NFAM1, an immunoreceptor tyrosine-based activation motif-bearing molecule that regulates B cell development and signaling

Abstract

A functional cDNA cloning system was developed by using a retrovirus library encoding CD8-chimeric proteins and a nuclear factor of activated T cells (NFAT)-GFP reporter cell line to identify molecules inducing NFAT activation. By using this strategy, NFAT activating molecule 1 (NFAM1) was cloned as an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cell surface molecule belonging to the Ig superfamily and is predominantly expressed in spleen B and T cells. NFAM1 crosslinking induced ITAM phosphorylation, ZAP-70͞Syk recruitment, NFAT activation, and cytokine production. The mechanism underlying ITAM-dependent signal transduction for lymphocyte activation is well established. Two tyrosines within ITAM are phosphorylated by Src-family kinases upon receptor engagement. Then, ZAP-70/Syk is recruited for binding to the phospho-ITAM and activated (6-8), which transduces a downstream signaling cascade by phosphorylating various adaptor͞effector molecules. Thus, ITAM represents a hallmark of signal modules that convert an Ag recognition event to initiate a signal cascade. The activation of ZAP-70͞Syk induces the activation of calcineurin and nuclear factor of activated T cells (NFAT). NFAT plays important roles in the activation of B, T, and NK cells (9-12). NFAT is rapidly dephosphorylated and translocated into the nucleus upon stimulation, and induces the transcription of various cytokine genes. Therefore, NFAT can be used as a standard readout for lymphocyte activation.We have developed a functional cDNA cloning system [NFAT-activation molecule cloning system (NACS)] for identifying molecules that induce NFAT activation by using a retrovirus cDNA library encoding CD8-chimeric molecules and reporter cells expressing NFAT-GFP, and have succeeded in identifying various cDNA clones that induce NFAT activation, including known surface molecules, kinases, adaptors, and unknown molecules. In particular, we have cloned an ITAM ϩ molecule, NFAM1 (NFAT activating molecule 1). We describe herein that NFAM1 crosslinking induces ITAM phosphorylation, Ca 2ϩ inf lux, and NFAT activation. NFAM1 co-crosslinking in B cells enhances BCR signaling, and its in vivo overexpression results in severe impairment of B cell development.The results suggest that NFAM1 belongs to a category of ITAM ϩ molecules that regulate B cell signaling and development. The successful cloning of various molecules by NACS indicates its applicability to the systematic cloning of functional molecules involved in a defined signaling pathway. Materials and MethodsMice. C57BL͞6 mice were purchased from Nihon SLC (Hamamatsu, Japan). NFAM1-transgenic (Tg) mice were generated by injecting the insert of the pHSE3Ј expression vector (provided by H. Pircher, University of Freiburg, Freiburg, Germany) (13) containing the H-2K b promoter and the IgH enhancer in which mouse NFAM1 cDNA was subcloned.NFAT-GFP Reporter Cell Line. NFAT-GFP construct was prepared by fusing three tandem NFAT-binding sites with enhanced GFP cDNA. T hybridoma 2B4 (14) was transfected ...

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