Elongation factor-1 alpha is a novel substrate of rho-associated kinase.
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.
Izawa
T
T
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Fukata
Y
Y
Kimura
T
T
Iwamatsu
A
A
Dohi
K
K
Kaibuchi
K
K
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D043263:Myosin-Light-Chain Phosphatase; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016622:Silver Staining; D013997:Time Factors; D054460:rho-Associated Kinases; D000099291:Adducins
D000199:Actins; D002148:Calmodulin-Binding Proteins; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D043263:Myosin-Light-Chain Phosphatase; D020648:Peptide Elongation Factor 1; D010749:Phosphoprotein Phosphatases; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D054460:rho-Associated Kinases; D000099291:Adducins