Reactive oxygen species sensitivity of angiotensin II-dependent translation initiation in vascular smooth muscle cells.
Translation initiation, the rate-limiting step in protein synthesis, is a key event in vascular smooth muscle cell growth, a major component of vascular disease. Translation initiation is regulated by interaction between PHAS-I and the eukaryotic initiation factor 4E (eIF4E). Although angiotensin II (Ang II)-induced vascular smooth muscle cell hypertrophy requires the generation of reactive oxygen species (ROS), the ROS sensitivity of these events and their upstream activators remain unclear. Here, we investigated the role of ROS in the regulation of PHAS-I phosphorylation on Thr-70 and Ser-65, an event required for the release of eIF4E from PHAS-I. Ang II-induced Ser-65 phosphorylation was ROS-dependent as assessed by pretreatment with ebselen (3.6 +/- 0.2 versus 1.1 +/- 0.2), diphenylene iodonium (3.6 +/- 0.2 versus 1.0 +/- 0.1), and N-acetyl cysteine (3.6 +/- 0.2 versus 1.2 +/- 0.1), but Ang II-stimulated phosphorylation of Thr-70 was ROS-insensitive. Although phosphatidylinositol 3-kinase pathway inhibition by LY294004 blocked both Ser-65 and Thr-70 phosphorylation (3.8 +/- 0.1 versus 0.8 +/- 0.1 and 3.2 +/- 0.2 versus 1.0 +/- 0.01, respectively), protein phosphatase 2A inhibition by okadaic acid selectively increased (3.3 +/- 0.1 versus 5.2 +/- 0.1) and p38 mitogen-activated protein kinase inhibition by SB203580 selectively decreased (3.8 +/- 0.1 versus 1.4 +/- 0.3) Ser-65 phosphorylation. Dominant negative Akt adenovirus also inhibited only Ser-65 phosphorylation (3.7 +/- 0.1 versus 1.0 +/- 0.03). These results demonstrate a unique differential ROS sensitivity of two separate residues on PHAS-I, which seems to be explained by the selective involvement of distinct signaling pathways in the regulation of these phosphorylation events.
Rocic
Petra
P
Division of Cardiology, Emory University, Atlanta, Georgia 30322, USA.
Seshiah
Puvi
P
Griendling
Kathy K
KK
D000111:Acetylcysteine / Q000494:pharmacology; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000166:cytology; D001393:Azoles / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism*; D005799:Genes, Dominant; D007093:Imidazoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D054833:Isoindoles; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D019319:Okadaic Acid / Q000494:pharmacology; D009861:Onium Compounds / Q000494:pharmacology; D016566:Organoselenium Compounds / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D054648:Protein Phosphatase 2; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D000111:Acetylcysteine; D000804:Angiotensin II; D001393:Azoles; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D054833:Isoindoles; D019319:Okadaic Acid; D009861:Onium Compounds; D016566:Organoselenium Compounds; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D054648:Protein Phosphatase 2; D011725:Pyridines; D017382:Reactive Oxygen Species; D012694:Serine; D013912:Threonine; C499526:Eif4ebp1 protein, rat; C042986:ebselen; C007517:diphenyleneiodonium; C093642:SB 203580