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Regulation of the invasion suppressor Arpin by Tankyrases

Abstract : The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. Recently, an inactivator of the Arp2/3 complex at the lamellipodium tip, a small protein, Arpin, was discovered and characterized. On its C-terminus, Arpin possesses an acidic (A) motif, which is homologous to the A-motif of various Nucleation Promoting Factors (NPFs). It was predicted that Arpin can bind at two binding sites to the Arp2/3 complex, similar to VCA domains of NPFs. Here, we used single particle electron microscopy to obtain a 3D reconstruction of the Arp2/3 complex bound to Arpin at a 25Å resolution. We showed that the binding of Arpin causes the standard open conformational of the Arp2/3 complex. We confirmed that there are two binding sites on the Arp2/3 complex for Arpin: one on the back of the Arp3 subunit, and the second is located between Arp2 and ARPC1 subunits. The distance between the Arp2/3 complex and Arpin (5 nm) supports the view that Arpin interacts with its partner via its unstructured C-terminal acidic tail.Next, using the pull-down assay, we identified the new Arpin binding partners, Tankyrases1/2. Interestingly, Tankyrases and the Arp2/3 complex possess overlapping amino acid sequences at Arpin binding sites. Hence, we demonstrated a competition between the ARC4 domain of Tankyrase1 and the Arp2/3 complex in a dose-dependent manner.To understand the principles of Tankyrases-Arpin interaction, we created a mutant Arpin (ArpinG218D) that lacks its ability to interact with Tankyrases, but not with the Arp2/3 complex in vitro. Interestingly, ArpinG218D was not able to inhibit the Arp2/3 complex in vivo, suggesting that Tankyrase may be necessary for Arpin-Arp2/3 complex interaction. Arpin is the turning factor of migrating cells, so we performed a migration analysis of MCF10-A cells expressing either wild type Arpin (ArpinWT) or mutant ArpinG218D in parallel with the depletion of endogenous Arpin. Cells expressing ArpinG218D had higher directional persistence, similar to the cells where the endogenous Arpin was knocked down. Thus, we suggested that mutant ArpinG218D cannot inactivate the Arp2/3 complex since it is not present at the lamellipodial tip. We compared the amount of protein for both ArpinWT and ArpinG218D in the membrane fraction of the migrating cells. A significant difference (44%) in the amount of ArpinWT and Arpin G218D was consistent with our hypothesis.Tankyrases are therapeutic targets in a variety of cancers, but currently there is no structural model available for these large and flexible proteins. In this work, we obtained for the first time two 3D reconstructions of full-length Tankyrase1 and Tankyrase1 bound to Arpin using single particle electron microscopy. The achieved resolution (27Å) was enough to detect a dramatic conformational change in Tankyrase SAM and PARP domains upon binding of Arpin molecules. In our reconstruction, three Arpins were bound to the ARC1, ARC4 and ARC5 domains of Tankyrase1. ARC5 was shown to be the most flexible part of the ARC cluster.Based on the obtained data, we suggested a model of regulation of the activity of Arpin by Tankyrases. According to our model, Tankyrases bind Arpin in the cytoplasm, change their conformational state and bring Arpin closer to the membrane in the lamellipodia. Deciphering the extracellular signals, Rac GTPase activates Arpin, which sequentially inactivates the Arp2/3 complex, while Tankyrases are released.
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  • HAL Id : tel-02931476, version 1

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Angelina Chemeris. Regulation of the invasion suppressor Arpin by Tankyrases. Cellular Biology. Université Paris Saclay (COmUE); Moskovskij gosudarstvennyj universitet imeni M. V. Lomonosova, 2018. English. ⟨NNT : 2018SACLX073⟩. ⟨tel-02931476⟩

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