Effect of Brain Ischemia and Reperfusion on the Localization of Phosphorylated Eukaryotic Initiation Factor 2α

119

107

Section: Methodsmentioning

confidence: 99%

Ultraviolet Light Activates NFκB through Translational Inhibition of IκBα Synthesis

Wu¹,

Tan

Hu³

et al. 2004

119

107

“…Polyclonal antibodies against GCN2 (serum HL2523) (32), phosphospecific antibodies against eIF2␣ phosphorylated on Ser-51 (34), and polyclonal antibodies against eIF2␣ (35) were described previously. Immunoblot analysis of yeast wheat cell extracts (WCEs) using the latter three antibodies was conducted as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

Serine 577 Is Phosphorylated and Negatively Affects the tRNA Binding and eIF2α Kinase Activities of GCN2

Garcia-Barrio

Dong

Cherkasova

et al. 2002

Protein kinase GCN2 regulates translation initiation by phosphorylating eukaryotic initiation factor 2␣ (eIF2␣), impeding general protein synthesis but specifically inducing translation of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. GCN2 activity is stimulated in amino acid-deprived cells through binding of uncharged tRNA to a domain related to histidyl tRNA synthetase. We show that GCN2 is phosphorylated by another kinase on serine 577, located N-terminal to the kinase domain. Mutation of Ser-577 to alanine produced partial activation of GCN2 in nonstarved cells, increasing the level of phosphorylated eIF2␣, derepressing GCN4 expression, and elevating the cellular levels of tryptophan and histidine. The Ala-577 mutation also increased the tRNA binding affinity of purified GCN2, which can account for the elevated kinase activity of GCN2-S577A in nonstarved cells where uncharged tRNA levels are low. Whereas Ser-577 remains phosphorylated in amino acidstarved cells, its dephosphorylation could mediate GCN2 activation in other stress or starvation conditions by lowering the threshold of uncharged tRNA required to activate the protein.

“…This assay was shown to be specific for p70 S6K (36). Phosphorylation State of eIF2␣-About 50 g of cell extract, prepared as for the eIF2B assay, was loaded on an SDS-gel and blotted to Immobilon-P. Phosphorylated eIF2␣ was detected with a rabbit antibody specific for the phosphorylated form of eIF2␣ (kindly provided by Dr. G. Krause) (37). eIF2␣ was detected with a monoclonal antibody (made by Dr. Henshaw, and obtained via Dr. Sarre, Freiburg, Germany).…”

Section: Eif2⅐[mentioning

confidence: 99%

Nerve and Epidermal Growth Factor Induce Protein Synthesis and eIF2B Activation in PC12 Cells

Kleijn¹,

Welsh²,

Scheper³

et al. 1998

The regulation of protein synthesis and of eukaryotic initiation factor eIF2B was studied in PC12 cells. An increase in protein synthesis was observed after nerve growth factor (NGF) and epidermal growth factor (EGF) treatment of PC12 cells, and this increase coincided with activation of eIF2B. Growth factor addition in the presence of the phosphatidylinositol-3-OH kinase inhibitor wortmannin showed that both NGF-and EGFinduced protein synthesis and eIF2B activation were phosphatidylinositol-3-OH kinase dependent. The EGFinduced stimulation of protein synthesis and activation of eIF2B was dependent upon FK506-binding proteinrapamycin-associated protein, as shown with the immunosuppressant rapamycin, whereas NGF induction was partially dependent upon FK506-binding protein-rapamycin-associated protein.The activities of two kinases that act on eIF2B, glycogen synthase kinase-3 and casein kinase II, were measured to assess their potential roles in the activation of eIF2B in PC12 cells. Inactivation of glycogen synthase kinase-3 was seen in response to both NGF and EGF and this coincided with activation of eIF2B. However, inactivation of glycogen synthase kinase-3 was not rapamycin sensitive, in contrast to the activation of eIF2B. This indicates the involvement of another protein kinase or regulatory mechanism in the eIF2B activation. Both growth factors activated casein kinase II. However, the time course of its activation and its insensitivity to wortmannin and rapamycin suggest that casein kinase II does not play a major regulatory role in eIF2B activation under these conditions.