Tyrosine Phosphorylation of 3BP2 Regulates B Cell Receptor-mediated Activation of NFAT

Shukla, Usha; Hatani, Tomoko; Nakashima, Kenichiro; Ogi, Kazuhiro; Sada, Kiyonao

doi:10.1074/jbc.m109.049999

Cited by 38 publications

(74 citation statements)

References 49 publications

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“…NFATc1 is regulated by the activation of PLCγ2 in osteoclasts (13). Moreover, PLCγ2 phosphorylation is increased following 3BP2 overexpression (14). We examined the phosphorylation status of PLCγ2 in osteoclasts and showed that the levels of phospho-PLCγ2 were similar in wild-type and Sh3bp2 -/-mice during in vitro osteoclastogenesis (Supplemental Figure 2C).…”

Section: Figurementioning

confidence: 99%

3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions

Levaot¹,

Simoncic²,

Dimitriou³

et al. 2011

J. Clin. Invest.

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A fine balance between bone resorption by osteoclasts and bone formation by osteoblasts maintains bone homeostasis. In patients with cherubism, gain-of-function mutations in 3BP2, which is encoded by SH3-domain binding protein 2 (SH3BP2), cause cystic lesions with activated osteoclasts that lead to craniofacial abnormalities. However, little is known about the function of wild-type 3BP2 in regulating bone homeostasis. Here we have shown that 3BP2 is required for the normal function of both osteoblasts and osteoclasts. Initial analysis showed that Sh3bp2 -/-mice developed osteoporosis as a result of reduced bone formation despite the fact that bone resorption was impaired. We demonstrated using reciprocal bone marrow chimeras, a cellintrinsic defect of the osteoblast and osteoclast compartments in vivo. Further, Sh3bp2 -/-osteoblasts failed to mature and form mineralized nodules in vitro, while Sh3bp2 -/-osteoclasts spread poorly and were unable to effectively degrade dentine matrix in vitro. Finally, we showed that 3BP2 was required for Abl activation in osteoblasts and Src activation in osteoclasts, and demonstrated that the in vitro defect of each cell type was restored by the respective expression of activated forms of these kinases. These findings reveal an unanticipated role for the 3BP2 adapter protein in osteoblast function and in coordinating bone homeostatic signals in both osteoclast and osteoblast lineages. Introduction3BP2 is an adapter protein that contains an N-terminal pleckstrin homology (PH) domain, a proline-rich stretch that binds to Src homology 3 (SH3) domain-containing proteins, and a C-terminal SH2 domain that binds to phosphotyrosine residues (1). 3BP2 was initially identified as a binding protein of the tyrosine kinase Abl (2). Work from our lab and others has identified the Src family kinases (SFKs), Syk, and the Vav family of Rho guanine nucleotide exchange factors (GEFs) as 3BP2-binding partners (1), all of which are known to play important roles in osteoclast function (3-5). Cherubism is a dominantly inherited syndrome characterized by excessive maxillary and mandibular bone resorption that is associated with activated osteoclasts and inflammatory cells creating interosseous cystic lesions (6). Single missense mutations in the gene encoding the adapter protein 3BP2 result in a gain-of-function alteration in the protein and is associated with the majority of cherubism patients (7). A mouse model that harbors 2 copies of a cherubism allele develops severe osteoporosis associated with highly activated osteoclasts (8).In order to elucidate the role of the wild-type form of 3BP2 in bone homeostasis, we analyzed loss-of-function mutant mice. As distinct from the osteoporotic phenotype of mice expressing the cherubism gain-of-function form of 3BP2 associated with active osteoclasts, we uncovered a complex bone phenotype in mice lacking 3BP2 characterized by loss-of-function in both the osteoclast and osteoblast lineages resulting in net decreased bone mineral density and reduced mechanical ...

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Section: Figurementioning

confidence: 99%

3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions

Levaot¹,

Simoncic²,

Dimitriou³

et al. 2011

J. Clin. Invest.

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“…After the stimulation, cells were washed with ice-cold PBS twice and solubilized in the lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na 3 VO 4 , 1 mM PMSF, and 2 g/ml aprotinin) containing 1% Triton X-100. Preparation of detergentsoluble cell lysates, immunoprecipitation, and immunoblotting were performed as described previously (32)(33)(34). Pull-down Assay-The GST-rat Syk-SH2 (both N-and C-terminal SH2 domains) expression construct was a gift from Dr. Reuben P. Siraganian.…”

Section: Methodsmentioning

confidence: 99%

“…5 ϫ 10 6 cells were incubated without or with 100 g/ml curdlan in medium and were solubilized with the lysis buffer containing 1% Triton X-100. In vitro binding experiments were performed as described previously (32)(33)(34).…”

Section: Methodsmentioning

confidence: 99%

Dectin-1-mediated Signaling Leads to Characteristic Gene Expressions and Cytokine Secretion via Spleen Tyrosine Kinase (Syk) in Rat Mast Cells

Kimura

Chihara

Honjoh

et al. 2014

Journal of Biological Chemistry

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Background: ␤-Glucan receptor Dectin-1 in dendritic cells and macrophages plays important roles in antifungal immunity. Results: Dectin-1 is expressed in rat mast cells, and its tyrosine phosphorylation induces characteristic gene expression of transcription factors and cytokines through protein-tyrosine kinase Syk. Conclusion: Dectin-1 functions in rat mast cells. Significance: Dectin-1-mediated signaling in mast cells may contribute to antifungal immunity.

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“…These scaffolds transduce signals to the cytoplasm, cytoskeleton, and nucleus to activate lipid and calcium signaling, gene expression, and metabolic changes involved in leukocyte proliferation, differentiation, and motility. We and others have identified a regulatory role for 3BP2 (c-Abl SH3 domainbinding protein-2, also known as SH3BP2) in immunoreceptor signaling, including T (21,22), B (23)(24)(25)(26), NK (27), and mast (28) cells. Importantly, 3BP2 associates several signaling proteins such as Src/Syk kinases, Vav proteins, and PLC␥, involved in NFAT activation (reviewed in Refs.…”

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confidence: 99%

3BP2 Adapter Protein Is Required for Receptor Activator of NFκB Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells

Guezguez

Prod’homme

Mouska

et al. 2010

Journal of Biological Chemistry

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The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.

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Tyrosine Phosphorylation of 3BP2 Regulates B Cell Receptor-mediated Activation of NFAT

Cited by 38 publications

References 49 publications

3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions

3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions

Dectin-1-mediated Signaling Leads to Characteristic Gene Expressions and Cytokine Secretion via Spleen Tyrosine Kinase (Syk) in Rat Mast Cells

3BP2 Adapter Protein Is Required for Receptor Activator of NFκB Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells

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