Dataset Information

Gas6-Axl pathway: the role of redox-dependent association of Axl with nonmuscle myosin IIB.

ABSTRACT: In vascular smooth muscle cells, Axl is a key receptor tyrosine kinase, because it is upregulated in injury, increases migration and neointima formation, and is activated by reactive oxygen species. Reaction of glutathione with cysteine residues (termed "glutathiolation") is an important posttranslational redox modification that may alter protein activity and protein-protein interactions. To investigate the mechanisms by which reactive oxygen species increase Axl-dependent vascular smooth muscle cell function we assayed for glutathiolated proteins that associated with Axl in a redox-dependent manner. We identified glutathiolated nonmuscle myosin heavy chain (MHC)-IIB as a novel Axl interacting protein. This interaction was specific in that other myosins did not interact with Axl. The endogenous ligand for Axl, Gas6, increased production of reactive oxygen species in vascular smooth muscle cells and also increased the association of Axl with MHC-IIB. Antioxidants ebselen and N-acetylcysteine decreased the association of Axl with MHC-IIB in response to both Gas6 and reactive oxygen species. Blocking the Axl-MHC-IIB interaction with the specific myosin II inhibitor blebbistatin decreased phosphorylation of Axl and activation of extracellular signal-regulated kinase 1/2 and Akt. Association of MHC-IIB with Axl was increased in balloon-injured rat carotid vessels. Finally, expression of MHC-IIB was upregulated in the neointima of the carotid artery after balloon injury similar to upregulation of Axl protein expression, as shown in our previous studies. These results demonstrate a novel interaction between Axl and MHC-IIB in response to reactive oxygen species. This interaction provides a direct link between Axl and molecular motors crucial for directed cell migration, which may mediate increased migration in vascular dysfunction.

SUBMITTER: Cavet ME

PROVIDER: S-EPMC2888491 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Gas6-Axl pathway: the role of redox-dependent association of Axl with nonmuscle myosin IIB.

Cavet Megan E ME Smolock Elaine M EM Menon Prashanthi P Konishi Atsushi A Korshunov Vyacheslav A VA Berk Bradford C BC

Hypertension (Dallas, Tex. : 1979) 20100517 1

In vascular smooth muscle cells, Axl is a key receptor tyrosine kinase, because it is upregulated in injury, increases migration and neointima formation, and is activated by reactive oxygen species. Reaction of glutathione with cysteine residues (termed "glutathiolation") is an important posttranslational redox modification that may alter protein activity and protein-protein interactions. To investigate the mechanisms by which reactive oxygen species increase Axl-dependent vascular smooth muscle ...[more]

PMID: 20479336

Similar Datasets

Project description:Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.Significance of the studyPhosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Dataset Information

Gas6-Axl pathway: the role of redox-dependent association of Axl with nonmuscle myosin IIB.

Publications

Gas6-Axl pathway: the role of redox-dependent association of Axl with nonmuscle myosin IIB.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets