Huang J Y, Umehara H, Inoue H, Tabassam F H, Okazaki T, Kono T, Minami Y, Tanaka Y, Domae N
Department of Internal Medicine, Osaka Dental University, 8-1 Kuzuha Hanazono-cho, Hirakata-shi, 573-1121, Osaka, Japan.
Mol Immunol. 2000 Dec;37(17):1057-65. doi: 10.1016/s0161-5890(01)00020-7.
Natural killer (NK) cells participate in both innate and adoptive immunity by their prompt secretion of cytokines and by their ability to lyse virally infected cells or tumor cells. CD2 is surface glycoprotein receptors and crucial for NK cell activation. However, molecular events involved in CD2-mediated NK cell activation have not been fully elucidated. Cbl-Grb2 and Cbl-CrkL interactions have been implicated in T cell and B cell receptor, and cytokine receptor signaling. Here we analyzed tyrosine phosphorylation and interactions of Cbl with adapter proteins, Grb2 and CrkL, in NK3.3 cells. CD2 crosslinking results in the marked tyrosine phosphorylation of Cbl in an antibody concentration- and time-dependent manner. Immunodepletion studies reveal that Grb2-associated tyrosine phosphorylated p120 kDa protein is Cbl. In vitro binding studies using GST-fusion proteins demonstrate that Cbl constitutively associates with the SH3 domains of Grb2, with a preference for the amino-terminal domain. In addition, we demonstrate that CrkL associates with a large portion of tyrosine phosphorylated Cbl after CD2 stimulation of NK3.3 cells. In contrast to constitutive Cbl association with Grb2, tyrosine phosphorylated Cbl interacts with CrkL via its SH2 domain only after CD2 stimulation. Although the precise roles of interactions of Cbl with Grb2 and CrkL in NK cell activation remains to be elucidated, their tyrosine phosphorylation, in addition to the multiple protein interactions described here, strongly suggest that interactions of Cbl with Grb2 and CrkL may play pivotal roles in CD2-mediated NK cell activation.
自然杀伤（NK）细胞通过迅速分泌细胞因子以及裂解病毒感染细胞或肿瘤细胞的能力，参与固有免疫和适应性免疫。CD2是表面糖蛋白受体，对NK细胞激活至关重要。然而，CD2介导的NK细胞激活所涉及的分子事件尚未完全阐明。Cbl - Grb2和Cbl - CrkL相互作用与T细胞和B细胞受体以及细胞因子受体信号传导有关。在此，我们分析了NK3.3细胞中Cbl与衔接蛋白Grb2和CrkL的酪氨酸磷酸化及相互作用。CD2交联以抗体浓度和时间依赖性方式导致Cbl显著的酪氨酸磷酸化。免疫耗竭研究表明，与Grb2相关的酪氨酸磷酸化的120 kDa蛋白是Cbl。使用GST融合蛋白的体外结合研究表明，Cbl与Grb2的SH3结构域组成性结合，优先结合氨基末端结构域。此外，我们证明在NK3.3细胞经CD2刺激后，CrkL与大部分酪氨酸磷酸化的Cbl相关联。与Cbl与Grb2的组成性结合相反，酪氨酸磷酸化的Cbl仅在CD2刺激后通过其SH2结构域与CrkL相互作用。尽管Cbl与Grb2和CrkL相互作用在NK细胞激活中的确切作用仍有待阐明，但它们的酪氨酸磷酸化，以及此处描述的多种蛋白质相互作用，强烈表明Cbl与Grb2和CrkL的相互作用可能在CD2介导的NK细胞激活中起关键作用。
