from global differentiation function.2006Ingår i: J Biol Chem, ISSN 0021-9258, Vol. 281, nr 35, s. 25659-69Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    RUNX1, or AML1, is a transcription factor that is the most frequent target for chromosomal gene translocations in acute leukemias. RUNX1 is essential for definitive hematopoiesis in embryos and profoundly influences adult steady-state hematopoiesis both positively and negatively. To investigate this wide range of normal activities and the pathological role of RUNX1, it is important to define the functions of different domains of the protein. RUNX1, RUNX2, and RUNX3 are highly conserved in their DNA binding runt homology domain and contain divergent sequences of unknown function N-terminal to this domain. Here we analyzed the role of the N-terminal sequence and the alpha-helix of the runt homology domain of Runx1 in DNA binding, transactivation, and megakaryocytopoiesis. Both the N terminus and the alpha-helix were found to reduce DNA binding of Runx1 and be essential for transactivation of the granulocyte-macrophage colony-stimulating factor and Ialpha1 promoters by Runx1. The N terminus of Runx1, including the alpha-helix, was also required for transactivation of a Gal4 reporter when expressed as fusion proteins with a Gal4 DNA binding domain, and the N terminus alone was capable of stimulating transcription when fused to the Gal4 DNA binding domain. The N terminus and the alpha-helix, however, were not required for megakaryocyte development from embryonic stem cells differentiated in vitro. Thus, our findings define a second transactivation domain of Runx1 that is differentially required for activation of transcription of some Runx1-dependent promoters and megakaryocytopoiesis.

  • 33.
    Liu, Hebin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets12002Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, nr 12, s. 4497-4507Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The multipotent cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in particular in the physiological response to infection and in inflammatory responses. GM-CSF is produced by many cell types, including T lymphocytes responding to T-cell receptor activation and mantle zone B lymphocytes. B-cell receptor and T-cell receptor activation generates two major signals: an increase in intracellular Ca(2+) concentration and a protein kinase cascade. Previous studies have shown that the Ca(2+)/calmodulin-dependent phosphatase calcineurin mediates stimulation of GM-CSF transcription in response to Ca(2+). In this study, we show that Ca(2+) signaling also regulates GM-CSF transcription negatively through Ca(2+)/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Wild-type Ets1 negatively affects GM-CSF transcription on Ca(2+) stimulation in the presence of cyclosporin A, which inhibits calcineurin. Conversely, Ets1 with mutated CaMK II target serines showed an increase in transactivation of the GM-CSF promoter/enhancer. Moreover, constitutively active CaMK II inhibited transactivation of GM-CSF by wild-type Ets1 but not by Ets1 with mutated CaMK II sites. Mutation of CaMK II target serines in Ets1 also relieves inhibition of cooperative transactivation of GM-CSF with the Runx1/AML1 transcription factor. In addition, the Ca(2+)-dependent phosphorylation of Ets1 reduces the binding of Ets1 to the GM-CSF promoter in vivo.

  • 34.
    Liu, Hebin
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Holm, Magnus
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Xie, Xiao-Qi
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Kemi.
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    AML1/Runx1 recruits calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1 activation.2004Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 28, s. 29398-29408Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Acute myeloid leukemia 1 (AML1), also denoted Runx1, is a transcription factor essential for hematopoiesis, and the AML1 gene is the most common target of chromosomal translocations in human leukemias. AML1 binds to sequences present in the regulatory regions of a number of hematopoiesis-specific genes, including certain cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) up-regulated after T cell receptor stimulation. Here we show that both subunits of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN), which is activated upon T cell receptor stimulation, interact directly with the N-terminal runt homology domain-containing part of AML1. The regulatory CN subunit binds AML1 with a higher affinity and in addition also interacts with the isolated runt homology domain. The related Runx2 transcription factor, which is essential for bone formation, also interacts with CN. A constitutively active derivative of CN is shown to activate synergistically the GM-CSF promoter/enhancer together with AML1 or Runx2. We also provide evidence that relief of the negative effect of the AML1 sites is important for Ca(2+) activation of the GM-CSF promoter/enhancer and that AML1 overexpression increases this Ca(2+) activation. Both subunits of CN interact with AML1 in coimmunoprecipitation analyses, and confocal microscopy analysis of cells expressing fluorescence-tagged protein derivatives shows that CN can be recruited to the nucleus by AML1 in vivo. Mutant analysis of the GM-CSF promoter shows that the Ets1 binding site of the promoter is essential for the synergy between AML1 and CN in Jurkat T cells. Analysis of the effects of inhibitors of the protein kinase glycogen synthase kinase-3beta and in vitro phosphorylation/dephosphorylation analysis of Ets1 suggest that glycogen synthase kinase-3beta-phosphorylated Ets1 is a target of AML1-recruited CN phosphatase at the GM-CSF promoter.

  • 35. Mårtensson, Annica
    et al.
    Xie, Xiao-Qi
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Persson, Christine
    Holm, Magnus
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Mårtensson, Inga-Lill
    PEBP2 and c-myb sites crucial for lambda5 core enhancer activity in pre-B cells.2001Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 31, nr 11, s. 3165-3174Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The lambda5 gene is expressed exclusively in precursor (pre-) B cells where its gene product, as part of the pre-B cell receptor, is crucial for the proliferation of these cells. Several DNA regions regulate the activity and expression pattern of the lambda5 gene. Amongst these is an enhancer, B(lambda5), located 5' of the gene. Here we analyze the lambda5 enhancer core, b(lambda5), which in earlier experiments was demonstrated to retain 50% of the enhancer activity, and show that this activity is restricted to pre-B cells. We identify a DNA element within b(lambda5), PEBP2(lambda5), which is essential for enhancer activity: mutation within this site dramatically reduces core enhancer activity in pre-B cells. The PEBP2(lambda5) site binds bacterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA). Furthermore, PEBP2 proteins present in nuclear extracts from murine pre-B cells bind to the PEBP2(lambda5) element. PEBP2 proteins in mature B cells also bind to the PEBP2(lambda5 )element, implying that if PEBP2 proteins are responsible for the stage-specific expression, they have to be non-activating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c-myb, binds to a sequence termed myb(lambda5) located just upstream of the PEBP2(lambda5) site in the core enhancer. The myb(lambda5) element is also crucial for enhancer activity, since mutating the myb site reduces core enhancer activity to the same extent as mutating the PEBP2 site. Earlier reports have shown that c-myb is expressed at high levels in pre-B cell lines whereas its expression is down-regulated in more mature B cell lines. Thus, c-myb may be involved in determining the stage-specific expression of the lambda5 gene.

  • 36.
    Onions, Jacqueline
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Hermann, Stefan
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    A novel type of calmodulin interaction in the inhibition of basic helix-loop-helix transcription factors.2000Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, nr 15, s. 4366-4374Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Calmodulin is the predominant intracellular receptor for Ca(2+) signals, mediating the regulation of numerous cellular processes. Previous studies have shown that calcium-loaded calmodulin can bind to and inhibit the activity of certain basic helix-loop-helix (bHLH) transcription factors. The basic sequence within the bHLH domain is the primary target for calmodulin binding, and sequences modulating the calmodulin interaction reside directly N-terminal to the basic sequence. Here we show that the interaction of calmodulin with bHLH proteins is of a novel type, displaying characteristics very different from those of previously characterized calmodulin-target complexes. We show that calmodulin interacts much stronger with a dimeric basic sequence than with the monomeric form. The calmodulin-bHLH protein complex contains equimolar amounts of calmodulin and bHLH chains. The interaction is unusual in being to a large extent polar in nature, and it is highly resistant to tested calmodulin inhibitors. Both the N-terminal and C-terminal domains of calmodulin can independently bind to and inhibit the DNA binding of bHLH proteins. The C-terminal domain preferentially binds to the basic sequence, whereas the N-terminal domain is essential for the effect of the modulatory sequence. We propose a model for the calmodulin-bHLH complex where two calmodulin molecules interact with one bHLH dimer, with one domain of calmodulin preferentially binding to the basic sequence of bHLH proteins and the other domain interacting with the modulatory sequence.

  • 37.
    Oruganti, Sreenivasa Rao
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Edin, Sofia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    CaMKII targets Bc110 in T-cell receptor induced activation of NF-κB2011Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 48, nr 12-13, s. 1448-1460Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recognition of antigen by T- or B-cell receptors leads to formation of an immunological synapse and initiation of signalling events that collaborate to determine the nature of the adaptive immune response. Activation of NF-κB transcription factors has a key role in regulation of numerous genes with important functions in immune responses and inflammation and is of great importance for lymphocyte activation and differentiation. The activation of NF-κB depends on changes in intracellular Ca2+ levels, and both calmodulin (CaM) and a CaM-dependent kinase, CaMKII, help regulate NF-κB activation after T-cell receptor (TCR) stimulation, but the mechanisms are not well characterized. Here we have analyzed the functional role of CaMKII in the signalling pathway from the TCR to activation of IKK, the kinase that phosphorylates the NF-κB inhibitor IκB. We show that CaMKII is recruited to the immunological synapse where it interacts with and phosphorylates the signalling adaptor protein Bcl10. Furthermore, phosphorylation of the CARD domain of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signalling complex and the essential signal induced ubiquitinations of Bcl10 and IKKγ. We propose a novel mechanism whereby Ca2+ signals can be integrated at the immunological synapse through CaMKII-dependent phosphorylation of Bcl10.

  • 38.
    Oruganti, Sreenivasa Rao
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Edin, Sofia
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Grundström, Christine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    CaMKII targets Bcl10 in T-cell receptor induced activation of NF-κB2011Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 48, nr 12-13, s. 1448-1460Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recognition of antigen by T- or B-cell receptors leads to formation of an immunological synapse and initiation of signalling events that collaborate to determine the nature of the adaptive immune response. Activation of NF-κB transcription factors has a key role in regulation of numerous genes with important functions in immune responses and inflammation and is of great importance for lymphocyte activation and differentiation. The activation of NF-κB depends on changes in intracellular Ca(2+) levels, and both calmodulin (CaM) and a CaM-dependent kinase, CaMKII, help regulate NF-κB activation after T-cell receptor (TCR) stimulation, but the mechanisms are not well characterized. Here we have analyzed the functional role of CaMKII in the signalling pathway from the TCR to activation of IKK, the kinase that phosphorylates the NF-κB inhibitor IκB. We show that CaMKII is recruited to the immunological synapse where it interacts with and phosphorylates the signalling adaptor protein Bcl10. Furthermore, phosphorylation of the CARD domain of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signalling complex and the essential signal induced ubiquitinations of Bcl10 and IKKγ. We propose a novel mechanism whereby Ca(2+) signals can be integrated at the immunological synapse through CaMKII-dependent phosphorylation of Bcl10.

  • 39.
    Saarikettu, Juha
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sveshnikova, Natalia
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.2004Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 39, s. 41004-41011Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Ca2+ sensor protein calmodulin can interact with the DNA binding basic helix-loop-helix (bHLH) domain of E12, E47, and SEF2-1 (E2-2), which belong to the E-protein subclass of bHLH transcription factors. This interaction inhibits the DNA binding of these bHLH proteins in vitro, and an ionophore that increases intracellular Ca2+ can inhibit transcriptional activation by the E-proteins. Here we have attempted to determine if these phenomena reflect a direct calmodulin-dependent inhibition of DNA binding by E-proteins in vivo. We show that calmodulin overexpression inhibits the transcriptional activity of E12, E47, and SEF2-1. We have compared calmodulin effects on DNA binding in vitro and on activation of transcription in vivo using a series of E12 mutants harboring defined alterations within the basic sequence of the bHLH domain that reduce their ability to bind calmodulin to varying degrees. We find a striking direct correlation between the ability of calmodulin to inhibit their DNA binding in vitro and the ability of overexpressed calmodulin or cellular Ca2+ mobilization to inhibit their transcriptional activity in vivo. Furthermore, E12 and overexpressed calmodulin were co-localized in the nucleus, and calmodulin pull-down experiments with cell extracts showed a Ca2+-dependent interaction between calmodulin and E12 but not with a calmodulin inhibition-deficient E12 mutant. Chromatin immunoprecipitation showed that calmodulin overexpression leads to decreased binding of E12 and E47, but not a calmodulin inhibition-deficient E12 mutant, to the DNA recognition sequence in vivo. The data suggest that Ca2+ signaling can inhibit the transcriptional activities of E-proteins through direct binding of Ca2+/calmodulin to the basic sequence of E-proteins, resulting in inhibition of their DNA binding. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

  • 40.
    Semenas, Julius
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wang, Tianyan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Khaja, Azharuddin Sajid Syed
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Mahmud, A. K. M. Firoj
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Simoulis, Athanasios
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Persson, Jenny L.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Division of Experimental Cancer Research, Department of Translational Medicine, Lund University, Clinical Research Centre in Malmö, Sweden; Department of Biomedical Science, Malmö University, Sweden.
    Targeted inhibition of ERα signaling and PIP5K1α/Akt pathways in castration‐resistant prostate cancer2021Ingår i: Molecular Oncology, ISSN 1574-7891, E-ISSN 1878-0261, Vol. 15, nr 4, s. 968-986Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Selective ERα modulator, tamoxifen, is well tolerated in a heavily pretreated castration‐resistant prostate cancer (PCa) patient cohort. However, its targeted gene network and whether expression of intratumor ERα due to androgen‐deprivation therapy (ADT) may play a role in PCa progression is unknown. In this study, we examined the inhibitory effect of tamoxifen on castration‐resistant PCa in vitro and in vivo. We found that tamoxifen is a potent compound that induced a high degree of apoptosis and significantly suppressed growth of xenograft tumors in mice, at a degree comparable to ISA‐2011B, an inhibitor of PIP5K1α that acts upstream of PI3K/AKT survival signaling pathway. Moreover, depletion of tumor‐associated macrophages using clodronate in combination with tamoxifen increased inhibitory effect of tamoxifen on aggressive prostate tumors. We showed that both tamoxifen and ISA‐2011B exert their on‐target effects on prostate cancer cells by targeting cyclin D1 and PIP5K1α/AKT network and the interlinked estrogen signaling. Combination treatment using tamoxifen together with ISA‐2011B resulted in tumor regression and had superior inhibitory effect compared with that of tamoxifen or ISA‐2011B alone. We have identified sets of genes that are specifically targeted by tamoxifen, ISA‐2011B or combination of both agents by RNA‐seq. We discovered that alterations in unique gene signatures, in particular estrogen‐related marker genes are associated with poor patient disease‐free survival. We further showed that ERα interacted with PIP5K1α through formation of protein complexes in the nucleus, suggesting a functional link. Our finding is the first to suggest a new therapeutic potential to inhibit or utilize the mechanisms related to ERα, PIP5K1α/AKT network and MMP9/VEGF signaling axis, providing a strategy to treat castration‐resistant ER‐positive subtype of prostate cancer tumors with metastatic potential.

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  • 41. Verma-Gaur, Jiyoti
    et al.
    Hauser, Jannek
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Negative feedback regulation of antigen receptors through calmodulin inhibition of E2AManuskript (preprint) (Övrigt vetenskapligt)
  • 42.
    Verma-Gaur, Jiyoti
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hauser, Jannek
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Negative feedback regulation of antigen receptors through calmodulin inhibition of E2A2012Ingår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 188, nr 12, s. 6175-6183Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Signaling from the BCR is used to judge Ag-binding strengths of the Abs of B cells. BCR signaling enables the selection for successive improvements in the Ag affinity over an extremely broad range of affinities during somatic hypermutation. We show that the mouse BCR is subject to general negative feedback regulation of the receptor proteins, as well as many coreceptors and proteins in signal pathways from the receptor. Thus, the BCR can downregulate itself, which can enable sensitive detection of successive improvements in the Ag affinity over a very large span of affinities. Furthermore, the feedback inhibition of the BCR signalosome and most of its proteins, as well as most other regulations of genes by BCR stimulation, is to a large extent through inhibition of the transcription factor E2A by Ca(2+)/calmodulin.

  • 43.
    Wallenius, Anders
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hauser, Jannek
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Aas, Per Arne
    Sarno, Antonio
    Kavli, Bodil
    Krokan, Hans E.
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Expression and recruitment of uracil-DNA glycosylase are regulated by E2A during antibody diversification2014Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 60, nr 1, s. 23-31Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID), which is positively regulated by the transcription factor E2A, is the key enzyme that initiates these processes by deaminating cytosine to uracil in Ig genes. Nuclear uracil-DNA glycosylase (UNG2) is subsequently required for uracil processing in the generation of high affinity antibodies of different isotypes. Here we show that the transcription factor E2A binds to the UNG2 promoter and represses UNG2 expression. Inhibition of E2A by binding of Ca2+-activated calmodulin alleviates this repression. Furthermore, we demonstrate that UNG2 preferentially accumulates in regions of the Ig heavy chain (IgH) gene containing AID hotspots. Calmodulin inhibition of E2A strongly enhances this UNG2 accumulation, indicating that it is negatively regulated by E2A as well. We show also that over-expression of E2A can suppress class switch recombination. The results suggest that E2A is a key factor in regulating the balance between AID and UNG2, both at expression and Ig targeting levels, to stimulate Ig diversification and suppress normal DNA repair processes. (c) 2014 Elsevier Ltd. All rights reserved.

  • 44.
    Wang, Tianyan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sarwar, Martuza
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Whitchurch, Jonathan B.
    School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
    Collins, Hilary M.
    School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
    Green, Tami
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Semenas, Julius
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Amjad, Ali
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Roberts, Christopher J.
    School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
    Morris, Ryan D.
    School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Department of Pharmacy, Uppsala University, Uppsala, Sweden.
    Chen, Sa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    El-Schich, Zahra
    Department of Biomedical Science, Malmö University, Malmö, Sweden.
    Wingren, Anette G.
    Department of Biomedical Science, Malmö University, Malmö, Sweden.
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Mongan, Nigel P.
    School of Veterinary Medicine and Science, University of Nottingham, Nottingham, United Kingdom; Department of Pharmacology, Weill Cornell Medicine, New York, NY, United States.
    Gunhaga, Lena
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Heery, David M.
    School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
    Persson, Jenny L.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Biomedical Science, Malmö University, Malmö, Sweden; Department of Translational Medicine, Lund University, Clinical Research Centre in Malmö, Malmö, Sweden.
    PIP5K1α is Required for Promoting Tumor Progression in Castration-Resistant Prostate Cancer2022Ingår i: Frontiers in Cell and Developmental Biology, E-ISSN 2296-634X, Vol. 10, artikel-id 798590Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    PIP5K1α has emerged as a promising drug target for the treatment of castration-resistant prostate cancer (CRPC), as it acts upstream of the PI3K/AKT signaling pathway to promote prostate cancer (PCa) growth, survival and invasion. However, little is known of the molecular actions of PIP5K1α in this process. Here, we show that siRNA-mediated knockdown of PIP5K1α and blockade of PIP5K1α action using its small molecule inhibitor ISA-2011B suppress growth and invasion of CRPC cells. We demonstrate that targeted deletion of the N-terminal domain of PIP5K1α in CRPC cells results in reduced growth and migratory ability of cancer cells. Further, the xenograft tumors lacking the N-terminal domain of PIP5K1α exhibited reduced tumor growth and aggressiveness in xenograft mice as compared to that of controls. The N-terminal domain of PIP5K1α is required for regulation of mRNA expression and protein stability of PIP5K1α. This suggests that the expression and oncogenic activity of PIP5K1α are in part dependent on its N-terminal domain. We further show that PIP5K1α acts as an upstream regulator of the androgen receptor (AR) and AR target genes including CDK1 and MMP9 that are key factors promoting growth, survival and invasion of PCa cells. ISA-2011B exhibited a significant inhibitory effect on AR target genes including CDK1 and MMP9 in CRPC cells with wild-type PIP5K1α and in CRPC cells lacking the N-terminal domain of PIP5K1α. These results indicate that the growth of PIP5K1α-dependent tumors is in part dependent on the integrity of the N-terminal sequence of this kinase. Our study identifies a novel functional mechanism involving PIP5K1α, confirming that PIP5K1α is an intriguing target for cancer treatment, especially for treatment of CRPC.

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  • 45. Wolf-Watz, M
    et al.
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Härd, T
    Structure and backbone dynamics of Apo-CBFbeta in solution.2001Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, nr 38, s. 11423-11432Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Runx proteins constitute a family of mammalian transcription factors that interact with DNA through their evolutionarily conserved Runt domain. CBFbeta, alternatively denoted PEBP2beta, is the non-DNA-binding heterodimer partner and acts to enhance the DNA binding affinity of Runx proteins. Runx proteins and CBFbeta are associated with a variety of biological functions and human diseases; they are, for example, together the most frequent targets for chromosomal rearrangements in acute human leukemias. We have determined the solution structure and characterized the backbone dynamics of C-terminally truncated fragments containing residues 1-141 of CBFbeta. The present apo-CBFbeta structure is very similar to that seen in a Runt-CBFbeta complex. An evaluation of backbone (15)N NMR relaxation parameters shows that CBFbeta is a rigid molecule with high order parameters throughout the backbone; the only regions displaying significant dynamics are a long loop and the C-terminal alpha-helix. A few residues display relaxation behavior indicating conformational exchange on microsecond to millisecond time scales, but only one of these is located at the Runt binding surface. Our structure and dynamics analysis of CBFbeta therefore suggests that the protein binds to Runt without large conformational changes or induced folding ("lock-and-key" interaction). The apo-CBFbeta structure presented here exhibits several significant differences with two other published NMR ensembles of very similar protein fragments. The differences are located in four regions outside of the central beta-barrel, whereas the beta-barrel itself is almost identical in the three NMR structures. The comparison illustrates that independently determined NMR structures may display rather large differences in backbone conformation in regions that appear to be well-defined in each of the calculated NMR ensembles.

  • 46.
    Wolf-Watz, Magnus
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Kemi.
    Bäckström, Stefan
    Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Grundström, Thomas
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sauer, Uwe
    Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Härd, Torleif
    Chloride binding by the AML1/Runx1 transcription factor studied by NMR2001Ingår i: FEBS Letters, ISSN 0014-5793, Vol. 488, nr 1-2, s. 81-4Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

  • 47.
    Xie, Xiao-Qi
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Pardali, Evangelia
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Holm, Magnus
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sideras, Paschalis
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    AML and Ets proteins regulate the I alpha1 germ-line promoter.1999Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 29, nr 2, s. 488-498Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The immunoglobulin heavy chain (IgH) class switch recombination of B lymphocytes preferentially targets unrearranged IgH genes that have already been rendered transcriptionally active. Transcription of the germ-line IgH genes is controlled by intervening (I) regions upstream of their switch regions. The I alpha1 promoter activates transcription of the human germ-line C alpha1 gene for IgA1 and mediates the transforming growth factor (TGF)-beta1 responsiveness of this locus. Here we show that the I alpha1 promoter contains several binding sites for the AML/PEBP2/CBF family of transcription factors and that AML and Ets proteins are major regulators of the basal and TGF-beta-inducible promoter activity. Our data constitute a starting point for studies to elucidate the molecular mechanism by which TGF-beta regulates IgA production.

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