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Calmodulin mediated regulation of NF-kappaB in lymphocytes
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

NF-κB transcription factors are regulators of a wide spectrum of genes involved in immune responses and inflammation as well as cellular proliferation and survival. Transcriptionally competent NF-κB dimers are retained in the cytoplasm of resting cells by binding to inhibitors of NF-κB (IκBs). Stimuli that activate NF-κB converge on the activation of the IκB kinase (IKK), resulting in phosphorylation and subsequent proteasomal degradation of IκB. This releases functional NF-κB dimers that rapidly move to the nucleus where they regulate transcription of NF-κB-dependent target genes. The study of signalling to NF-κB from T and B lymphocyte antigen receptors is a field of intense investigation, and much attention is focused on the complex of the molecular scaffolding proteins Carma1, Bcl10 and MALT1. Together, these are crucial for the organisation of a structure beneath the activated receptor, termed the immunological synapse. IKK is recruited to this structure and becomes activated, subsequently leading to activation of NF-κB.

Calcium (Ca2+) is a ubiquitous intracellular messenger that is involved in the regulation of numerous aspects of cellular function, including transcription. NF-κB activity is known to be regulated by changes in intracellular Ca2+ levels, such as those created by antigen receptor activation, but the mechanisms are to a large extent undefined. Ca2+ signals in cells are transmitted predominantly by the ubiquitous Ca2+ sensor protein calmodulin (CaM). Signalling that increases the intracellular Ca2+ concentration leads to binding of Ca2+ to CaM, which changes its structure, thereby allowing it to interact with a new range of target proteins.

The studies of NF-κB signalling in lymphocytes presented here reveal that CaM is involved, both directly and indirectly, in the regulation of NF-κB. CaM was found to interact directly and in a Ca2+-dependent manner with the NF-κB proteins RelA and c-Rel after their signal-induced release from IκB. The interaction of CaM with c-Rel, but not RelA, was found to be inhibitory for its nuclear accumulation and transcriptional activity on Ca2+-regulated IL-2 and GM-CSF promoters; thus, CaM binding was found to differentially regulate c-Rel and RelA in lymphocytes. CaM was also shown to interact directly and in a Ca2+-dependent manner with Bcl10. The interaction was mapped to the Carma1-interacting CARD domain of Bcl10 and was found to have a negative effect on the ability of Bcl10 to bind to Carma1. Binding of CaM to Bcl10 also had a negative effect on activation of NF-κB after T cell receptor stimulation, since a point mutant of Bcl10 with reduced binding to CaM showed increased activation of an NF-κB reporter in Jurkat T cells, which was further enhanced by TCR-activating stimuli.

In addition, CaM was found to positively regulate NF-κB activation indirectly through CaM-dependent kinase II (CaMKII). Inhibitors of CaM and CaMKII were shown to inhibit IκBα degradation in lymphocytes induced by phorbol ester or T cell receptor stimulation. The actions of CaMKII were mapped to a point upstream of IKK activation and further studies revealed that CaMKII is recruited to the immunological synapse, where it inducibly interacts with and phosphorylates Bcl10 at multiple sites. Phosphorylation of Bcl10 by CaMKII was shown to be important for the ability of Bcl10 to activate NF-κB, since mutation of the phosphorylation sites of Bcl10 inhibited Bcl10-induced transcriptional activity of NF-κB, in part by preventing signalinduced ubiquitination and degradation of Bcl10.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Medicinska fakulteten) , 2008. , 63 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1217
Keyword [en]
calcium, CaM, CaMKII, NF-kappaB, Bc110, immunological synapse
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1895ISBN: 978-91-7264-659-9 (print)OAI: oai:DiVA.org:umu-1895DiVA: diva2:142339
Public defence
2008-11-14, Major Groove, 6L, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-10-29 Created: 2008-10-29 Last updated: 2010-01-18Bibliographically approved
List of papers
1. Regulation of c-Rel Nuclear Localization by Binding of Ca2+/Calmodulin
Open this publication in new window or tab >>Regulation of c-Rel Nuclear Localization by Binding of Ca2+/Calmodulin
2003 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, no 4, 1418-1427 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
American Society for Microbiology, 2003
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-3008 (URN)10.1128/MCB.23.4.1418-1427.2003 (DOI)
Available from: 2003-11-25 Created: 2003-11-25 Last updated: 2012-05-10Bibliographically approved
2. Calmodulin-dependent Kinase II Mediates T Cell Receptor/CD3- and Phorbol Ester-induced Activation of IκB Kinase
Open this publication in new window or tab >>Calmodulin-dependent Kinase II Mediates T Cell Receptor/CD3- and Phorbol Ester-induced Activation of IκB Kinase
2001 (English)In: Journal of Biological Chemistry, ISSN 1083-351X, Vol. 276, no 38, 36008 - 36013 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-3010 (URN)
Available from: 2003-11-25 Created: 2003-11-25 Last updated: 2009-04-03Bibliographically approved
3. Interaction of calmodulin with Bc110 modulates NF-κB activation
Open this publication in new window or tab >>Interaction of calmodulin with Bc110 modulates NF-κB activation
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-3571 (URN)
Available from: 2008-10-29 Created: 2008-10-29 Last updated: 2010-01-13Bibliographically approved
4. CaMKII targets Bc110 in T-cell receptor induced activation of NF-κB
Open this publication in new window or tab >>CaMKII targets Bc110 in T-cell receptor induced activation of NF-κB
2011 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 48, no 12-13, 1448-1460 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Elsevier, 2011
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-3572 (URN)10.1016/j.molimm.2011.03.020 (DOI)
Available from: 2008-10-29 Created: 2008-10-29 Last updated: 2012-03-08Bibliographically approved

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