PAG1 directs SRC-family kinase intracellular localization to mediate receptor tyrosine kinase-induced differentiation

Abstract

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 TMcells exhibited higher amounts of active SFKs and increased growth rate. PAG1 TMcells were unresponsive to TRKA and RET signaling, two RTKs that induce neuronal differentiation, but retained responses to EGFR and KIT. Under differentiation conditions, PAG1 TMcells continued to proliferate and did not extend neurites or increase b-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 TMcells. In particular, activated FYN was sequestered in PAG1 TMcells, 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.

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“…At first, most of the above researches define SFKs as the plasma membrane, interestingly, SFKs are also found to localize to organelle membranes including Golgi membranes and lysosomal membranes and mitochondrial membranes [89]. The intracellular location is dynamic, such as the enrichment of activated Fyn in multivesicular bodies led to a defect in cell differentiation in a neuroblastoma cell line [90]. Next, the study on the pathological mechanism of SFKs in the above-mentioned diseases is not in-depth enough, SFKs can phosphorylate multiple sites of relative proteins in mammalian cells [91], or the presence of bi-phosphorylated SFKs was found in triple-negative breast cancer model [92].…”

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“…Activated FYN was sequestered in PAG1™ − cells, suggesting that disruption of FYN localization led to the observed defects in differentiation. Overall, PAG1 is an additional example of how a scaffold protein may control SFK intracellular localization, impacting on their activity and signaling that induces differentiation events, that may be crucial in the control of NB aggressiveness [76].…”