Changes in Ribosomal Binding Activity of eIF3 Correlate with Increased Translation Rates during Activation of T Lymphocytes

Suzanne Miyamoto,Purvi Patel,John W.B Hershey

doi:10.1074/jbc.m414129200

Suzanne Miyamoto, Purvi Patel + Show 1 more

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https://doi.org/10.1074/jbc.m414129200

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Abstract

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. The stimulation of translation is due to an increase in the rate of initiation caused by the regulation of initiation factor activities. Here, we focus on eIF3, a large multiprotein complex that plays a central role in the formation of the 40 S initiation complex. Using sucrose density gradient centrifugation to analyze ribosome complexes, we find that most eIF3 is not bound to 40 S ribosomal subunits in unactivated T lymphocytes but becomes ribosome-bound following activation. Immunoblot analyses of sucrose gradient fractions for individual eIF3 subunits show that the small eIF3j subunit is unassociated with the eIF3 complex in quiescent T lymphocytes, but upon activation joins the other eIF3 subunits and binds 40 S ribosomal subunits. Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and then to the 40 S ribosomes. The association of eIF3j with the other eIF3 subunits appears to be inhibited by rapamycin, suggesting a mechanism that lies downstream from the mammalian target of rapamycin kinase. This association requires ionomycin together with a phorbol ester, which also suggests that calcium signaling is involved. We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation.

Highlights

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation
Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and to the 40 S ribosomes
The objectives of this study are to investigate if eIF3 subunit expression and eIF3 activity change in IϩP- or PMA-activated T lymphocytes, elucidate how eIF3 interacts with other translational components to form the 40 S preinitiation complex, and determine if eIF3-associated translation initiation events are affected by rapamycin or wortmannin

Summary

Introduction

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. It is suggested that the mechanism responsible for increased translation is complex and involves a number of regulated initiation steps [7] These include the phosphorylation state of eIF2␣1 [7, 8] and increased eIF2B exchange activity [7, 9], both of which affect the level of the ternary complex (eIF21⁄7GTP1⁄7MettRNAiMet), an intermediate in the binding of the initiator methionyl-tRNA to the 40 S ribosomal subunit (10 –12). EIF3 is the largest of the mammalian initiation factors and is implicated in a number of steps in the initiation pathway It affects ribosome dissociation/re-association, promotes or stabilizes ternary complex binding to 40 S subunits, helps position mRNA on the 40 S ribosome through its interaction with eIF4G, and may contribute to AUG recognition by affecting eIF5 stimulation of eIF2 GTPase activity Yeast eIF3 interacts with eIF1, eIF2␤, and eIF5 to form a multifactor

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