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Regulation of activation of NF-κB by Calmodulin in T-lymphocytes
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nuclear factor kappa B (NF-kB) is a widely expressed family of transcription factors that are involved in a diverse number of processes. These include inflammation or differentiation, survival or apoptosis, and proliferation or cell cycle arrest. NF-kB is usually associated with inhibitory kB proteins (IkB), which mask the nuclear localisation sequence (NLS) of NF-kB and renders it in the cytoplasm. Various stimuli result in the activation of the I kappa B kinase (IKK) protein complex, which phosphorylates IκB proteins and thereby marks them for degradation by the ubiquitin-proteasome pathway. Thereby NF-kB enters the nucleus and acts on its target genes. The study of T- and B-lymphocyte antigen receptor signalling to NF-kB is a field of intense investigation, with much attention being focused on the molecular scaffolding proteins Carma1, Bcl10 and MALT1 and their post-translational modifications. These have been shown to be crucial for the organization of the immunological synapse structure under the activated receptor, to which IKK is recruited and becomes activated, which subsequently leads to the activation of NF-kB.

T cell receptor (TCR) activation results in a rapid increase in the intracellular Ca2+ level and NF-kB activation is known to be regulated by those increases, but the mechanisms have remained unclear. Calmodulin (CaM) is a calcium sensory protein that responds to increases in intracellular Ca2+ levels. When CaM binds Ca2+ ions, it leads to structural changes that directly as well as indirectly, through CaM dependent kinases (CaMKs), phosphatases and other enzymes, alters a variety of cellular processes, among them transcriptional regulation. Here CaM is shown to interact directly with Bcl10 in a Ca2+ dependent manner. Increases in the intracellular Ca2+ level are shown to induce the proximity of Bcl10 and CaM in vivo. Carma1 associates with Bcl10 through a CARD-CARD domain interaction that is known to be crucial for TCR signalling to NF-kB. The interaction of CaM with Bcl10 was mapped to the CARD domain and was shown to be a negative regulator for the Bcl10-Carma1 interaction. Inhibition of the CaM interaction by a point mutation within the CaM binding site of Bcl10 results in decreased binding of CaM to Bcl10 in vivo, as well as an increased ability of Bcl10 to induce NF-kB transcriptional activity, which is further enhanced by TCR activating stimuli.

NF-kB activation is also shown here to be regulated by CaM indirectly through actions of CaMKII. The CaMKII is recruited to the immunological synapse where it interacts with Bcl10 in an inducible fashion and phosphorylates Bcl10. Phosphorylations of Bcl10 by CaMKII are shown to be important for the ability of Bcl10 to induce NF-κB transcriptional activity. Upon mutation of its most important CaMKII site, Bcl10 fails to activate an NF-kB reporter and an NF-kB target gene (IL-2). This mutated Bcl10 also fails to induce activating phosphorylations of IKKa/b and the kinase JNK2 but not JNK1. Furthermore, phosphorylation of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signaling complex and the essential signal induced ubiquitinations of Bcl10 and IKKg. Phosphorylation of IKK by TAK1 is also regulated by CaMKII, and serine 82 is a putative CaMKII target site of TAK1 that appears to be important for IκBα degradation.

In summary, this thesis explores that not only NF-kB but also CaM is a double-edged sword, since the multi-functional NF-kB family of transcription factors is regulated by CaM both negatively and positively.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2011. , 75 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1445
Keyword [en]
Calcium, Calmodulin, CaMKII, TCR, Bcl10, NF-κB
Identifiers
URN: urn:nbn:se:umu:diva-46561ISBN: 978-91-7459-278-8 (print)OAI: oai:DiVA.org:umu-46561DiVA: diva2:438864
Public defence
2011-09-27, Major Groove, Byggnad 6L, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-09-06 Created: 2011-09-05 Last updated: 2011-09-06Bibliographically approved
List of papers
1. 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
2. Interaction of calmodulin with Bcl10 modulates NF-kappaB activation.
Open this publication in new window or tab >>Interaction of calmodulin with Bcl10 modulates NF-kappaB activation.
2010 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 47, no 11-12, 2057-2064 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Elsevier, 2010
Keyword
Gene regulation, Signal transduction, NF-κB, Bcl10, Carma1, Calmodulin, Calcium signaling
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:umu:diva-35923 (URN)10.1016/j.molimm.2010.04.005 (DOI)000279578800014 ()20439115 (PubMedID)
Available from: 2010-09-09 Created: 2010-09-09 Last updated: 2011-09-06Bibliographically approved

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Oruganti, Sreenivasa Rao

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