Pauls Samantha D, Ray Arnab, Hou Sen, Vaughan Andrew T, Cragg Mark S, Marshall Aaron J
Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada;
Department of Immunology, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada; and.
J Immunol. 2016 Sep 1;197(5):1587-96. doi: 10.4049/jimmunol.1600105. Epub 2016 Jul 25.
SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB, it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study, we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly, fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation, suggesting that although BCR and FcγRIIB can both recruit SHIP, this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation, but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP, whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk, as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP, but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling.
SHIP是免疫细胞信号传导的重要调节因子，其功能是使质膜上的磷酸肌醇磷脂酰肌醇3,4,5-三磷酸去磷酸化，并介导蛋白质-蛋白质相互作用。SHIP激活的一个既定模式涉及将其募集到抑制性受体FcγRIIB的磷酸化免疫受体酪氨酸抑制基序。尽管SHIP对FcγRIIB的抑制功能至关重要，但它在由激活免疫受体（如B细胞抗原受体）引发的信号传导中也具有关键的调节功能。在本研究中，我们发现，在人细胞系和原代细胞中，无论是否存在FcγRIIB共连接，在BCR刺激后SHIP均无差别地募集到质膜上。有趣的是，光漂白后荧光恢复分析显示，根据刺激模式不同，SHIP增强型绿色荧光蛋白（SHIP-enhanced GFP）具有不同的迁移率，这表明尽管BCR和FcγRIIB都能募集SHIP，但这是通过不同的分子复合物实现的。SHIP增强型绿色荧光蛋白融合蛋白的诱变显示，SHIP的Src同源2结构域在两种情况下都是必需的，而C末端对于通过BCR刺激进行募集是必需的，但在FcγRIIB共连接时不太重要。药理学抑制剂实验表明，Syk活性是SHIP最佳刺激诱导的膜定位所必需的，而PI3K或Src激酶活性都不是必需的。BCR诱导的SHIP与结合伴侣Shc1的结合依赖于Syk，两个伴侣的酪氨酸磷酸化也是如此。我们的结果表明，FcγRIIB并非唯一能够促进SHIP膜募集的因子，而是通过形成不同的信号复合物来调节其功能。通过Syk依赖机制进行的SHIP膜募集可能是调节免疫受体信号传导的一个重要因素。
