Centaurin-α₂ interacts with β-tubulin and stabilizes microtubules.

Paola Zuccotti,Daniele Cartelli,Vittorio Pandini,Elena Battaglioli,Marco Venturin,Michela Stroppi,Paola Riva,Graziella Cappelletti,Alessandro Aliverti,Yanmin Yang

doi:10.1371/journal.pone.0052867

Abstract

Centaurin-α2 is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α2 can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α2 interacting protein, β-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of β-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α2 overexpression in HeLa cells and extraction of soluble (αβ dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α2 mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α2 remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α2 and tubulin confirmed that Centaurin-α2 promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α2 overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α2 role on MT stabilization. Centaurin-α2 interacts with β-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α2 as a new microtubule-associated protein (MAP) increasing MT stability.

Highlights

Human Centaurin-a2, recently renamed ‘‘ARFGAP protein with dual PH domain-containing protein 2’’ (ADAP2) to stress the systematic relationships within the superfamily of ARFGAP proteins, is characterized by a C4-type zinc finger and two PH domains
Centaurin-a2 interacts with b-Tubulin With the aim of identifying novel proteins interacting with human Centaurin-a2, a yeast two-hybrid assay has been carried out using as bait a fusion protein between LexA DNA binding domain and the full length human Centaurin-a2
Centaurin-a2 is an ARFGAP protein showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols, and displaying a GTPaseactivating protein activity towards ADP-ribosylation factor 6 (ARF6) [3]

Summary

Introduction

Human Centaurin-a2, recently renamed ‘‘ARFGAP protein with dual PH (pleckstrin homology) domain-containing protein 2’’ (ADAP2) to stress the systematic relationships within the superfamily of ARFGAP proteins, is characterized by a C4-type zinc finger and two PH domains. Centaurin-a2 has been shown to bind phosphatidylinositol-trisphosphate in vitro [2]. It has a diffuse cytoplasmic localization and has been found to translocate, after Epidermal growth factor (EGF) stimulation, from the cytoplasm to the plasma membrane, where it preferentially binds phosphatidylinositol-3,4-bisphosphate (PIP2), through its C-PH domain [3]. Centaurin-a2 displays a GTPase-activating protein activity on the ADP-ribosylation factor 6 (ARF6), a small GTPase involved in actin cytoskeleton remodelling. The distribution of Centaurin-a2 in intact cells is predominantly cytosolic and, in this context, its biological function is not known

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Conclusion