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Nck, a Missing Adaptor Between the B-Cell Receptor Complex and the BCAP/PI3K/Akt Pathway

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Nck, a Missing Adaptor Between the B-Cell Receptor Complex and the BCAP/PI3K/Akt Pathway Cellular & Molecular Immunology (2014) 11, 120–122 ß 2014 CSI and USTC. All rights reserved 1672-7681/14 $32.00 www.nature.com/cmi RESEARCH HIGHLIGHT Nck, a missing adaptor between the B-cell receptor complex and the BCAP/PI3K/Akt pathway Chuen-Miin Leu Cellular & Molecular Immunology (2014) 11, 120–122; doi:10.1038/cmi.2013.53; published online 25 November 2013 he signaling pathways activated by the recruitment of the Syk tyrosine reductions in protein tyrosine phospho- T the B-cell antigen receptor (BCR) kinase (Figure 1) to the BCR complex rylation and calcium influx in the Nck- play a crucial role in the development, and induces Syk phosphorylation and deficient cell line (Ref. 9, Figure 1c–e), activation, and differentiation of B cells. activation, which subsequently induces suggesting an important role for Nck in Among them, the activation of phospha- the phosphorylation of an adaptor pro- BCR signaling. tidylinositol 3-kinase (PI3K)/Akt is par- tein, SLP-65 (also known as BLNK).10,11 To study whether Nck is recruited to ticularly important.1–5 The recruitment Phosphorylated SLP-65 provides docking BCR signalosomes, which are platforms of PI3K to the plasma membrane is sites for phospholipase C-c2, Bruton’s for efficient B-cell activation, the authors necessary for the generation of phospha- tyrosine kinase,12,13 and another adaptor used total internal reflection fluorescence tidyl-inositol-3,4,5-triphosphate, which protein, Grb2,10 which initiates the activa- microscopy to observe the dynamic activates PDK1 (Akt/PKB kinase); in tion of the Ras/Raf/MEK/ERK kinase cas- interactions of the surface molecules on turn, PDK1 activates Akt (Figure 1). cade (Figure 1). B cells. Because the resolution of total The BCR coreceptor CD19 and a cyto- The Nck family is another group of internal reflection fluorescence micro- plasmic adaptor, BCAP (B-cell adaptor adaptors that contains three Src-homo- scopy can reach the single-molecule level, for PI3K), are major molecules respons- logy 3 (SH3) domains at its N-terminus it can be used to quantitate the asso- ible for the recruitment of PI3K to the and 1 SH2 domain at its C-terminus. ciation of Nck with other molecules in BCR complex through direct binding Two members in the Nck family exist BCR signalosomes. Meanwhile, to mimic with PI3K.6–8 For more than a decade, in mice and humans (Nck1 and Nck2). the interaction of B cells with antigen- how BCAP approaches the BCR complex Nck proteins control the cytoskeletal presenting cells, Castello and colleagues and brings PI3K to the plasma membrane rearrangement and mobility of kidney used a planar lipid bilayer containing a has remained unknown. Recently, Castello podocytes and neurons.14,15 In the fluorochrome-labeled anti-IgM antibody et al.9 identified the Nck proteins as key immune system, Nck is known to assoc- to induce BCR signaling. The analyses adaptors linking BCAP/PI3K to the BCR iate with the T-cell receptor and regulate showed the colocalization of Nck with complex.9 T-cell receptor signaling and T-cell SLP-65, Syk and antigens in a Lyn- Antigen-triggered BCR crosslinking development.16 However, the role of dependent manner in B cells. In summary, induces the tyrosine phosphorylation of Nck in B cells is largely unknown, Nck is recruited to BCR signalosomes and immunoglobulin-a (Iga, CD79A) and although SLP-65 is known to bind Nck.10 is required for efficient activation of signal immunoglobulin-b (Igb, CD79B) by Based on these previous observations, transduction. Src family kinases such as Lyn. BCR Castello and colleagues proposed that The next key question was how Nck engagement also stimulates the tyrosine Nck might play a role in BCR signaling, was recruited to the BCR complex. phosphorylation of CD19. The phos- and they tested this hypothesis using bio- Analytical mass spectrometry demon- phorylation of Iga and Igb stimulates chemical and genetic methods. First, strated the association of Iga and Igb they purified Nck-interacting proteins with Nck. To confirm this result, the Institute of Microbiology and Immunology, after BCR activation by affinity purifica- authors used His-tagged Nck proteins National Yang-Ming University, Taipei, Taiwan Correspondence: Dr CM Leu, Institute of tion and identified the co-precipitated as probes to perform a binding assay with Microbiology and Immunology, National Yang- proteins by mass spectrometry. They immobilized peptides corresponding to Ming University, 155 Sec. 2, Li-Nong St., Taipei found that the m-heavy chain of the the human Iga cytoplasmic domain. City 11221, Taiwan. BCR associated with Nck. Furthermore, They found that Nck proteins bound E-mail: [email protected] Received: 26 September 2013; Revised: 30 they deleted the NCK gene in the chicken Iga peptides containing phosphorylated September 2013; Accepted: 1 October 2013 DT40 B-cell line and observed significant Tyr204, which is a non-ITAM tyrosine Research Highlight 121 s-lgM s-lgM CD19 Antigen lgα/β lgα/β Lyn Lyn Lyn PI3K Syk Syk Btk Nck P Y204 SH3SH3 SH3 SH2 PIP3 SLP-65 BCAP PLC-γ2 Grb2 PI3K Sos PDK1 IP DAG 3 Ras Akt β PKC- Ca2+ Metabolism MAPK activation NF-κB activation Protein Foxo1 Bad Proliferation synthesis Survival Figure 1 Nck proteins as the newly identified adaptors between the BCR complex and BCAP/PI3K/Akt pathway. After BCR crosslinking, Nck proteins bind to phosphorylated Tyr204 at the tail of Iga via their SH2 domains to recruit BCAP through their SH3 domains. Nck may control BCR signaling by activating the BCAP/PI3K/Akt pathway and cross-talking with other pathways. BCAP, B-cell adaptor for PI3K; BCR, B-cell antigen receptor; Iga, immunoglobulin-a; PI3K, phosphatidylinositol 3-kinase; SH3, Src-homology 3. residue. Castello et al. further used an lineage. B-cell development appeared to the affinity maturation or the memory assay called ‘biolayer interferometry’ to be normal in Nck1-KO and Nck1–Nck2- responses were observed in the Nck-KO measure the avidity of Nck proteins KO mice, although a decrease in B1a cells mice. Overall, these experiments demon- and phosphorylated Tyr204 peptides. was observed. However, Nck-deficient B strated the importance of Nck in B-cell Biolayer interferometry is a method used cells were demonstrated to have defects function in vivo, particularly in the T- to assess the affinity and kinetics of pro- in BCR-induced calcium influx, prolif- independent response. tein–protein interactions in real time. In eration, and cell survival. In contrast, To delineate the mechanism whereby this method, one protein is coated on the IL-4- and BAFF-mediated cell survival Nck controls B-cell activation, the tip of a biosensor, and the other is added and the antigen presentation ability of authors re-examined their mass spectro- to the solution. Any protein binding to Nck-deficient B cells were comparable metry data and observed an association the immobilized protein will generate a to that of wild-type B cells in vitro. between BCAP and Nck. Using the pep- wavelength shift in the interference pat- More importantly, T-independent and tide-binding assay, they found that the tern of white light that is reflected from early T-dependent antibody responses Nck SH3 domains, particularly the sec- the inside and outside surfaces of the bio- were suppressed in the Nck1- and ond domain, interacted with the proline- sensor. Using this technique, this group Nck1–Nck2-KO mice after immuniza- rich domain of BCAP. The analyses of confirmed that Nck proteins could bind tion. In accordance with the reduced biolayer interferometry and total internal to phosphorylated Tyr204 on Iga in humoral response, the number of hap- reflection fluorescence microscopy con- vitro. ten-specific, IgM-secreting cells was sig- firmed the binding of BCAP to Nck after To study the role of Nck in B cells in nificantly reduced in the KO mice. BCR stimulation. Notably, they showed vivo, the authors generated conventional However, no substantial differences in that Nck was necessary for BCAP recruit- Nck1-knockout (KO) and conditional the frequencies of antigen-specific, IgG- ment to BCR signalosomes and for Nck2-KO mice lacking Nck2 in the B secreting cells or germinal center B cells, the tyrosine phosphorylation of BCAP Cellular & Molecular Immunology Research Highlight 122 (Ref. 9, Figure 6e–g). This discovery cells, macrophages, dendritic cells and insert region to bind phosphatidylinositol 3- reveals how BCAP is linked to the BCR kinase. Science 1993; 260: 986–989. NK cells. In the study conducted by 7 Okada T, Maeda A, Iwamatsu A, Gotoh K, complex for the first time. Castello et al., the ablation of Nck1 in Kurosaki T. BCAP: the tyrosine kinase To consolidate their findings, the other cell types may have disturbed their substrate that connects B cell receptor to phosphorylation of Akt and its substrate, interactions with B cells, which could phosphoinositide 3-kinase activation. Foxo-1, was examined. As expected, the Immunity 2000; 13: 817–827. have led to the observed reductions in 8 Aiba Y, Kameyama M, Yamazaki T, Tedder phosphorylation of Akt and Foxo-1 was the T-dependent and T-independent TF, Kurosaki T. Regulation of B-cell partially reduced in Nck1-deficient B humoral responses. Because Nck pro- development by BCAP and CD19 through cells; however, it was nearly completely teins regulate T-cell signaling and syn- their binding to phosphoinositide 3-kinase. abolished in Nck1–Nck2-deficient B cells 16 Blood 2008; 111: 1497–1503. apse formation, we should be careful 9 Castello A, Gaya M, Tucholski J, Oellerich T, (Ref. 9, Figure 7d and e). These results when interpreting these results from Lu KH, Tafuri A et al.Nck-mediated were somewhat striking because BCAP- Nck1–Nck2-KO mice.
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