Coulpier Muriel, Anders Jonas, Ibáñez Carlos F
Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.
J Biol Chem. 2002 Jan 18;277(3):1991-9. doi: 10.1074/jbc.M107992200. Epub 2001 Nov 16.
The catalytic and signaling activities of RET, a tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF), are controlled by the autophosphorylation of several tyrosine residues in the RET cytoplasmic domain. To analyze the phosphorylation state of individual tyrosines, we generated antibodies recognizing specific phosphotyrosine sites involved in the catalytic (Tyr(905)) and downstream signaling (Tyr(1015), Tyr(1062), and Tyr(1096)) activities of this receptor. Stimulation with GDNF induced coordinated phosphorylation of the 4 tyrosine residues in neuronal cell lines and in primary cultures of sympathetic neurons isolated from rat superior cervical ganglia. Neurturin and artemin, two other members of the GDNF ligand family, also induced synchronized phosphorylation of RET tyrosines with kinetics comparable to those observed with GDNF. Tyrosine phosphorylation was maximal 15 min after ligand stimulation, decaying thereafter with similar kinetics in all 4 residues. Co-stimulation with a soluble form of the GFRalpha1 co-receptor potentiated ligand-dependent phosphorylation of different intracellular tyrosines to a similar extent and increased the survival of superior cervical ganglion neurons compared with treatment with GDNF alone. In vivo, high levels of phosphorylated Tyr(905), Tyr(1015), and Tyr(1062) were detected in embryonic mouse dorsal root ganglia, with a sharp decline at early postnatal stages. Protein transduction of anti-Tyr(P)(1062) antibodies into cultured cells reduced activation of MAPKs ERK1 and ERK2 and the AKT kinase in response to GDNF and diminished GDNF-dependent neuronal differentiation and survival of embryonic sensory neurons from the nodose ganglion. These results demonstrate synchronized utilization of individual RET tyrosine residues in neurons in vivo and reveal an important role for RET Tyr(1062) in mediating neuronal survival by GDNF.
RET是胶质细胞系源性神经营养因子（GDNF）的酪氨酸激酶受体，其催化和信号传导活性受RET胞质结构域中多个酪氨酸残基的自磷酸化作用控制。为了分析单个酪氨酸的磷酸化状态，我们制备了能识别该受体催化活性（Tyr(905)）和下游信号传导活性（Tyr(1015)、Tyr(1062)和Tyr(1096)）中特定磷酸酪氨酸位点的抗体。用GDNF刺激可诱导神经细胞系以及从大鼠颈上神经节分离的交感神经元原代培养物中4个酪氨酸残基的协同磷酸化。神经营养因子（Neurturin）和艺术蛋白（artemin）是GDNF配体家族的另外两个成员，它们也能诱导RET酪氨酸的同步磷酸化，其动力学与GDNF诱导的相似。配体刺激后15分钟酪氨酸磷酸化达到最大值，此后所有4个残基的磷酸化以相似的动力学衰减。与单独用GDNF处理相比，用可溶性形式的GFRα1共受体共同刺激可在相似程度上增强不同细胞内酪氨酸的配体依赖性磷酸化，并提高颈上神经节神经元的存活率。在体内，胚胎小鼠背根神经节中检测到高水平的磷酸化Tyr(905)、Tyr(1015)和Tyr(1062)，在出生后早期阶段急剧下降。将抗Tyr(P)(1062)抗体蛋白转导到培养细胞中，可降低对GDNF反应时丝裂原活化蛋白激酶ERK1和ERK2以及AKT激酶的激活，并减少GDNF依赖的结节神经节胚胎感觉神经元的神经元分化和存活。这些结果证明了体内神经元中单个RET酪氨酸残基的同步利用，并揭示了RET Tyr(1062)在介导GDNF促进神经元存活中的重要作用。
