Phosphorylation site of eukaryotic initiation factor 4E.

Rychlik, Wojciech; Russ, Mike; Rhoads, Robert E.

doi:10.1016/s0021-9258(18)60978-0

Cited by 83 publications

(9 citation statements)

References 24 publications

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“…The data summarized in Figure 10 indicate that eIF-4E* begins around residue 47 and ends at around residue 182. Such a fragment would contain the major phosphorylation site, (Rychlik et al, 1987b). This is supported by the observations that two forms of eIF-4E* are detected by isoelectric focusing (Rychlik et al, 1986) and that extracts of cultured lymphocytes labeled with 32P yield labeled forms of both eIF-4E and eIF-4E* when passed over m7GTP-Sepharose (W. Rychlik and R. Rhoads, unpublished work).…”

Section: Discussionmentioning

confidence: 75%

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

Chavan¹,

Rychlik²,

Blaas³

et al. 1990

Biochemistry

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Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

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Section: Discussionmentioning

confidence: 75%

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

Chavan¹,

Rychlik²,

Blaas³

et al. 1990

Biochemistry

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“…For example, at least two major phosphopeptides identified on a two-dimensional map of the insulin receptor isolated from COS cells (Carter et al, 1995) were not present in maps of the insulin receptor from HepG2 cells (Tavare et al, 1991). Similarly, the phosphorylation site in eIF-4E isolated from CHO cells (Flynn & Proud, 1995) was different from the site phosphorylated in reticulocytes (Rychlik et al, 1987). Since the modulation of calmodulin activity is dependent on the site of phosphorylation (Sacks et al, 1995), it is possible that cell type specific phosphorylation may be a mechanism to differentially regulate calmodulin in various cells.…”

Section: Discussionmentioning

confidence: 97%

Identification of Insulin-Stimulated Phosphorylation Sites on Calmodulin

et al. 1996

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Insulin enhances calmodulin phosphorylation in vivo. To determine the insulin-sensitive phosphorylation sites, phosphocalmodulin was immunoprecipitated from Chinese hamster ovary cells expressing human insulin receptors (CHO/IR). Calmodulin was constitutively phosphorylated on serine, threonine, and tyrosine residues, and insulin enhanced phosphate incorporation on serine and tyrosine residues. Phosphocalmodulin immunoprecipitated from control and insulin-treated CHO/IR cells, and calmodulin phosphorylated in vitro by the insulin receptor kinase and casein kinase II were resolved by two-dimensional phosphopeptide mapping. Several common phosphopeptides were detected. The phosphopeptides from the in vitro maps were eluted and phosphoamino acid analysis, manual sequencing, strong cation exchange chromatography, and additional proteolysis were performed. This strategy demonstrated that Tyr-99 and Tyr-138 were phosphorylated in vitro by the insulin receptor kinase and Thr-79, Ser-81, Ser-101 and Thr-117 were phosphorylated by casein kinase II. In vivo phosphorylation sites were identified by comigration of phosphopeptides on two-dimensional maps with phosphopeptides derived from calmodulin phosphorylated in vitro and by phosphoamino acid analysis. This approach revealed that Tyr-99 and Tyr-138 of calmodulin were phosphorylated in CHO/IR cells in response to insulin. Additional sites remain to be identified. The identification of the insulin-stimulated in vivo tyrosine phosphorylation sites should facilitate the elucidation of the physiological role of phosphocal-modulin.

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“…It is likely to be the component of the complex which confers the specific recognition of caps. Its gene has been cloned and sequenced from several sources (Altmann et al, 1987;Rychlik et al, 1987a;Sonenberg, 1988), and a site of serine phosphorylation has been determined (Rychlik et al, 1987b). Dephosphorylation of p25 correlates with altered translation rates during mitosis (Bonneau & Sonenberg, 1987) and heat shock (Duncan et al, 1987), but not poliovirus infection (Buckley & Ehrenfeld, 1986) .…”

mentioning

confidence: 99%

The p220 component of eukaryotic initiation factor 4F is a substrate for multiple calcium-dependent enzymes

Wyckoff¹,

Croall²,

Ehrenfeld³

1990

Biochemistry

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Eukaryotic initiation factor 4F (eIF-4F) is a multisubunit protein that functions in the first step of the binding of capped mRNAs to the small ribosomal subunit. Its largest polypeptide component, p220, is cleaved following poliovirus infection. This is thought to inactivate eIF-4F function, thereby preventing cap-dependent initiation of translation of cellular mRNAs. In this report, we show that p220 in extracts of uninfected HeLa cells is specifically lost in the presence of calcium. The responsible activities have been partially purified and identified as the calcium-dependent, neutral, cysteine proteases calpains I and II. In addition, a third calcium-dependent activity was resolved from the calpains and also results in the loss of p220. This activity has properties similar to a transglutaminase and copurifies with tissue transglutaminase through several chromatographic steps. None of these calcium-dependent activities appears to mediate p220 cleavage in poliovirus-infected cells.

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Phosphorylation site of eukaryotic initiation factor 4E.

Cited by 83 publications

References 24 publications

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

Identification of Insulin-Stimulated Phosphorylation Sites on Calmodulin

The p220 component of eukaryotic initiation factor 4F is a substrate for multiple calcium-dependent enzymes

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