Kendrick Tulene S, Lipscombe Richard J, Rausch Oliver, Nicholson Sandra E, Layton Judith E, Goldie-Cregan Lauren C, Bogoyevitch Marie A
Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Western Australia 6009, Australia.
J Biol Chem. 2004 Jan 2;279(1):326-40. doi: 10.1074/jbc.M310144200. Epub 2003 Oct 13.
We have evaluated the contribution of intracellular tyrosine residues of the granulocyte colony-stimulating factor receptor (GCSF-R) to its signaling and cellular outcomes. We began with stable BaF3 cell lines overexpressing wild-type or mutant GCSF-Rs. When all four intracellular tyrosines of the GCSF-R were replaced with phenylalanine (FFFF GCSF-R), cell proliferation and survival were compromised. Replacement of only the membrane-distal tyrosine (YYYF GCSF-R) also showed reduced survival following a GCSF withdrawal/replacement protocol, suggesting a role for this tyrosine. Proliferation by FFFY GCSF-R cells was attenuated by approximately 70%. In evaluating the biochemical steps involved in signaling, we then showed that the membrane-distal tyrosine was necessary and sufficient for c-Jun N-terminal kinase (JNK) activation. With the use of a cell-permeable JNK-inhibitory peptide, JNK was implicated in the proliferation of the FFFY GCSF-R mutant. To further define the events linking the membrane-distal tyrosine and JNK activation, the Src homology 2 domains of Shc, Grb2, and 3BP2 were shown to bind the full-length GCSF-R and a phosphopeptide encompassing the membrane-distal tyrosine. When binding to variant phosphopeptides based on this membrane-distal tyrosine was tested, altering the amino acids immediately following the phosphotyrosine could selectively abolish the interaction with Shc or Grb2, or the binding to both Grb2 and 3BP2. When these changes were introduced into the full-length GCSF-R and new cell lines created, only the mutant that did not interact with Grb2 and 3BP2 did not activate JNK. Our results suggest that direct binding of Shc by the GCSF-R is not essential for JNK activation.
我们评估了粒细胞集落刺激因子受体（GCSF-R）的细胞内酪氨酸残基对其信号传导和细胞结果的贡献。我们从过表达野生型或突变型GCSF-R的稳定BaF3细胞系开始研究。当GCSF-R的所有四个细胞内酪氨酸都被苯丙氨酸取代（FFFF GCSF-R）时，细胞增殖和存活受到损害。仅替换膜远端酪氨酸（YYYF GCSF-R）也显示在GCSF撤除/替换方案后存活率降低，表明该酪氨酸具有一定作用。FFFFY GCSF-R细胞的增殖减弱了约70%。在评估信号传导所涉及的生化步骤时，我们随后表明膜远端酪氨酸对于c-Jun氨基末端激酶（JNK）的激活是必要且充分的。使用细胞可渗透的JNK抑制肽，发现JNK参与了FFFFY GCSF-R突变体的增殖。为了进一步确定连接膜远端酪氨酸和JNK激活的事件，研究表明Shc、Grb2和3BP2的Src同源2结构域可结合全长GCSF-R以及包含膜远端酪氨酸的磷酸肽。当测试与基于该膜远端酪氨酸的变体磷酸肽的结合时，改变磷酸酪氨酸之后的氨基酸可选择性地消除与Shc或Grb2的相互作用，或与Grb2和3BP2两者的结合。当将这些变化引入全长GCSF-R并创建新的细胞系时，只有不与Grb2和3BP2相互作用的突变体未激活JNK。我们的结果表明，GCSF-R直接结合Shc对于JNK激活并非必不可少。
