CD40 translocation to lipid rafts: Signaling requirements and downstream biological events

Abstract

CanadaCD40, a member of the TNF receptor family, is expressed on a variety of immune and nonimmune cells. Its interaction with its ligand, CD154, plays a pivotal role in humoral and cellmediated immunity. A low level of CD40 is constitutively associated within membrane lipid rafts and, upon engagement, this level is significantly enhanced. In this study, our objective is to evaluate the process of CD40/lipid raft association in terms of the signals required for its initiation and the resulting biological outcomes. Here, we show the CD40/lipid raft association to be independent of PI-3-kinase, Src family kinases and p38 MAPK pathways. Moreover, CD40 lacking its intracellular domain, which is usually required for CD40-mediated signaling, still localizes to lipid rafts upon engagement, confirming that the CD40/ lipid raft association is independent of signaling events. As to the biological outcomes of the CD40/lipid raft association, we show that disrupting lipid raft integrity selectively abolishes CD40-mediated Akt phosphorylation. In addition, replacing the transmembrane domain of CD40 with that of CD45 (a protein excluded from lipid rafts) dramatically reduced CD40-mediated Akt phosphorylation and B7.1 upregulation, while not influencing p38, ERK and JNK activation. Together, these findings clarify the requirements for CD40/lipid raft association and the signals triggered upon CD40 engagement by CD154.Keywords: CD40 . Lipid rafts . Signaling . Transmembrane domain Supporting Information available online IntroductionInteraction between the TNFR, CD40 and its ligand CD154 drives crucial steps in humoral immune responses [1][2][3]. Notably, the CD40/CD154 interaction is critical for the affinity maturation of immunoglobulins (Ig), the development of long-lived plasma B cells and the clonal expansion of memory B cells [1,4,5]. In the absence of intrinsic tyrosine kinase activity, CD40 transmits its intracellular signal via recruitment of several adaptor proteins, such as JAK3 [6] and TNFR-associated factor (TRAF) proteins [5], to specific domains in the CD40 cytoplasmic tail [4,[7][8][9]. This results in the activation of members of the Src kinase family (such as Lyn and Fyn), and other protein tyrosine kinases (such as Syk and Btk) [10,11]; the activation of PI-3 kinase and phospholipase Cg2 [11] and the activation of the MAP kinases p38, JNK and ERK [12][13][14].Lipid rafts are detergent-resistant plasma membrane microdomains that are enriched in cholesterol and sphingolipids. Being 2358scaffolds of many signaling effectors including lipid-anchored proteins (such as Thy-1 and alkaline phosphatase) and signaling molecules (such as some Src kinase family members), lipid rafts act as platforms to initiate and regulate signal transduction processes in many cellular models [15,16]. Engagement of CD40 molecules leads to CD40 clustering into ceramide-enriched lipid rafts of living human B cells [17,18]. Common early events following CD40 clustering in mouse B cells [19] and in human dendritic cells [20] include Lyn act...

MHC class II association with lipid rafts on the antigen presenting cell surface

“…CD40 weakly associates with MHC-II in resting B cells, and either B cell activation or cross-linking with MHC-II or CD40 mAb greatly increases their association [66]. Unlike MHC-II, only a small portion of the total pool of CD40 is lipid raft associated in resting APCs, but engagement of CD40 with CD40 Ligand (CD154) dramatically enhances CD40 clustering [67] and raft association [68, 69]. Similarly, shortly after CD40-mediated B cell activation total MHC-II and CD80 cell surface expression remains unchanged but there is a rapid increase in clustering of MHC-II with CD80 at the plasma membrane that is blocked by cholesterol depletion [70].…”

Semaphorin 4C: A Novel Component of B-Cell Polarization in Th2-Driven Immune Responses

“…Since lipid rafts frequently contain the proteins and surface molecules that migrate to synapses (19–21), we evaluated if Sema4C and lipid rafts colocalized in B-cells using immunofluorescent staining. Lipid rafts were identified by Cholera Toxin B that binds the lipid raft constituent ganglioside GM1.…”

“…Lipid rafts were isolated according to previously published protocols (19–21). Stimulated and unstimulated isolated murine B-cells (10 8 ) were washed twice in cold serum-free RPMI 1640 and lysed in 600 μL of ice-cold TNE buffer (10 mM Tris, pH 7.5, 150 mM NaCl, and 5 mM EDTA) containing 1% Triton X-100, 2 mM Na 3 VO 4 , and protease inhibitors (Roche Molecular Biochemicals, Montreal, QC, Canada) on ice for 30 min.…”

Scaffold Protein JLP Is Critical for CD40 Signaling in B Lymphocytes

“…Activation of the Akt (but not MAPK or NF-B) signaling pathway has been documented to be dependent on CD40-lipid raft association, which is independent of any signaling events (42). The formation of a signaling complex containing CD40, TRAFs, and other components is required but not sufficient in CD40-dependent MAPK ϩ/ϩ and jlp Ϫ/Ϫ mice were left untreated (0 min) or were treated with rCD154 (1 g/ml) at the indicated time points.…”