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Calmodulin-dependent Kinase II Mediates T Cell Receptor/CD3- and Phorbol Ester-induced Activation of IκB Kinase
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Biology.
2001 (English)In: Journal of Biological Chemistry, ISSN 1083-351X, Vol. 276, no 38, 36008 - 36013 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2001. Vol. 276, no 38, 36008 - 36013 p.
Identifiers
URN: urn:nbn:se:umu:diva-3010OAI: oai:DiVA.org:umu-3010DiVA: diva2:141433
Available from: 2003-11-25 Created: 2003-11-25 Last updated: 2009-04-03Bibliographically approved
In thesis
1. Regulation of NF-κB by Calmodulin
Open this publication in new window or tab >>Regulation of NF-κB by Calmodulin
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells experience numerous external signals which they must respond to. Such signals arriving at the cell surface are transduced via various signal transduction pathways and often ultimately result in regulation of transcription. NF-κB is a family of transcription factors involved in the regulation of genes important for processes such as immune and inflammatory responses, cell growth, development and cell survival. NF-κB proteins are normally kept inactive in the cytoplasm due to masking of their nuclear localisation signal (NLS) by inhibitory IκB proteins. A large number of stimuli lead to the activation of IκB-kinase (IKK). Active IKK phosphorylates IκB and thereby labels it for ubiquitination and, subsequently, degradation by the proteasome. Liberated NF-κB enters the nucleus, where it takes part in the regulation of its target genes.

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein which is considered to be the predominant intracellular Ca2+ sensor. CaM plays a major role in the Ca2+-dependent regulation of a wide variety of cellular processes, including transcription. CaM regulates transcription both indirectly through CaM-dependent kinases and phosphatases and directly through interaction with transcription factors.

CaM was found to bind directly and in a Ca2+-dependent fashion to the two NF-κB family members c-Rel and RelA. The CaM-NF-κB interactions were strongly enhanced by NF-κB activating stimuli and this enhancement was blocked by the addition of IκB, suggesting that c-Rel and RelA can bind CaM after their signal-induced release from IκB. Compared to wild-type c-Rel, CaM binding-deficient mutants were shown to exhibit an increased nuclear accumulation and transcriptional activity on Ca2+-regulated cytokine promoters. The results suggest that CaM can inhibit transport of c-Rel, but not of RelA, to the nucleus and thereby differentially regulate the activation of NF-κB proteins following cell stimulation. CaM was also found to affect NF-κB activity indirectly through the action of a CaM-dependent kinase (CaMK). Studies of the events leading to IκBα phosphorylation revealed that CaM and CaMKII inhibitors blocked phorbol ester induced activation of IKK. Furthermore, CaM and CaMKII inhibitors also blocked T cell receptor/CD3 induced IκBα degradation, and expression of an inhibitor-resistant derivative of the γ isoform of CaMKII caused the inhibitors lose their effect on phorbol ester induced IκBα degradation. Finally, expression of a constitutively active CaMKII resulted in the activation of NF-κB. These results identify CaMKII as a mediator of IKK activation, specifically in response to T cell receptor/CD3 and phorbol ester stimulation.

In conclusion, this thesis describes the identification of CaM as a dual regulator of NF-κB proteins, acting both directly and indirectly to affect the activity of this family of transcription factors.

Publisher
69 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; ISSN 0346-6612; 864
Keyword
Molecular biology, NF-κB, calmodulin, transcription, calcium, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-158 (URN)91-7305-549-2 (ISBN)
Public defence
2003-12-05, Major Groove, 6L, NUS, Umeå, 12:00
Opponent
Supervisors
Available from: 2003-11-25 Created: 2003-11-25Bibliographically approved
2. Calmodulin mediated regulation of NF-kappaB in lymphocytes
Open this publication in new window or tab >>Calmodulin mediated regulation of NF-kappaB in lymphocytes
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
calcium, CaM, CaMKII, NF-kappaB, Bc110, immunological synapse
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1895 (URN)978-91-7264-659-9 (ISBN)
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

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