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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^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 ?-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.

SUBMITTER: Foltz L

PROVIDER: S-EPMC7550700 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Foltz Lauren L Palacios-Moreno Juan J Mayfield Makenzie M Kinch Shelby S Dillon Jordan J Syrenne Jed J Levy Tyler T Grimes Mark M

Molecular biology of the cell 20200729 20

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 f ...[more]

PMID: 32726167

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Project description:Embryonic stem (ES) cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein-tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent Src-family kinase inhibitor A-419259 retained the morphology of domed, pluripotent colonies and continued to express the self-renewal marker TRA-1-60 despite culture under differentiation conditions. Taken together, these observations support a role for Src-family kinase signaling in the regulation of human ES cell fate, and suggest that the activities of individual Src-family members are required for the initiation of the differentiation program.

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Dataset Information

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

Publications

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

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