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mTORc1 activity is necessary and sufficient for phosphorylation of eNOS^S1177

Brandee Decker¹, Kevin Pumiglia¹

¹Department of Regenerative and Cancer Cell Biology, Albany Medical College, Albany, New York.

Physiological Reports

|June 23, 2018

Abstract:

Nitric oxide, produced by eNOS, plays critical roles in the regulation of vascular function and maintenance. Chronic PI3K signaling has recently been associated with vascular malformations. A well described substrate downstream of PI3K signaling is eNOS. Another critical downstream target of PI3K is the metabolic regulator, mTORc1. The relationship between mTORc1 and eNOS regulation, has not been determined. We generated cells with manipulated PI3K signaling by expressing the activating mutation, PIK3CA^H1047R , or knocking down PTEN expression. We investigated eNOS^S1177 phosphorylation, a major activating regulatory site, following mTORC1 inhibition. We also tested the sufficiency of mTORc1 activation to stimulate eNOS^S1177 phosphorylation. Our data indicate mTORc1 activity is required for the phosphorylation of eNOS^S1177 , even in the presence of robust AKT activation. Moreover, we found that expression of RHEB, which functions in the absence of AKT activation to activate mTORc1, is sufficient to phosphorylate this site. Our data indicate that mTORc1, rather than AKT, may be the critical determinant of eNOS^S1177 phosphorylation. As mTORc1 is a central regulator of cellular metabolism, the finding that this regulatory complex can directly participate in the regulation of eNOS provides new insights into metabolic uncoupling and vascular disease that often accompanies diabetes, high fat diets, and aging.

Insights

Mammalian target of rapamycin complex 1 (mTORC1) signaling is essential for endothelial nitric oxide synthase (eNOS) phosphorylation, independent of AKT activation. This discovery offers new insights into metabolic dysregulation and vascular diseases.

Area of Science:

Vascular Biology
Cellular Metabolism
Signal Transduction

Background:

Endothelial nitric oxide synthase (eNOS) regulates vascular function.
Chronic PI3K signaling is linked to vascular malformations.
The interplay between PI3K, mTORC1, and eNOS is not fully understood.

Purpose of the Study:

To investigate the role of mTORC1 in regulating eNOS phosphorylation.
To determine if mTORC1 activation is sufficient to stimulate eNOS phosphorylation.
To clarify the relationship between PI3K signaling, mTORC1, and eNOS activity.

Main Methods:

Manipulated PI3K signaling by expressing PIK3CA^H1047R or knocking down PTEN.
Assessed eNOS^S1177 phosphorylation following mTORC1 inhibition.
Tested the sufficiency of mTORc1 activation to stimulate eNOS^S1177 phosphorylation by expressing RHEB.

Main Results:

mTORC1 activity is required for eNOS^S1177 phosphorylation, even with high AKT activation.
RHEB expression, activating mTORC1 independently of AKT, is sufficient to induce eNOS^S1177 phosphorylation.
mTORC1 appears to be a critical determinant of eNOS^S1177 phosphorylation, more so than AKT.

Conclusions:

mTORC1 directly regulates eNOS phosphorylation, suggesting a novel mechanism linking cellular metabolism and vascular function.
This finding provides new perspectives on metabolic uncoupling in vascular diseases associated with diabetes, high-fat diets, and aging.
Targeting the mTORC1-eNOS pathway may offer therapeutic strategies for metabolic and vascular disorders.

Read full analysis

Keywords:

Endothelial cell endothelial nitric oxide synthase mammalian target of rapamycin phosphatidylinositide 3-kinase rapamycin ras homolog expressed in brain

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