Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphatase.
BACKGROUND
Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined.


OBJECTIVE
We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity.


METHODS
Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation.


RESULTS
We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity.


CONCLUSIONS
Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1.
Patel
B
B
Medical Biosciences, University of Bradford, Bradford, UK.
Sharifi
M
M
Milward
A D
AD
Oberprieler
N G
NG
Gibbins
J M
JM
Parkin
S
S
Naseem
K M
KM
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006490:Hemostatics / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology
D006490:Hemostatics; D014665:Vasodilator Agents; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D013917:Thrombin; D001920:Bradykinin