Scharenberg A M, Lin S, Cuenod B, Yamamura H, Kinet J P
Molecular Allergy and Immunology Section/NIAID/NIH, Rockville, MD 20852, USA.
EMBO J. 1995 Jul 17;14(14):3385-94. doi: 10.1002/j.1460-2075.1995.tb07344.x.
High affinity IgE receptor (Fc epsilon RI) signaling after contact with antigen occurs in response to receptor clustering. This paper describes methodology, based on vaccinia virus driven protein expression, for probing signaling pathways and its application to Fc epsilon RI interactions with the lyn and syk tyrosine kinases. Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non-hematopoietic 'null' cell line is sufficient to reconstruct clustering-controlled receptor tyrosine phosphorylation and activation of syk, without apparent requirement for hematopoietic specific phosphatases. The src family kinase lyn phosphorylates Fc epsilon RI in response to receptor clustering, resulting in syk binding to the phosphorylated Fc epsilon RI. Lyn also participates in the tyrosine phosphorylation and activation of syk in a manner which is dependent on phosphorylated Fc epsilon RI. Using overexpression of active and dominant negative syk proteins in a mast cell line which naturally expresses Fc epsilon RI, we corroborate syk's role downstream of receptor phosphorylation, and demonstrate that syk SH2 domains protect receptor ITAMs from ongoing dephosphorylation. Based on these results, we propose that receptor clustering controls lyn-mediated Fc epsilon RI tyrosine phosphorylation by shifting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI tyrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allowing clustering to indirectly control syk activity.
与抗原接触后，高亲和力IgE受体（FcεRI）信号因受体聚集而产生。本文描述了基于痘苗病毒驱动的蛋白质表达来探究信号通路的方法及其在FcεRI与lyn和syk酪氨酸激酶相互作用中的应用。在非造血“缺失”细胞系中重建完整的四聚体FcεRI受体、lyn和syk足以重建聚集控制的受体酪氨酸磷酸化以及syk的激活，而显然不需要造血特异性磷酸酶。src家族激酶lyn响应受体聚集使FcεRI磷酸化，导致syk与磷酸化的FcεRI结合。Lyn还以依赖磷酸化FcεRI的方式参与syk的酪氨酸磷酸化和激活。通过在天然表达FcεRI的肥大细胞系中过表达活性和显性负性syk蛋白，我们证实了syk在受体磷酸化下游的作用，并证明syk SH2结构域可保护受体免疫受体酪氨酸激活基序（ITAM）免于持续去磷酸化。基于这些结果，我们提出受体聚集通过将磷酸化和去磷酸化之间的平衡转向酪氨酸磷酸化的FcεRI积累来控制lyn介导的FcεRI酪氨酸磷酸化。FcεRI酪氨酸磷酸化的作用是将syk带入一个微环境，在那里它被酪氨酸磷酸化并激活，从而使聚集能够间接控制syk活性。
