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.
Highlights
Endothelial cells are constantly subjected to shear stress generated by blood flow
To examine whether protein kinase A (PKA) is stimulated by shear stress, I first explored the phosphorylation of cAMP responsive element binding protein (CREB), one of the well known regulatory targets of PKA (Daniel et al, 1998; Park et al, 2003; Min et al, 2004)
When the Bovine aorta endothelial cells (BAECs) were exposed to shear stress, there was a significant increase in CREB phosphorylation at Ser133, maximizing at 30 min from the onset of shear stress (Figure 1A)
Summary
Endothelial cells are constantly subjected to shear stress generated by blood flow. Shear stress regulates vascular tone and diameter, inflammatory responses, hemostasis, vessel wall remodeling and other vascular functions (Resnick et al, 2003). In an attempt to determine in situ PKA activation by shear stress in endothelial cells, I chose to monitor phosphorylation of known and unknown endogenous targets of PKA. Shear stress stimulates phosphorylation of putative PKA substrates As an independent approach to examine in situ PKA activation in cells, the putative endogenous targets of PKA were monitored for their phosphorylation by using the phospho-PKA substrate antibody.
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