Serine 48 in Initiation Factor 2 (eIF2) Is Required for High-Aff
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- 作者：Akulapalli Sudhakar ; Thanuja Krishnamoorthy ; Anjali Jain ; Udayan Chatterjee ; Seyed E. Hasnain ; Randal J. Kaufman ; and Kolluru V. A. Ramaiah
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：November 16, 1999
- 年：1999
- 卷：38
- 期：46
- 页码：15398 - 15405
- 全文大小：261K
- 年卷期：v.38,no.46(November 16, 1999)
- ISSN：1520-4995
文摘
Phosphorylation of the serine 51 residue in the 
-subunit of translational initiation factor 2 ineukaryotes (eIF2) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentamericguanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDPbinary complex. To further understand the importance of eIF2 phosphorylation in the interaction betweeneIF2(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type(wt) and a mutant eIF2 in which the serine 48 residue was replaced with alanine (48A mutant) in thebaculovirus system. The findings reveal that the expression of both of these recombinant subunits wasvery efficient (15-20% of the total protein) and both proteins were recognized by an eIF2 monoclonalantibody and were phosphorylated to the same extent by reticulocyte eIF2 kinases. However, partiallypurified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit thephosphorylation of eIF2 of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activityand formation of the eIF2(P)-eIF2B complex that occurs due to eIF2 phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extractsexpressing the 48A mutant eIF2 compared to those for wt. These findings support the hypothesis thatthe serine 48 residue is required for high-affinity interaction between eIF2(P) and eIF2B.
-subunit of translational initiation factor 2 ineukaryotes (eIF2) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentamericguanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDPbinary complex. To further understand the importance of eIF2 phosphorylation in the interaction betweeneIF2(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type(wt) and a mutant eIF2 in which the serine 48 residue was replaced with alanine (48A mutant) in thebaculovirus system. The findings reveal that the expression of both of these recombinant subunits wasvery efficient (15-20% of the total protein) and both proteins were recognized by an eIF2 monoclonalantibody and were phosphorylated to the same extent by reticulocyte eIF2 kinases. However, partiallypurified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit thephosphorylation of eIF2 of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activityand formation of the eIF2(P)-eIF2B complex that occurs due to eIF2 phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extractsexpressing the 48A mutant eIF2 compared to those for wt. These findings support the hypothesis thatthe serine 48 residue is required for high-affinity interaction between eIF2(P) and eIF2B.