Watcharasit P, Tucholski J, Jope R S
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, 35294-0017, USA.
Neurochem Res. 2001 Jul;26(7):809-16. doi: 10.1023/a:1011612118779.
Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.
在分化的人神经母细胞瘤SH-SY5Y细胞中检测了毒蕈碱受体介导的蛋白酪氨酸磷酸化变化。用1 mM卡巴胆碱处理分化细胞导致粘着斑激酶（FAK）、Cas和桩蛋白的酪氨酸磷酸化迅速增加。src家族激酶选择性抑制剂PP1使卡巴胆碱刺激的FAK、Cas和桩蛋白的酪氨酸磷酸化降低了50%至75%。相比之下，卡巴胆碱刺激的ERK1/2激活不受PP1影响。卡巴胆碱诱导的src底物p120的酪氨酸磷酸化增加以及src家族激酶激活相关的自磷酸化位点的酪氨酸磷酸化进一步证明了卡巴胆碱对src家族激酶的激活作用。使用位点特异性FAK磷酸酪氨酸抗体来确定卡巴胆碱刺激的FAK自磷酸化增加不受PP1预处理的影响，而卡巴胆碱刺激的src家族激酶介导的FAK磷酸酪氨酸增加被PP1预处理完全阻断。在稳定过表达Fyn的SH-SY5Y细胞系中，FAK的磷酸酪氨酸免疫反应性是对照细胞的625%。因此，毒蕈碱受体在分化细胞中激活蛋白酪氨酸磷酸化，并且FAK、Cas和桩蛋白的酪氨酸磷酸化，而非ERK1/2的酪氨酸磷酸化，是由响应毒蕈碱受体刺激而激活的src家族酪氨酸激酶介导的。
