Role of a preformed signalosome in GnRH-stimulated FAK and paxillin phosphorylation by ERK at focal adhesions: possible role in gonadotropes migration
L. Rahamim-Ben Navi, M. Dobkin-Bekman & Z. Naor
Introduction: We have recently described a preformed signalosome associated with the GnRH receptor (GnRHR), which undergoes dynamic rearrangement upon stimulation with GnRH. This signaling complex included c-Src, focal adhesion kinase (FAK), paxillin, vinculin, tubulin, caveolin-1, protein kinase C (PKC) δ, PKCε, PKCα, Ras, kinase suppressor of Ras-1 (KSR), MAPK kinase (MEK) 1/2, ERK1/2 and the GnRHR. To sort out the role of the signalosomes associated with the GnRHR we investigated the activation of ERK1/2 and its substrates in the complex. First, caveolin-1, FAK, vinculin, and paxillin were phosphorylated on Tyr residues by the GnRH-activated c-Src. Then, GnRH activated ERK1/2 in the complex in a c-Src-dependent manner, and the activated ERK1/2 subsequently phosphorylated FAK and paxillin.
Methods: LβT2 gonadotrope cells were transfected with GnRHR-mCherry and paxillin-GFP, or GnRHR-mCherry and ERK-GFP, stimulated with GnRH and visualized in live imaging.
Results: Addition of GnRH to LβT2 gonadotrope cells transfected with GnRHR-mCherry and ERK-GFP resulted in bleb formation and ERK accumulation in the blebs. Furthermore, the cells seem to be migrating after GnRH stimulation. Also, addition of GnRH to LβT2 gonadotrope cells transfected with GnRHR-mCherry and paxillin-GFP resulted in enrichment of paxillin in focal adhesions in the newly formed blebs.
Conclusion: We therefore propose that the role of the signalosome is to sequester a cytosolic pool of activated ERK1/2 to phosphorylate FAK and paxillin at focal adhesions apparently to mediate gonadotropes migration.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.