REGULATION OF ACTIVATION OF NF-?B

Regulation of activation of NF-?B by calmodulin in T-lymphocytes

Abstract

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 (Ca2+) signals in cells, is required in the pathway from the T cell receptor (TCR) to activation of the key transcription factor NF-?B. 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 Ca2+ dependent and of high affinity, with a Kd of approximately 160 nM. Proximity of CaM and Bcl10 in vivo is induced by increases in the intracellular Ca2+ 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-?B. Furthermore, a mutant of Bcl10 with reduced binding to CaM shows increased activation of an NF-?B reporter, which is further enhanced by activating stimuli. We propose a novel mechanism whereby the Ca2+ sensor CaM regulates T cell responses to antigens by binding to Bcl10, thereby modulating its interaction with Carma1 and subsequent activation of NF-?B.

Table of Contents

Abstract2

1. INTRODUCTION4

2. MATERIALS AND METHODS8

2.1. Plasmids and mutagenesis8

2.2. Expression and purification of proteins8

2.3. In vitro binding experiments9

2.4. Cell culture and transient transfections10

2.5. Proximity ligation assays10

3. RESULTS11

3.1. CaM interacts with Bcl1011

3.2. Identification of the CaM binding domain of Bcl1014

3.3. A Bcl10 mutant with reduced CaM binding15

3.4. Binding of CaM to Bcl10 inhibits the interaction between Bcl10 and Carma119

3.5. In vivo proximity of CaM and Bcl10 is induced by an increased Ca2+ level19

3.6. Binding of CaM to Bcl10 is inhibitory for NF-?B activation in Jurkat T cells23

4. DISCUSSION25

References34

Regulation of activation of NF-?B by calmodulin in T-lymphocytes

1. Introduction

Antigen receptor activation results in the recruitment and activation of a number of signaling mediators and lipid metabolizing enzymes, building up an immunological synapse at the T cell receptor (TCR) or B cell receptor (BCR) ([Schulze-Luehrmann and Ghosh, 2006] and [Weil and Israel, 2004]). The immunological synapse triggers downstream signaling pathways, leading to activation of transcription factors such as the NF-?B family, which is important for the regulation of activation, proliferation, and differentiation of T and B lymphocytes (Caamano and Hunter, 2002). In a resting cell, NF-?B dimers mainly reside in the cytoplasm, sequestered by inhibitory I?B proteins. Most signals to NF-?B converge on the activation of the I?B kinase (IKK), resulting in signal-induced phosphorylation and degradation of I?B, which allows NF-?B to enter the nucleus and activate gene transcription (Hayden and Ghosh, 2008).

T cell receptor-induced activation of NF-?B is dependent on the activation of protein kinase C (PKC)? and the subsequent recruitment and activation of a signaling complex containing the proteins Carma1, Bcl10, and Malt1, which is often referred to as the CBM ...