Protocol for structural and biochemical analyses of RhoA GTPase.
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ABSTRACT:
SUBMITTER: Lin Y
PROVIDER: S-EPMC8138865 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding.
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Project description:The Rho family of GTP-binding proteins plays critical roles during myogenesis induction. To elucidate their role later during myogenesis, we have analyzed RhoA function during myoblast fusion into myotubes. We find that RhoA activity is rapidly and transiently increased when cells are shifted into differentiation medium and then is decreased until myoblast fusion. RhoA activity must be down-regulated to allow fusion, because expression of a constitutively active form of RhoA (RhoAV14) inhibits this process. RhoAV14 perturbs the expression and localization of M-cadherin, a member of the Ca2+-dependent cell-cell adhesion molecule family that has an essential role in skeletal muscle cell differentiation. This mutant does not affect N-cadherin and other proteins involved in myoblast fusion, beta1-integrin and ADAM12. Active RhoA induces the entry of M-cadherin into a degradative pathway and thus decreases its stability in correlation with the monoubiquitination of M-cadherin. Moreover, p120 catenin association with M-cadherin is decreased in RhoAV14-expressing cells, which is partially reverted by the inhibition of the RhoA effector Rho-associated kinase ROCK. ROCK inhibition also restores M-cadherin accumulation at the cell-cell contact sites. We propose that the sustained activation of the RhoA pathway inhibits myoblast fusion through the regulation of p120 activity, which controls cadherin internalization and degradation.
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