Sillman A L, Monroe J G
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
J Biol Chem. 1995 May 19;270(20):11806-11. doi: 10.1074/jbc.270.20.11806.
Phospholipase C gamma-catalyzed inositol phospholipid hydrolysis, a critical step in B cell antigen receptor signaling leading to second messenger generation and proliferation, depends upon tyrosine kinase activation. The B cell antigen receptor-associated tyrosine kinases p53/56lyn, p59fyn, p55blk, and p72syk are assumed to participate in receptor-initiated signaling. It is unknown, however, which of these kinases is involved in the tyrosine phosphorylation and resulting activation of phospholipase C gamma in response to antigen receptor cross-linking. We have used a fusion protein containing the tandem src homology-2 (SH2) domains of phospholipase C gamma 1 (PLC gamma 1) to identify B cell kinases which associate with PLC gamma 1. Using an in vitro kinase assay, we demonstrate SH2-dependent association of tyrosine kinase activity from anti-mu-stimulated B cells. The PLC gamma 1 SH2 domains associate with a prominent 70-72-kDa tyrosine phosphoprotein from anti-mu-stimulated, but not resting, B cells. Immunoblotting and secondary immunoprecipitation studies definitively identify this protein as p72syk. These results imply a physical interaction between PLC gamma 1 and p72syk in antigen receptor-stimulated B cells. This conclusion is confirmed by our ability to co-immunoprecipitate p72syk and PLC gamma 1 from lysates of anti-mu-stimulated B cells. These results implicate p72syk in the activation of phospholipase C gamma 1 during B cell antigen receptor signaling.
磷脂酶Cγ催化的肌醇磷脂水解是B细胞抗原受体信号传导中导致第二信使生成和增殖的关键步骤，这一过程依赖于酪氨酸激酶的激活。B细胞抗原受体相关的酪氨酸激酶p53/56lyn、p59fyn、p55blk和p72syk被认为参与受体启动的信号传导。然而，尚不清楚这些激酶中哪一种参与了酪氨酸磷酸化以及由此导致的磷脂酶Cγ在抗原受体交联时的激活。我们使用了一种包含磷脂酶Cγ1（PLCγ1）串联src同源2（SH2）结构域的融合蛋白来鉴定与PLCγ1相关的B细胞激酶。通过体外激酶测定，我们证明了来自抗μ刺激的B细胞的酪氨酸激酶活性存在SH2依赖性关联。PLCγ1的SH2结构域与抗μ刺激而非静止的B细胞中一种突出的70 - 72 kDa酪氨酸磷酸化蛋白相关联。免疫印迹和二次免疫沉淀研究明确将该蛋白鉴定为p72syk。这些结果表明在抗原受体刺激的B细胞中PLCγ1和p72syk之间存在物理相互作用。我们能够从抗μ刺激的B细胞裂解物中共免疫沉淀p72syk和PLCγ1，这证实了这一结论。这些结果表明p72syk在B细胞抗原受体信号传导过程中参与磷脂酶Cγ1的激活。
