Mariko Okumura

Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, United States

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Publications (4)37.7 Total impact

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    ABSTRACT: Signaling pathways leading to natural killer (NK) cell effector function are complex and incompletely understood. Here, we investigated the proximal signaling pathways downstream of immunotyrosine-based activation motif (ITAM) bearing activating receptors. We found that the adaptor molecule SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is recruited to microclusters at the plasma membrane in activated NK cells, and this is required for initiation of downstream signaling and multiple NK cell effector functions in vitro and in vivo. Surprisingly, we found that two types of proximal signaling complexes involving SLP-76 were formed. In addition to the canonical membrane complex formed between SLP-76 and linker for activation of T cells (LAT) family members, a novel LAT family-independent SLP-76-dependent signaling pathway was identified. The LAT family-independent pathway involved the SH2 domain of SLP-76 and adhesion and degranulation-promoting adaptor protein (ADAP). Both the LAT family-dependent and ADAP-dependent pathway contributed to IFNγ production and cytotoxicity, but was not essential for other SLP-76-dependent events including phosphorylation of AKT and extracellular signal related kinase and cellular proliferation. These results demonstrate that NK cells possess an unexpected bifurcation of proximal ITAM-mediated signaling, each involving SLP-76 and contributing to optimal NK cell function.
    Blood 02/2013; · 9.78 Impact Factor
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    ABSTRACT: Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1.
    The EMBO Journal 07/2011; 30(15):3160-72. · 9.82 Impact Factor
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    ABSTRACT: Activation through FcepsilonRI, a high-affinity IgE-binding receptor, is critical for mast cell function during allergy. The formation of a multimolecular proximal signaling complex nucleated by the adaptor molecules SLP-76 and LAT1 is required for activation through this receptor. Based on previous T-cell studies, current dogma dictates that LAT1 is required for plasma membrane recruitment and function of SLP-76. Unexpectedly, we found that the recruitment and phosphorylation of SLP-76 were preserved in LAT1(-/-) mast cells and that SLP-76(-/-) and LAT1(-/-) mast cells harbored distinct functional and biochemical defects. The LAT1-like molecule LAT2 was responsible for the preserved membrane localization and phosphorylation of SLP-76 in LAT1(-/-) mast cells. Although LAT2 supported SLP-76 phosphorylation and recruitment to the plasma membrane, LAT2 only partially compensated for LAT1-mediated cell signaling due to its decreased ability to stabilize interactions with phospholipase Cgamma (PLCgamma). Comparison of SLP-76(-/-) LAT1(-/-) and SLP-76(-/-) mast cells revealed that some functions of LAT1 could occur independently of SLP-76. We propose that while SLP-76 and LAT1 depend on each other for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions also mediate a positive signaling pathway downstream of FcepsilonRI in mast cells.
    Molecular and cellular biology 09/2010; 30(17):4188-96. · 6.06 Impact Factor
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    ABSTRACT: Lymphocyte cytosolic protein 2, also known as Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76), is an essential adaptor molecule in myeloid cells, where it regulates FcepsilonRI-induced mast cell (MC) and FcgammaR- and integrin-induced neutrophil (polymorphonuclear leukocyte [PMN]) functions. SLP-76 contains 3 N-terminal tyrosines at residues 112, 128, and 145 that together are critical for its function. We sought to explore the relative importance of tyrosines 112, 128, and 145 of SLP-76 during MC and PMN activation. We examined in vitro MC and PMN functions using cells isolated from knock-in mice harboring phenylalanine substitution mutations at tyrosines 112 and 128 (Y112/128F) or 145 (Y145F). We also examined the effects of these mutations on in vivo MC and PMN activation using models of anaphylaxis, dermal inflammation, and serum-induced arthritis. Mutations at Y112/Y128 and Y145 both interfered with SLP-76 activity; however, Y145F had a greater effect than Y112/128F on most in vitro FcR-induced functions. In vitro functional defects were recapitulated in vivo, where mice expressing Y145F exhibited greater attenuation of MC-dependent passive systemic anaphylaxis and PMN-mediated inflammatory responses. Notably, the Y145F mutation completely protected mice against development of joint-specific inflammation in the MC and PMN-dependent K/B x N model of arthritis. Our data indicate that Y145 is the most critical tyrosine supporting SLP-76 function in myeloid cells. Future efforts to dissect how Y145 mediates SLP-76-dependent signaling in MCs and PMNs will increase our understanding of these lineages and provide insights into the treatment of allergy and inflammation.
    The Journal of allergy and clinical immunology 11/2009; 124(5):1088-98. · 12.05 Impact Factor