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RhoA-dependent phosphorylation and relocalization of ERM proteins into apical membrane/actin protrusions in
1Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 12139, USA.
Abstract:
The ERM proteins (ezrin, radixin, and moesin) are a group of band 4. 1-related proteins that are proposed to function as membrane/cytoskeletal linkers. Previous biochemical studies have implicated RhoA in regulating the association of ERM proteins with their membrane targets. However, the specific effect and mechanism of action of this regulation is unclear. We show that lysophosphatidic acid stimulation of serum-starved NIH3T3 cells resulted in relocalization of radixin into apical membrane/actin protrusions, which was blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, was sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment led to phosphorylation of radixin preceding its redistribution into apical protrusions. Significantly, cotransfection of RhoAV14 or C3 transferase with radixin and moesin revealed that RhoA activity is necessary and sufficient for their phosphorylation. These findings reveal a novel function of RhoA in reorganizing the apical actin cytoskeleton and suggest that this function may be mediated through phosphorylation of ERM proteins.
Insights
RhoA signaling is crucial for reorganizing the cell
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The ezrin, radixin, and moesin (ERM) proteins link the cell membrane to the actin cytoskeleton.
- RhoA signaling is known to regulate ERM protein function, but the precise mechanism is unclear.
Purpose of the Study:
- To elucidate the specific role and mechanism of RhoA in regulating ERM protein localization and function.
- To investigate the effect of RhoA on the actin cytoskeleton and ERM protein phosphorylation.
Main Methods:
- Utilized NIH3T3 and Rat1 cell lines.
- Stimulated cells with lysophosphatidic acid (LPA).
- Employed C3 transferase to inhibit RhoA and activated RhoA alleles (RhoAV14).
Main Results:
- LPA stimulation induced radixin relocalization to apical actin protrusions, dependent on RhoA activity.
- Activated RhoA alone was sufficient to induce these protrusions and recruit ERM proteins.
- RhoA activity was necessary and sufficient for ERM protein phosphorylation, preceding their redistribution.
Conclusions:
- RhoA plays a novel role in reorganizing the apical actin cytoskeleton.
- RhoA-mediated phosphorylation of ERM proteins is a key mechanism for this cytoskeletal reorganization.