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Spotlight: GloboZymes eIF4E and Related Products

Introduction

Eukaryotic Initiation Factor 4E (eIF4E) is a rate-limiting initiation factor in protein synthesis. It directs ribosomes to the cap structure of mRNAs. eIF4E is a 217 amino acid ~ 24-kD polypeptide that exists as a free form, as part of the active eIF4F pre-initiation complex, or as an inactive complex with eIF4E binding proteins (4E-BPs). In their dephosphorylated form, 4E-BPs inhibit assembly of the eIF4F complex and hence cap-dependent translation. Once phosphorylated by the mTOR kinase (see figure below), the 4EBPs release eIF4E which can then associate with the eIF4F complex. The other subunits of the eIF4F complex are a 47-kD polypeptide, termed eIF4A, that possesses ATPase and RNA helicase activities, and a 220-kD scaffolding polypeptide, eIF4G.
 

Phosphorylation of eIF4E

eIF4E is phosphorylated at Ser209 in cells in response to insulin and other growth factors. This phosphorylation increases the affinity of eIF4E for capped mRNA. The extent of eIF4E Phosphorylation at Ser209 changes with the stage of the cell cycle. Low level phosphorylation of eIF4E is seen during the G0 and M phases. In contrast, significant phosphorylation of eIF4E occurs during the G1 and S phases. Elevated eIF4E activity has been implicated in several cancers including breast, lung, and prostate cancers.  Transcriptional profiling of metastatic human tumors indicates that eIF4E is consistently up-regulated. 
 

 eIF4E-Independent Translation

Interestingly, some viruses are able to translate their proteins by eliminating the eIF4E binding site on eIF4F. Heat shock and some other protein mRNAs also do not require eIF4E in order to be translated. Both viruses and the heat shock proteins mRNAs achieve this eIF4E-independent translation through an internal ribosome entry site in the RNA.

Figure 1 : GloboZymes Purified eIF4E Preparations

Typical SDS PAGE Pattern of GloboZymes Affinity-Purified Recombinant Preparation of Human eIF4E. The gel was stained with Coomassie Blue.

NOTE:  In addition to wild type eIF4E, GloboZymes provides several phosphorylation and other site-directed mutants of this initiation factor including eIF4E(S209A)eIF4E(T210A)eIF4E(S209AT210A),   eIF4E(S53A)eIF4E(S124A).

Figure 2:  GloboZymes Anti-eIF4E

Extract of bovine kidney (30 ug) was subjected to Western Blotting with GloboZymes Anti-eIF4E.  This antiserum was produced in rabbits to  affinity-purified recombinant eIF4E protein extracted following SDS-PAGE electrophoresis of the preprations. Western blotting with preimmune serum showed no reaction.  

Signaling Pathway Regulating eIF4E Phosphorylation

 

 

Figure 3: Signaling Pathways Regulating eIF4E Phosphorylation – GloboZymes cPK

GloboZymes provides purified preparations of insulin-stimulated cPK from bovine kidney. These preparations phosphorylate eIF4E at Ser209 and Thr210. Interestingly, Thr210 is rapidly and preferentially dephosphorylated by PP2A which may be one reason this phosphorylation has not been detected in cell cultures.  PP2A may thus, at least in part, confer the site specificity of eIF4E phosphorylation at Ser209 in response to mitogenic stimuli. 

eIF4E and Fragile X Mental Retardation Protein

Fragile X mental retardation protein (FMRP) acts to regulate translation of specific mRNAs through its binding of eIF4E via CYFIP1. This binding of eIF4E occurs at a domain on CYFIP1 that is structurally similar to those present in 4EBPs. The FMRP/CYFIP1/eIF4E complex prevents the eIF4E-eIF4G interaction, which is necessary for translation to occur. The FMRP/CYFIP1/eIF4E interaction is strengthened by the presence of mRNA(s). In particular, BC1 RNA, which is a non-translatable dendritic mRNA, allows for an optimal interaction between FMRP and CYFIP1 and specific mRNAs with the FMRP/CYFIP1/eIF4E complex at synapses. Dissociation of eIF4E from CYFIP1 occurs in response to increased synaptic stimulation of neurons, which then allows for the eIF4E-eIF4G interaction to occur and hence the initiation of translation.

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