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1.
Figure 12

Figure 12. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

A two-step model for the phosphorylation of 4E-BP1. FRAP/mTOR phosphorylates 4E-BP1 (in a 4E-BP1/eIF4E complex) on Thr-37 and Thr-46. Phosphorylation of Thr-37 and Thr-46 is a prerequisite for an unidentified serum-sensitive kinase (acting downstream of Akt) to phosphorylate 4E-BP1 on the serum-sensitive sites. The latter phosphorylation triggers the release of 4E-BP1 from eIF4E. A direct link between Akt and FRAP/mTOR remains to be proven.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
2.
Figure 3

Figure 3. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

In vitro kinase assay of Thr-37–Ala and Thr-46–Ala mutant proteins confirm that they are the primary FRAP/mTOR phosphorylation sites. Histidine-tagged murine 4E-BP1 was phosphorylated in vitro in an immune-complex kinase assay with FRAP/mTOR, and tryptic/chymotryptic maps were performed. (A) Wild type; (B) Thr-37–Ala; (C) Thr-46–Ala.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
3.
Figure 7

Figure 7. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

Phosphorylated Thr-37 and Thr-46 are present in the fastest migrating phosphorylated 4E-BP1 isoform. 293 cells were serum starved for 30 hr, incubated with [32P]orthophosphate, stimulated with 10% dialyzed FCS, and extracts were subjected to SDS-PAGE and autoradiography (A, inset). The slowest (A) and fastest-migrating (B) 4E-BP1 isoforms were isolated and phosphopeptide maps were performed.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
4.
Figure 6

Figure 6. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

Phosphorylated Thr-37 and Thr-46 are present at a high stoichiometry in serum-starved 293 cells. 293 cells were grown to confluence and serum starved for 30 hr. 32P-Labeling was performed for 4 hr and phosphopeptide maps were generated from starved cells (A) or cells stimulated for 30 min with 10% dialyzed FCS (B). Phosphopeptides containing phospho-Thr-37 or -Thr-46 are identified. Serum-induced phosphopeptides are labeled a–d.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
5.
Figure 11

Figure 11. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

Effects of mutations of Thr-37 and/or Thr-46 on other phosphorylation sites. (A–E) Phosphopeptide maps were performed on HA-tagged 4E-BP1 proteins from Fig. 10. (F) Thr-37 and Thr-46 affect 4E-BP1 phosphorylation. The arrows indicate (1) a mutually positive effect of Thr-37 and Thr-46 phosphorylation, and (2) a positive effect of Thr-37 and Thr-46 phosphorylation on the phosphorylation of the serum-sensitive sites. The fraction of samples from Fig. 10 loaded onto each map is shown (upper left). An equivalent quantity in cpm was loaded for each mutant.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
6.

Figure 5. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

FRAP/mTOR phosphorylation of Thr-37 and Thr-46 does not disrupt the 4E-BP1/eIF4E complex. (A) 4E-BP1 alone, or a 4E-BP1/eIF4E complex, was incubated with FRAP/mTOR, as in Fig. 4. The kinase reaction was stopped and one-half of the material was immediately subjected to SDS-PAGE (bottom). The remaining material was incubated with m7GDP-agarose beads to purify eIF4E and associated proteins. The bound (top) and unbound (middle) fractions were analyzed by SDS-PAGE. (B) Phosphorylated 4E-BP1 (A, lane 2; bound, unbound, and total) was analyzed by two-dimensional phosphopeptide mapping.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
7.
Figure 9

Figure 9. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

Sensitivity of Thr-37 and Thr-46 phosphorylation to rapamycin. Serum-starved 293 cells were incubated with rapamycin (20 ng/ml). One set of plates was lysed immediately (lanes 1–5); the other was serum stimulated for 30 min prior to lysis (lanes 6–10). Extracts were heat treated and analyzed by Western blotting, as described in Materials and Methods. The membranes were incubated with either an anti-phosphospecific antibody (top) or with an anti-4E-BP1 antibody (bottom).

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
8.

Figure 4. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

FRAP/mTOR phosphorylates 4E-BP1 complexed with eIF4E. (A) 4E-BP1 was pre-incubated without eIF4E, with an equimolar quantity or with a twofold molar excess of eIF4E, and subjected to a kinase assay using FRAP/mTOR immunoprecipitated from rat brain (lanes 1–3) or recombinant ERK2 (lanes 4–8). Phosphorylated proteins were separated via SDS-15%-PAGE, transferred to nitrocellulose membranes, and subjected to autoradiography. The substrates used were as follows. (Lanes 1–6) A thrombin-cleaved and HPLC-purified GST–4E-BP1; (lane 7) an uncleaved GST–4E-BP1 wild-type protein; (lane 8) an uncleaved GST–4E-BP1 mutant lacking the eIF4E-binding site. (B) Tryptic/chymotryptic map of 4E-BP1 alone (A, lane 1). (C) Tryptic/chymotryptic map of 4E-BP1 complexed with a twofold molar excess eIF4E (A, lane 3).

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
9.

Figure 8. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

Phosphospecific antibodies confirm the relative lack of serum sensitivity of Thr-37 and Thr-46. (A) 293 cells were transfected with wild-type HA–4E-BP1 (wt), HA–4E-BP1 Thr-37–Ala/Thr-46–Ala (mut), or vector alone (vec). Cell extracts were prepared and analyzed by Western blotting with either the phosphospecific antibody to Thr-37 and Thr-46 or with an anti-HA antibody. (B) A time course of serum stimulation (after 30 hr of starvation) of 293 cells was performed for the indicated times. Extracts were prepared for Western blotting (see Materials and Methods) and duplicate blots were performed. Phosphospecific antibody to Thr-37 and Thr-46 (see Materials and Methods; top) and an anti-4E-BP1 antibody raised against the entire protein (bottom) were used to probe the membranes.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
10.
Figure 1

Figure 1. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

FRAP/mTOR phosphorylates 4E-BP1 in vitro on two sites that are also phosphorylated in vivo. (A) FRAP/mTOR immunoprecipitated from rat brain was utilized in an in vitro kinase assay with human 4E-BP1 (expressed as a GST fusion protein, which was then cleaved and HPLC purified) as a substrate. Phosphopeptide mapping was performed on phosphorylated 4E-BP1. The two most intense phosphopeptides are numbered 1 and 2. (+) The origin of migration. (B) Tryptic/chymotryptic map of 4E-BP1 phosphorylated in 293 cells. The positions of phosphopeptides that comigrate with the FRAP/mTOR phosphorylated peptides 1 and 2 are indicated. (C) An equal number of cpm from the in vitro- and the in vivo-phosphorylated 4E-BP1 were combined and phosphopeptide mapping was performed. (D) Tryptic/chymotryptic map of 4E-BP1 phosphorylated in vitro with purified FRAP/mTOR produced using the baculovirus system.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
11.

Figure 2. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

Thr-37 and Thr-46 are phosphorylated in vivo on phosphopeptides that comigrate with FRAP/mTOR phosphorylated peptides. 4E-BP1 immunoprecipitated from 2 × 108 cells (1/20 labeled with [32P]orthophosphate) was separated by SDS-15% PAGE, electroblotted onto a nitrocellulose membrane, and proteolytically cleaved with trypsin/chymotrypsin as described in Materials and Methods. (A) Tryptic/chymotryptic two-dimensional phosphopeptide map of 4E-BP1. Phosphopeptides (labeled 1–5) were isolated from the plates and identified by mass spectrometry. (B) Tandem mass spectrum of m/z 428.9 revealing the sequence and phosphorylation site of the phosphopeptide contained in spot 1 of the map shown in A. The position of the phosphorylated threonine residue (Thr-46) is shown. (C) Identification of the phosphorylated peptides in spots 1–5 from the phosphopeptide map shown in A. Phosphorylated amino acids are in boldface type; the amino acid amino terminal to the cleavage site for trypsin or chymotrypsin is indicated in parentheses. (D) The positions of Thr-37 and Thr-46 in 4E-BP1, relative to the eIF4E-binding site.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.
12.
Figure 10

Figure 10. From: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

Mutation of Thr-37 or Thr-46 drastically reduces 4E-BP1 phosphorylation in vivo. 4E-BP1 wild type (wt) and mutants Thr-37–Ala (T37A), Thr-46–Ala (T46A), Thr-37–Ala/Thr-46–Ala (TTAA), and Thr-37–Glu/Thr-46–Glu (TTEE) in a pcDNA3-3HA vector were transfected into 293T cells (six plates/construct). Three transfected plates were subjected to 32P-labeling, and HA-tagged 4E-BP1 was immunoprecipitated using an anti-HA antibody (HA.11). Precipitated proteins were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and autoradiographed. The intensity of the HA–4E-BP1 bands was quantified by PhosphorImager. The three other plates were used to analyze the expression levels of the HA-tagged proteins by Western blotting, using anti-HA as the primary antibody and 125I-coupled anti-mouse IgG as the secondary antibody. Expression level was quantified by PhosphorImager. The average of the 32P incorporation into the mutant proteins was normalized to the expression level of each mutant. (A) A representative autoradiograph is shown; arrows indicate 32P-labeled HA–4E-BP1 and coprecipitated eIF4E. (B) A representative Western blot showing the levels of the mutants; this Western blot was developed by chemiluminescence, which produces a brighter signal. (C) Incorporation of 32P into each mutant, normalized to the expression level. The incorporation in the wild-type protein is set to 100%.

Anne-Claude Gingras, et al. Genes Dev. 1999 June 1;13(11):1422-1437.

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