All receptor tyrosine kinases (RTKs) activate similar downstream signaling pathways through a common set of effectors, yet it is not fully understood how different receptors elicit distinct cellular responses to cause cell proliferation, differentiation, or other cell fates. We tested the hypothesis that regulation of SRC Family Kinase (SFK) signaling by the scaffold protein, PAG1, influences cell fate decisions following RTK activation. We generated a neuroblastoma cell line expressing a PAG1 fragment that lacks the membrane spanning domain (PAG1^TM-) and localized to the cytoplasm. PAG1^TM- cells exhibited higher amounts of active SFKs and increased growth rate. PAG1^TM- cells were unresponsive to TRKA and RET signaling, two RTKs that induce neuronal differentiation, but retained responses to EGFR and KIT. Under differentiation conditions, PAG1^TM- cells continued to proliferate and did not extend neurites or increase β-III tubulin expression. FYN and LYN were sequestered in multivesicular bodies (MVBs), and dramatically more FYN and LYN were in the lumen of MVBs in PAG1^TM- cells. In particular, activated FYN was sequestered in PAG1^TM- cells, suggesting that disruption of FYN localization led to the observed defects in differentiation. The results demonstrate that PAG1 directs SFK intracellular localization to control activity and to mediate signaling by RTKs that induce neuronal differentiation.

所有受体酪氨酸激酶（RTK）通过一组共同的效应子激活类似的下游信号传导途径，但尚未完全了解不同的受体如何引发不同的细胞反应以引起细胞增殖，分化或其他细胞命运。我们测试了一个假设，即支架蛋白PAG1对SRC家族激酶（SFK）信号的调节会影响RTK激活后的细胞命运决定。我们生成了神经母细胞瘤细胞系，该细胞系表达缺少膜跨结构域（PAG1 ^TM-）并定位于细胞质的PAG1片段。PAG1 ^TM细胞表现出更高数量的活性SFKs和增加的生长速率。PAG1 ^TM-细胞对TRKA和RET信号无反应，这两个RTK诱导神经元分化，但保留了对EGFR和KIT的反应。在分化条件下，PAG1 ^TM-细胞继续增殖并且不延伸神经突或不增加β-III微管蛋白表达。FYN和LYN被隔离在多囊泡体（MVB）中，并且在PAG1 ^TM细胞的MVB内腔中显着增加了FYN和LYN 。特别地，活化的FYN被隔离在PAG1 ^TM细胞中，表明FYN定位的破坏导致观察到的分化缺陷。结果表明，PAG1指导SFK细胞内定位，以控制活性并通过诱导神经元分化的RTK介导信号传导。