Abstract
RhoGAP proteins control the precise regulation of the ubiquitous small RhoGTPases. The Drosophila Crossveinless-c (Cv-c) RhoGAP is homologous to the human tumour suppressor proteins Deleted in Liver Cancer 1–3 (DLC1-3) sharing an identical arrangement of SAM, GAP and START protein domains. Here we analyse in Drosophila the requirement of each Cv-c domain to its function and cellular localization. We show that the basolateral membrane association of Cv-c is key for its epithelial function and find that the GAP domain targeted to the membrane can perform its RhoGAP activity independently of the rest of the protein, implying the SAM and START domains perform regulatory roles. We propose the SAM domain has a repressor effect over the GAP domain that is counteracted by the START domain, while the basolateral localization is mediated by a central, non-conserved Cv-c region. We find that DLC3 and Cv-c expression in the Drosophila ectoderm cause identical effects. In contrast, DLC1 is inactive but becomes functional if the central non-conserved DLC1 domain is substituted for that of Cv-c. Thus, these RhoGAP proteins are functionally equivalent, opening up the use of Drosophila as an in vivo model to analyse pharmacologically and genetically the human DLC proteins.
Highlights
RhoGAP proteins control the precise regulation of the ubiquitous small RhoGTPases
We find that the DLC3 human homolog, which in vertebrate cells localizes at the adherens junctions[18], associates to the basolateral membrane of Drosophila epidermal cells and behaves as Cv-c
No effects on embryo viability or polarity are observed after expression of the Cv-cR601Q-GFP protein where a single conserved catalytic arginine in the GTPase Activating Proteins (GAP) domain is mutated to glutamine, showing that the loss of polarity requires Cv-c’s GAP function[20] (Fig. 2C)
Summary
The Drosophila Crossveinless-c (Cv-c) RhoGAP is homologous to the human tumour suppressor proteins Deleted in Liver Cancer 1–3 (DLC1-3) sharing an identical arrangement of SAM, GAP and START protein domains. Active Rho GTPases control multiple cellular aspects including actin cytoskeleton organization, microtubule dynamics, cell adhesion, cell polarity, endocytosis, progression through cell cycle, differentiation and gene transcription (Reviewed in)[1,2,3,4,5] Such functional diversity for a ubiquitously expressed single regulator requires the tight spatial and temporal control of its activity. Comparison of the Cv-c and DLC RhoGAP sequences reveal they are large proteins with three conserved domains: the GAP domain involved in RhoGTP binding, a protein-protein interaction SAM domain and a lipid binding START domain[9] These domains, organized in the same order, are present in the human DLC1, DLC2 and DLC3 proteins suggesting that they are all functional homologs. DLC1 can function efficiently in Drosophila only if fused to the Drosophila subcellular localization domain
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