Exp Mol Med.  2006 Feb;38(1):63-71.

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

• Boo YC

Abstract

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.

Keyword

cyclic AMP responsive element-binding protein; endothelial cells; nitric oxide synthase type III; protein kinase A

MeSH Terms

Animals
Aorta, Thoracic/cytology
Blotting, Western
Cattle
Cell Culture Techniques
Cell Extracts
Cells, Cultured
Comparative Study
Cyclic AMP-Dependent Protein Kinases/analysis/*metabolism
Endothelium, Vascular/cytology/*enzymology/*metabolism
Nitric Oxide Synthase Type III/analysis/*metabolism
Phosphorylation
Precipitin Tests
Research Support, Non-U.S. Gov't
Stress, Mechanical
Time Factors