Abstract
The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and (1)H,(13)C,(15)N-based NMR spectroscopy. The two domains assume a "chair" shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.
Highlights
The lymphoid blast crisis (Lbc) oncoprotein stimulates deregulated GTPase activity in RhoA
The minimal structural unit that is stably folded spans residues Gly2186–Glu2346. This represents what we term the dbl homology (DH)␣pleckstrin homology (PH) fold in recognition of the obligate integration of the PH fold with the last helix of the DH domain
The binding of RhoA-GTP by the PH domain does not compete with the guanine nucleotide exchange factor (GEF) activity of the DH domain but rather constitutes a possible mechanism of regulation by orientation of onco-Lbc on the membrane by a PH domain that does not itself contain membrane-interacting sites
Summary
The Lbc oncoprotein stimulates deregulated GTPase activity in RhoA. Results: the Lbc DH domain can independently activate GTP exchange by RhoA, its PH domain presents surfaces for DH and activated RhoA interaction. The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). Relative to AKAP-Lbc, the oncogenic form, onco-Lbc, contains only the DH-PH tandem as well as a 70-residue N-terminal extension comprising residues 1922–2346 and induces constitutive GEF activity It induces cell transformation in a Rho-dependent manner [11]. Cardiac hypertrophy and remodeling of the heart following stress involve AKAP-Lbc signaling [13] Together these findings suggest that the Lbc family forms a critical trigger for mitogenic signaling, deregulation of which has dire consequences. The tandem DH-PH module is a prime target as it provides the core functionality required for RhoGEF activation It captures the GDP-bound RhoA and stabilizes the nucleotide-free form until GTP is loaded and released. By mapping and mutating the key residues, the mechanisms by which DH and PH domains communicate and integrate signals to control GTPase activity are revealed
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