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The adaptor protein 3BP2 (c-Abl Src homology 3 domain-binding protein-2, also referred to SH3BP2) is known to play a regulatory role in signaling from immunoreceptors. In mast cells, 3BP2 is rapidly tyrosine phosphorylated by the aggregation of the high affinity IgE receptor and the overexpression of its SH2 domain results in the dramatic suppression of IgE-mediated tyrosine phosphorylation of PLC-alpha, Ca2+ mobilization and degranulation. 3BP2 is a substrate of the protein-tyrosine kinase Syk, which phosphorylates it on Tyr174, Tyr183, and Tyr446 (in the mouse protein). Phosphorylation of Tyr183 promotes the activation of Rac1 through the interaction with the SH2 domain of Vav1. Phosphorylation of Tyr446 induces the binding to the SH2 domain of the upstream protein-tyrosine kinase Lyn and enhances its kinase activity. Thus, 3BP2 has a positive regulatory role in IgE-mediated mast cell activation. In lymphocytes, engagement of T cell or B cell receptors triggers tyrosine phosphorylation of 3BP2. Suppression of the 3BP2 expression by siRNA results in the inhibition of T cell or B cell receptor-mediated activation of NFAT. Genetic analyses reveal that 3BP2 is required for the proliferation of B cells and B cell receptor signaling. Point mutations of the 3BP2 gene cause the rare human inherited disorder cherubism, characterized by excessive bone resorption in the jaw bones. These mutations include substitution and deletion mutations of 3BP2. "Cherubism" mice exhibit increased myeloid cell responses to M-CSF and RANKL leading to the activation of osteoclasts. Further analysis could demonstrate that inhibition of 3BP2 might have therapeutic potential.

Ubiquitin-protein ligase Cbl-b negatively regulates high affinity IgE receptor (FcepsilonRI)-mediated degranulation and cytokine gene transcription in mast cells. In this study, we have examined the role of a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus using the mast cell signaling model. Overexpression of the truncated Cbl-b that lacks the C-terminal region did not suppress the activation of proximal and distal signaling molecules leading to degranulation. FcepsilonRI-mediated tyrosine phosphorylation of Syk, Gab2, and phospholipase C-gamma1, and activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAP kinase), and inhibitor of nuclear factor kappaB kinase (IKK), and generation of Rac1 are unaffected in cells overexpressing the truncated Cbl-b in the lipid raft. On the other hand, FcepsilonRI-mediated transcriptional activation of nuclear factor of activated T cells (NFAT), and transcription of interleukin-3 (IL-3) and IL-4 mRNA are inhibited by overexpression of the truncated variant of Cbl-b. This suppression parallels the re-compartmentalization of specific effector molecules in the lipid raft. These structural and functional analyses reveal the mechanism underlying the selective inhibition of cellular signaling by the truncated variant of Cbl-b related to insulin-dependent diabetes mellitus.

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2, also referred to SH3BP2) regulates immune receptor-mediated signal transduction. In this report we focused on the molecular mechanism of 3BP2 function in B cell receptor (BCR) signaling. Engagement of BCR induces tyrosine phosphorylation of 3BP2. Genetic analysis demonstrated that Syk is critical for BCR-mediated tyrosine phosphorylation of 3BP2. Mutational analysis of 3BP2 revealed that both Tyr(183) and Src homology 2 (SH2) domain are necessary for 3BP2-mediated BCR-induced activation of nuclear factor of activated T cells (NFAT). Point mutation of Tyr(183) or Arg(486) in the SH2 domain of 3BP2 diminished BCR-mediated tyrosine phosphorylation of 3BP2. Endogenous 3BP2 forms a complex with tyrosine-phosphorylated cellular signaling molecules. Peptide binding experiments demonstrated that only phosphorylated Tyr(183) in 3BP2 could form a complex with the SH2 domain(s) of phospholipase Cgamma2 and Vav1 from B cell lysates. These interactions were represented by using bacterial glutathione S-transferase-phospholipase Cgamma2 or -Vav1 SH2 domain. Furthermore, pulldown and Far Western experiments showed that the 3BP2-SH2 domain directly binds to B cell linker protein (BLNK) after BCR stimulation. These results demonstrated that 3BP2 induces the protein complex with cellular signaling molecules through phosphorylation of Tyr(183) and SH2 domain leading to the activation of NFAT in B cells.

Sodium zinc dihydrolipoylhistidinate (DHLHZn) is a compound of Zn(2+)/dihydrolipoic acid derivate complex, which was developed for cosmetic/medical use. To characterize DHLHZn as a novel skin-lightening agent, inhibitory actions of DHLHZn on tyrosinase (including its reaction pathway) have been elucidated in this study. In a B16 melanoma cell system, DHLHZn was active in suppressing the synthesis of melanins as well as alpha-arbutin, well known as a depigmenting drug. Furthermore, in a tyrosinase assay, DHLHZn showed stronger inhibitory effect on DOPAchrome formation than other tyrosinase inhibitors such as kojic acid. Our previous report demonstrated that the sulfhydryl groups of lipoyl motif react with DOPAquinone to form lipoyl DOPA conjugates. We therefore postulated that conjugated products between DHLHZn and DOPAquinone might be formed. Upon reaction of DHLHZn with L-DOPA following tyrosinase-catalyzed oxidation, the formation of DHLH DOPA conjugated products was confirmed by HPLC-tandem mass spectrometry using reserpine as the internal standard. In addition, the inhibitory kinetics analyzed by a Lineweaver-Burk plot exhibited the reversibility of DHLHZn as a competitive inhibitor with a KI value of 0.35 microM. These results indicate that this covalent reaction might contribute to alternating DOPAquinone, which is a tyrosinase reaction product, and result in the competitive inhibitory effect of DHLHZn on DOPAchrome formation. DHLHZn may thus serve as a potentially effective skin-lightening agent, an effectiveness that is based on the compound's covalent scavenging of DOPAquinone resulting in depigmentation.

Previous studies have examined the conjugation of sulfhydryl compounds such as l -cysteine and glutathione with DOPA-quinone following the oxidation of tyrosine and DOPA by tyrosinase. These covalent reactions play a key role in the regulation and metabolism of pigment cells. We report on the first direct evidence for the formation of lipoyl adducts in reactions of thiol groups with DOPA-quinone in dihydrolipoic acid (6,8-dimercaptooctanoic acid [DHLA]). Incubating DHLA with DOPA-quinone followed by tyrosinase-catalyzed oxidation resulted in the three products predicted by HPLC-UV and LC-ESI−-MS analyses for DHLA DOPA conjugates. In the current study, we identified 5-S-lipoyl-DOPA among the principal products isolated by HPLC and characterized by FAB−-MS, ESI−-MS/MS, and 1H NMR, 2D-COSY studies. Collectively, these results suggest that DHLA undergoes sulfhydryl conjugation with DOPA-quinone, pointing to the involvement of thiol-reactive metabolites.

Molecular adaptors/scaffolds have indispensable roles in the activation of lymphocytes. In this report, we have demonstrated the role of tyrosine phosphorylation of an adaptor protein 3BP2 (c-Abl-SH3 domain binding protein-2, also known as SH3BP2) in T cell receptor (TCR)-mediated activation of transcription factor. Short interfering RNA for 3BP2 suppresses the expression level of endogenous 3BP2 and inhibits TCR-mediated activation of interleukin (IL)-2 promoter and nuclear factor of activated T cells (NFAT) element. Engagement of TCR induces tyrosine phosphorylation and lipid raft translocation of 3BP2. The overexpression studies reveal that substitution of 3BP2-Tyr(183), Tyr(446), or Arg(486) in the SH2 domain suppresses TCR-mediated activation of NFAT. Point mutations of 3BP2 cannot affect the translocation of 3BP2 into the lipid raft. Phosphorylation of Tyr(183) is required for the interaction with Vav1, the guanine nucleotide exchanging factor of Rac1. In fact, overexpression of dominant-negative form of Rac1 inhibits TCR-mediated activation of NFAT. Phosphorylation of Tyr(446) recruits the SH2 domain of Lck for the optimal activation of transcription factors. Furthermore, point mutation of Arg(486) in the 3BP2-SH2 domain that couples ZAP-70 to LAT dramatically reduces NFAT activation. These results suggest that the site-directed functions of 3BP2 induce the activation of transcription factors.

Adaptor protein 3BP2 positively regulates the high affinity IgE receptor (FcepsilonRI)-mediated activation of degranulation in mast cells. Genetic study identified the point mutations of 3BP2 gene in human-inherited disease cherubism. The multiple cysts in cherubism lesion of jaw bones are filled with the activated osteoclasts and stromal cells, including mast cells. By over-expression study using rat basophilic leukaemia RBL-2H3 mast cells, we have analysed the effect of the point mutations on the function of 3BP2 protein, which plays a positive regulatory role on FcepsilonRI-mediated mast cell activation. Over-expression of 3BP2 mutants suppressed the antigen-induced degranulation and cytokine gene transcription. Antigen-induced phosphorylation of Vav1, activation of Rac1, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), inhibitor of nuclear factor kappaB kinase (IKK) and nuclear factor of activated T cells (NFAT) were all impaired in the cells over-expressing the cherubism mutants of 3BP2. Furthermore, cherubism mutations of 3BP2 may abrogate the binding ability to interact with chaperone protein 14-3-3. These results demonstrate that over-expression of the mutant form of 3BP2 inhibits the antigen-induced mast cell activation. It suggests that point mutations of 3BP2 gene cause the dysfunction of 3BP2 in vivo.

Antigen-induced aggregation of the high affinity IgE receptor (FcεRI) on mast cells induces degranulation to release chemical mediators, leading to acute allergic inflammation. We have demonstrated that the treatment of rat mast cells, RBL-2H3, with a phenoxazine derivative Phx-1 (2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one) suppresses the antigen-induced degranulation. Biochemical analysis reveals that the complementary signaling pathway through Gab2 and Akt is inhibited by this compound in mast cells. These findings suggest that phenoxazine derivatives may have a therapeutic potential for allergic diseases by inhibiting mast cell degranulation. Keywords:: phenoxazine derivative, mast cell, degranulation