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Elongation factor-1 alpha is a novel substrate of rho-associated kinase
1Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Abstract:
Rho-associated kinase (Rho-kinase), which is activated by the Rho small GTPase, phosphorylates the myosin-binding subunit (MBS) of myosin phosphatase, myosin light chain (MLC), the ERM family proteins, and adducin, thereby regulating the formation of stress fibers, focal adhesions, microvillus formation, and cell motility. Here, to further understand the role of Rho-kinase in the regulation of the numerous cellular processes by Rho, we purified a novel substrate of Rho-kinase having a molecular mass of 48 kDa (p48) from a rat liver cytosol extract. Mass spectral analysis revealed p48 to be elongation factor-1 alpha (EF-1 alpha), which is known as an actin-binding protein besides a cofactor of polypeptide elongation. Rho-kinase directly phosphorylated recombinant EF-1alpha in vitro. A high- speed cosedimentation assay revealed that phosphorylation of EF-1 alpha by Rho-kinase decreased the binding activity of EF-1 alpha to filamentous actin (F-actin). A low-speed sedimentation assay revealed that phosphorylation of EF-1 alpha by Rho-kinase decreased the F-actin-bundling activity. In addition, EF-1 alpha bound to MBS of myosin phosphatase, suggesting that both Rho-kinase and myosin phosphatase regulate the phosphorylation state of EF-1 alpha downstream of Rho as other substrates of Rho-kinase, i.e., MLC, adducin, and the ERM family. These results suggest that the Rho/Rho-kinase pathway regulates the organization of actin cytoskeleton via the phosphorylation of EF-1 alpha.
Insights
Rho-kinase phosphorylates elongation factor-1 alpha (EF-1 alpha), reducing its actin-binding and bundling activities. This suggests the Rho/Rho-kinase pathway regulates actin cytoskeleton organization through EF-1 alpha.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Rho-associated kinase (Rho-kinase) is activated by Rho GTPase and regulates cellular processes.
- Rho-kinase phosphorylates substrates like myosin light chain (MLC) and ERM proteins, affecting cell motility and cytoskeleton organization.
Purpose of the Study:
- To identify novel substrates of Rho-kinase.
- To investigate the role of Rho-kinase in regulating elongation factor-1 alpha (EF-1 alpha) and its impact on actin cytoskeleton organization.
Main Methods:
- Purification of a 48 kDa protein (p48) from rat liver cytosol.
- Mass spectral analysis to identify p48 as EF-1 alpha.
- In vitro phosphorylation assays using recombinant EF-1 alpha.
- High-speed and low-speed sedimentation assays to assess F-actin binding and bundling activities.
Main Results:
- p48 was identified as elongation factor-1 alpha (EF-1 alpha), an actin-binding protein.
- Rho-kinase directly phosphorylated EF-1 alpha in vitro.
- Phosphorylation by Rho-kinase decreased EF-1 alpha's binding affinity to F-actin and its F-actin bundling activity.
- EF-1 alpha was found to bind to the myosin-binding subunit (MBS) of myosin phosphatase.
Conclusions:
- The Rho/Rho-kinase pathway regulates actin cytoskeleton organization.
- Phosphorylation of EF-1 alpha by Rho-kinase modulates its interaction with F-actin.
- EF-1 alpha is a novel substrate of Rho-kinase involved in actin cytoskeleton regulation.