Proteasome Inhibition Down-regulates Endothelial Nitric-oxide Synthase Phosphorylation and Function

Qin Wei,Yong Xia

doi:10.1074/jbc.m602105200

Abstract

Endothelial nitric-oxide synthase (eNOS) function is fundamentally modulated by protein phosphorylation. In particular, phosphorylation of serine 1179 (bovine)/1177 (human) by Akt has been shown to be the central mechanism of eNOS regulation. Here we revealed a novel role of proteasome in controlling eNOS serine 1179 phosphorylation and function. Rather than affecting eNOS turnover, proteasomal inhibition specifically dephosphorylated eNOS serine 1179, leading to decreased enzymatic activity. Blocking protein phosphatase 2A (PP2A) by okadaic acid or PP2A knockdown restored eNOS serine 1179 phosphorylation and activity in proteasome-inhibited cells. Although total PP2A expression and activity in cells were not affected by proteasome inhibitors, proteasomal inhibition induced PP2A ubiquitination and ubiquitinated PP2A translocated from cytosol to membrane. Further biochemical analyses demonstrated that eNOS associated with PP2A on cell membranes. Proteasomal inhibition markedly enhanced PP2A association to eNOS, and this increase of PP2A dephosphorylated eNOS and its upstream kinase Akt. Taken together, these studies identified a novel pathway in which proteasome modulates eNOS phosphorylation by inducing intracellular PP2A translocation.

Highlights

Appears to be most multifaceted [5]
In an effort to define the roles of the ubiquitin-proteasome system in regulating Endothelial nitric-oxide synthase (eNOS) turnover, we unexpectedly found that proteasome profoundly affected eNOS serine 1179
In consistent with the eNOS serine 1179 was reported to be dephosphorylated by results from eNOS-HEK 293 cells, MG132 selectively reduced phosphatase 2A (PP2A) [13], we sought to determine the roles of PP2A in proteaeNOS serine 1179 phosphorylation in a time-dependent man- some inhibition-induced eNOS dephosphorylation

Summary

EXPERIMENTAL PROCEDURES

Materials—Cell culture materials were obtained from Invitrogen. MG132 and lactacystin were purchased from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). Target proteins in cytosolic and membrane fractions were resuspended in radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 50 mM NaF, 1 mM Na3VO4, 5 mM sodium pyrophosphate, 1 mM EDTA, and protease inhibitor tablet), and immunoprecipitations were carried out with appropriate antibodies. Co-immunoprecipitation and Pulldown Assay—Cells were harvested and lysed on ice for 30 min in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 5 mM sodium pyrophosphate, and protease inhibitor tablet). Western Blotting—Cells were lysed on ice for 30 min in modified radioimmune precipitation assay buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 50 mM NaF, 1 mM Na3VO4, 5 mM sodium pyrophosphate, and protease inhibitor tablet. Differences were considered to be statistically To reconfirm that proteasome function was inhibited by significant at p Ͻ 0.05

RESULTS

Proteasomal Inhibition Enhanced

DISCUSSION