Phospho-4E-BP1 (Thr37) Antibody [L9H8]

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000-1:2000

Application
WB

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
13 kDa 17 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Datasheet & SDS

Biological Description

Specificity
Phospho-4E-BP1 (Thr37) Antibody [L9H8] detects endogenous levels of total 4E-BP1 protein only when it is phosphorylated at Thr37.

Clone
L9H8

Synonym(s)
Eukaryotic translation initiation factor 4E-binding protein 1, 4E-BP1, eIF4E-binding protein 1, Phosphorylated heat- and acid-stable protein regulated by insulin 1, PHAS-I, EIF4EBP1

Background
Phospho‑4E‑BP1 (Thr37) represents an early, mTOR‑dependent regulatory state of the eIF4E‑binding protein 1 within the cap‑dependent translation pathway, functioning as a priming modification that integrates growth factor and nutrient signals from PI3K–Akt–mTOR into hierarchical multisite phosphorylation and control of translation initiation. Hypophosphorylated 4E‑BP1 binds tightly to the mRNA cap‑binding protein eIF4E via a canonical YXXXXLΦ motif adjacent to the central region containing Thr37 and Thr46, occluding eIF4G docking and preventing assembly of the eIF4F complex that recruits 40S ribosomal subunits to the 5′ cap, while N‑ and C‑terminal regions stabilize this repressor conformation and respond to upstream kinase inputs. FRAP/mTOR directly phosphorylates 4E‑BP1 at Thr37 and Thr46 when 4E‑BP1 is complexed with eIF4E, generating phosphopeptides that co‑migrate with the two most prominent in vivo 4E‑BP1 phosphopeptides and identifying Thr37 and Thr46 as primary mTOR target sites; these residues are highly phosphorylated even in serum‑starved cells and are present in all electrophoretically distinct phosphorylated 4E‑BP1 isoforms, including forms that remain bound to eIF4E. Phosphorylation at Thr37 and Thr46 does not disrupt the 4E‑BP1–eIF4E complex or shift electrophoretic mobility, but mutational analysis shows that Thr37/Thr46 phosphorylation is required for efficient subsequent phosphorylation of downstream “serum‑sensitive” sites in the C‑terminal half (Thr70 and Ser65), since Thr37Ala, Thr46Ala, or double Thr37Ala/Thr46Ala substitutions reduce total 4E‑BP1 phosphate incorporation by 10–20‑fold and abolish appearance of C‑terminal phosphopeptides without impairing basal eIF4E binding, defining phospho‑Thr37/Thr46 as obligatory priming events for hierarchical phosphorylation and release from eIF4E. Thr37 and Thr46 phosphorylation is rapamycin‑ and LY294002‑sensitive under starvation conditions and is driven by the PI3K–Akt–mTOR axis: PI3K or Akt inhibition, or rapamycin‑sensitive mTOR blockade, reduces Thr37/Thr46 phosphorylation, whereas constitutively active Akt promotes phosphorylation on the same sites targeted during serum stimulation, placing Thr37‑phosphorylated 4E‑BP1 as a proximal reporter of mTORC1 output immediately downstream of Akt. In Drosophila, the ortholog d4E‑BP exhibits conserved regulatory interplay between Thr37 and Thr46, with insulin‑stimulated phosphorylation at Thr46 (detected by phospho‑Thr37/46 antibodies) acting as the dominant event that converts d4E‑BP from an eIF4E‑bound translational repressor to a more weakly binding form; this regulation depends on the insulin–PI3K–Akt–TSC–TOR cascade, as RNAi against Dp110 (PI3K), dAkt, dTSC1, or dTOR respectively decreases or increases d4E‑BP Thr37/Thr46 phosphorylation and shifts d4E‑BP isoform patterns in a manner consistent with a linear Akt→TSC→TOR→4E‑BP pathway. Hierarchical analysis in flies indicates that d4E‑BP carries several phosphate groups in serum‑starved cells on sites that do not prevent eIF4E interaction, with insulin triggering additional phosphorylation at Thr46 in a rapamycin‑sensitive manner that coincides with the appearance of more acidic isoforms and reduced eIF4E binding, functionally mirroring the requirement for primed Thr37/Thr46 phosphorylation in mammalian 4E‑BP1 to enable subsequent modifications that release eIF4E and permit eIF4F assembly. These findings place phospho‑4E‑BP1 (Thr37) within a two‑step mechanism where mTORC1 first phosphorylates Thr37/Thr46 on eIF4E‑bound 4E‑BP1 to create a primed intermediate, and additional kinases—still operating within the mTORC1 signaling context—then modify Thr70 and Ser65, culminating in loss of eIF4E binding, transition from repressive to permissive translation states, and increased synthesis of growth‑ and survival‑related proteins.

Background

Phospho‑4E‑BP1 (Thr37) represents an early, mTOR‑dependent regulatory state of the eIF4E‑binding protein 1 within the cap‑dependent translation pathway, functioning as a priming modification that integrates growth factor and nutrient signals from PI3K–Akt–mTOR into hierarchical multisite phosphorylation and control of translation initiation. Hypophosphorylated 4E‑BP1 binds tightly to the mRNA cap‑binding protein eIF4E via a canonical YXXXXLΦ motif adjacent to the central region containing Thr37 and Thr46, occluding eIF4G docking and preventing assembly of the eIF4F complex that recruits 40S ribosomal subunits to the 5′ cap, while N‑ and C‑terminal regions stabilize this repressor conformation and respond to upstream kinase inputs. FRAP/mTOR directly phosphorylates 4E‑BP1 at Thr37 and Thr46 when 4E‑BP1 is complexed with eIF4E, generating phosphopeptides that co‑migrate with the two most prominent in vivo 4E‑BP1 phosphopeptides and identifying Thr37 and Thr46 as primary mTOR target sites; these residues are highly phosphorylated even in serum‑starved cells and are present in all electrophoretically distinct phosphorylated 4E‑BP1 isoforms, including forms that remain bound to eIF4E. Phosphorylation at Thr37 and Thr46 does not disrupt the 4E‑BP1–eIF4E complex or shift electrophoretic mobility, but mutational analysis shows that Thr37/Thr46 phosphorylation is required for efficient subsequent phosphorylation of downstream “serum‑sensitive” sites in the C‑terminal half (Thr70 and Ser65), since Thr37Ala, Thr46Ala, or double Thr37Ala/Thr46Ala substitutions reduce total 4E‑BP1 phosphate incorporation by 10–20‑fold and abolish appearance of C‑terminal phosphopeptides without impairing basal eIF4E binding, defining phospho‑Thr37/Thr46 as obligatory priming events for hierarchical phosphorylation and release from eIF4E. Thr37 and Thr46 phosphorylation is rapamycin‑ and LY294002‑sensitive under starvation conditions and is driven by the PI3K–Akt–mTOR axis: PI3K or Akt inhibition, or rapamycin‑sensitive mTOR blockade, reduces Thr37/Thr46 phosphorylation, whereas constitutively active Akt promotes phosphorylation on the same sites targeted during serum stimulation, placing Thr37‑phosphorylated 4E‑BP1 as a proximal reporter of mTORC1 output immediately downstream of Akt. In Drosophila, the ortholog d4E‑BP exhibits conserved regulatory interplay between Thr37 and Thr46, with insulin‑stimulated phosphorylation at Thr46 (detected by phospho‑Thr37/46 antibodies) acting as the dominant event that converts d4E‑BP from an eIF4E‑bound translational repressor to a more weakly binding form; this regulation depends on the insulin–PI3K–Akt–TSC–TOR cascade, as RNAi against Dp110 (PI3K), dAkt, dTSC1, or dTOR respectively decreases or increases d4E‑BP Thr37/Thr46 phosphorylation and shifts d4E‑BP isoform patterns in a manner consistent with a linear Akt→TSC→TOR→4E‑BP pathway. Hierarchical analysis in flies indicates that d4E‑BP carries several phosphate groups in serum‑starved cells on sites that do not prevent eIF4E interaction, with insulin triggering additional phosphorylation at Thr46 in a rapamycin‑sensitive manner that coincides with the appearance of more acidic isoforms and reduced eIF4E binding, functionally mirroring the requirement for primed Thr37/Thr46 phosphorylation in mammalian 4E‑BP1 to enable subsequent modifications that release eIF4E and permit eIF4F assembly. These findings place phospho‑4E‑BP1 (Thr37) within a two‑step mechanism where mTORC1 first phosphorylates Thr37/Thr46 on eIF4E‑bound 4E‑BP1 to create a primed intermediate, and additional kinases—still operating within the mTORC1 signaling context—then modify Thr70 and Ser65, culminating in loss of eIF4E binding, transition from repressive to permissive translation states, and increased synthesis of growth‑ and survival‑related proteins.

References
https://pubmed.ncbi.nlm.nih.gov/10364159/ https://pubmed.ncbi.nlm.nih.gov/14645523/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%