The C-terminus of the oncoprotein TGAT is necessary for plasma membrane association and efficient RhoA-mediated signaling

M Postma,M A Hink,Y I Wu,J Van Unen,J Goedhart,D Botman,T Yin,T W J Gadella

doi:10.1186/s12860-018-0155-2

M Postma, M A Hink + Show 6 more

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https://doi.org/10.1186/s12860-018-0155-2

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Abstract

BackgroundRho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity.MethodsSince plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest.ResultsThe analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGATΔ15, lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGATΔ15 variant. Synthetic recruitment of TGATΔ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane.ConclusionTogether, these results show that membrane association of TGAT is critical for its activity.

Highlights

Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation
The Dbl homology (DH) domain of p63RhoGEF is highly homologous to the second DH domain from Trio, from which the splice variant Trio-related transforming gene in Adult T-cell leukemia (TGAT) is derived (Additional file 1: Fig. S1)
The location of Yellow fluorescent protein (YFP)-TGAT differed between cells, but we consistently observed that the fluorescence was excluded from the nucleus and increased in a perinuclear compartment (Additional file 2: Fig. S2)

Summary

Introduction

Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. Through remodeling of the F-actin network they regulate several important cellular processes like cell migration, cell adhesion, proliferation and cell shape [3,4,5]. Rho GTPases function as molecular switches that cycle between an active GTPbound form and an inactive GDP-bound form [6]. Rho guanine exchange factors (RhoGEFs) activate Rho GTPases by accelerating the exchange of GDP for GTP [7]. Rho GTPase activating or accelerating proteins (RhoGAPs) are responsible for turning Rho GTPases off by promoting the hydrolysis of the bound GTP to GDP [8]. Deregulation of the Rho GTPase cycle has been mainly investigated within the context of cancer [11] and metastasis [12], but is implicated in other pathologies like neurodegeneration [13], hypertension [14] and hemopathies [15]

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