Uckun F M, Tuel-Ahlgren L, Waddick K G, Jun X, Jin J, Myers D E, Rowley R B, Burkhardt A L, Bolen J B
Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota Health Sciences Center, Minneapolis 55455, USA.
J Biol Chem. 1996 Mar 15;271(11):6389-97. doi: 10.1074/jbc.271.11.6389.
Exposure of human B-cell precursors (BCP) to ionizing radiation results in cell cycle arrest at the G2-M checkpoint as a result of inhibitory tyrosine phosphorylation of p34cdc2 . Here, we show that ionizing radiation promotes physical interactions between p34cdc2 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of human BCP leading to tyrosine phosphorylation of p34cdc2. Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as a full-length glutathione S-transferase (GST)-Lyn fusion protein-phosphorylated recombinant human p34cdc2 on tyrosine 15. Furthermore, Lyn kinase physically associated with and tyrosine-phosphorylated p34cdc2 kinase in vivo when co-expressed in COS-7 cells. Binding experiments with truncated GST-Lyn fusion proteins suggested a functional role for the SH3 rather than the SH2 domain of Lyn in Lyn-p34cdc2 interactions in BCP. The first 27 residues of the unique amino-terminal domain of Lyn were also essential for the ability of GST-Lyn fusion proteins to bind to p34cdc2 from BCP lysates. Ionizing radiation failed to cause tyrosine phosphorylation of p34cdc2 or G2 arrest in Lyn kinase-deficient BCP, supporting an important role of Lyn kinase in radiation-induced G2 phase-specific cell cycle arrest. Our findings implicate Lyn as an important cytoplasmic suppressor of p34cdc2 function.
人B细胞前体（BCP）暴露于电离辐射会导致细胞周期在G2-M期检查点停滞，这是p34cdc2发生抑制性酪氨酸磷酸化的结果。在此，我们表明电离辐射会促进人BCP细胞质中p34cdc2与Src家族蛋白酪氨酸激酶Lyn之间的物理相互作用，从而导致p34cdc2的酪氨酸磷酸化。从受辐照BCP的裂解物中免疫沉淀的Lyn激酶以及全长谷胱甘肽S-转移酶（GST）-Lyn融合蛋白使重组人p34cdc2的酪氨酸15位点发生磷酸化。此外，当在COS-7细胞中共表达时，Lyn激酶在体内与p34cdc2激酶发生物理结合并使其酪氨酸磷酸化。用截短的GST-Lyn融合蛋白进行的结合实验表明，Lyn的SH3结构域而非SH2结构域在BCP的Lyn-p34cdc2相互作用中发挥功能作用。Lyn独特的氨基末端结构域的前27个残基对于GST-Lyn融合蛋白与BCP裂解物中的p34cdc2结合的能力也至关重要。在Lyn激酶缺陷的BCP中，电离辐射未能导致p34cdc2的酪氨酸磷酸化或G2期停滞，这支持了Lyn激酶在辐射诱导的G2期特异性细胞周期停滞中的重要作用。我们的研究结果表明Lyn是p34cdc2功能的重要细胞质抑制因子。
