Tridandapani S, Pradhan M, LaDine J R, Garber S, Anderson C L, Coggeshall K M
Department of Microbiology, Ohio State University, OH 43210, USA.
J Immunol. 1999 Feb 1;162(3):1408-14.
Our recent studies revealed that the inositol phosphatase Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP) is phosphorylated and associated with Shc exclusively under negative signaling conditions in B cells, which is due to recruitment of the SHIP SH2 domain to the FcgammaRIIb. In addition, we reported that SHIP-Shc interaction involves both SHIP SH2 and Shc phosphotyrosine binding domains. These findings reveal a paradox in which the single SH2 domain of SHIP is simultaneously engaged to two different proteins: Shc and FcgammaRIIb. To resolve this paradox, we examined the protein interactions of SHIP. Our results demonstrated that isolated FcgammaRIIb contains SHIP but not Shc; likewise, Shc isolates contain SHIP but not FcgammaRIIb. In contrast, SHIP isolates contain both proteins, revealing two separate pools of SHIP: one bound to FcgammaRIIb and one bound to Shc. Kinetic studies reveal rapid SHIP association with FcgammaRIIb but slower and more transient association with Shc. Affinity measurements using a recombinant SHIP SH2 domain and phosphopeptides derived from FcgammaRIIb (corresponding to Y273) and Shc (corresponding to Y317) revealed an approximately equal rate of binding but a 10-fold faster dissociation rate for FcgammaRIIb compared with Shc phosphopeptide and yielding in an affinity of 2.1 microM for FcgammaRIIb and 0.26 microM for Shc. These findings are consistent with a model in which SHIP transiently associates with FcgammaRIIb to promote SHIP phosphorylation, whereupon SHIP binds to Shc and dissociates from FcgammaRIIb.
我们最近的研究表明，含肌醇磷酸酶Src同源2（SH2）结构域的肌醇磷酸酶（SHIP）仅在B细胞的负信号条件下被磷酸化并与Shc结合，这是由于SHIP的SH2结构域被招募到FcγRIIb。此外，我们报道SHIP与Shc的相互作用涉及SHIP的SH2结构域和Shc的磷酸酪氨酸结合结构域。这些发现揭示了一个矛盾之处，即SHIP的单个SH2结构域同时与两种不同的蛋白质结合：Shc和FcγRIIb。为了解决这个矛盾，我们研究了SHIP的蛋白质相互作用。我们的结果表明，分离出的FcγRIIb含有SHIP但不含有Shc；同样，Shc分离物含有SHIP但不含有FcγRIIb。相比之下，SHIP分离物同时含有这两种蛋白质，揭示了SHIP的两个独立池：一个与FcγRIIb结合，一个与Shc结合。动力学研究表明，SHIP与FcγRIIb的结合迅速，但与Shc的结合较慢且更短暂。使用重组SHIP SH2结构域和源自FcγRIIb（对应于Y273）和Shc（对应于Y317）的磷酸肽进行的亲和力测量显示，结合速率大致相等，但FcγRIIb的解离速率比Shc磷酸肽快10倍，FcγRIIb的亲和力为2.1微摩尔，Shc的亲和力为0.26微摩尔。这些发现与一个模型一致，即SHIP与FcγRIIb短暂结合以促进SHIP磷酸化，随后SHIP与Shc结合并从FcγRIIb解离。
