Aman M J, Walk S F, March M E, Su H P, Carver D J, Ravichandran K S
Beirne B. Carter Center for Immunology Research and the Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA.
Mol Cell Biol. 2000 May;20(10):3576-89. doi: 10.1128/MCB.20.10.3576-3589.2000.
The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator.
肌醇磷酸酶SHIP与FcγRIIB1受体结合，并在FcγRIIB1介导的抑制B细胞增殖和免疫球蛋白合成过程中发挥关键作用。SHIP功能的分子细节尚未完全明确。虽然肌醇磷酸酶结构域中特征基序的点突变会消除SHIP抑制B细胞中钙流的能力，但对于SHIP在体内进化保守的假定非催化区域的功能却知之甚少。在本研究中，我们通过系统诱变方法确定了SHIP位于氨基酸400至866之间的肌醇磷酸酶结构域。通过用野生型和突变型SHIP重建SHIP缺陷的B细胞系，我们证明仅催化结构域不足以介导FcγRIIB1/SHIP依赖性对B细胞受体信号传导的抑制。表达具有完整磷酸酶活性但缺少最后190个氨基酸的SHIP截短突变体表明，C末端的非催化区域对于抑制性信号传导至关重要。先前确定在与Shc结合中起重要作用的该C末端区域内两个酪氨酸的突变显示对钙流的抑制作用降低。然而，对缺乏Shc的B细胞系的研究表明，不需要形成Shc-SHIP复合物，并且结合这些酪氨酸的其他蛋白质可能在FcγRIIB1/SHIP介导的钙抑制中起重要作用。有趣的是，缺少C末端的SHIP的膜靶向能够恢复这种抑制作用，表明C末端在SHIP在膜上的定位或稳定相互作用中起作用。综上所述，这些数据表明SHIP的非催化性羧基末端190个氨基酸在B细胞中SHIP功能中起关键作用，并且可能在SHIP作为负调节因子发挥作用的其他几个受体系统中起类似作用。
