Figure 2. Phosphorylation of 4EBP1, but Not rpS6, Is Required for Increased Protein Synthesis and Cell Size Control Downstream of Oncogenic Akt Signaling.

(A) Schematic representation of the PI3K-Akt-mTOR pathway and the genetic strategy to specifically inhibit rpS6 phosphorylation or eIF4E hyperactivation.

(B) Representative western blot analysis showing the hyperphosphorylation status of pAkt, p4EBP1, and prpS6 in AKT^T thymocytes. Western blot analysis of total Akt, 4EBP1, and rpS6 in AKT^T thymocytes is included.

(C) A representative cap-binding assay to analyze eIF4E-eIF4G complex formation in thymocyte cell lysates of WT, AKT^T, AKT^T;4EBP1^M, and 4EBP1^M mice and densitometry of eIF4G/eIF4E ratio (n = 3/genotype) (*p < 0.05; n.s., no statistical significance). The eIF4E complex bound to the cap analog m⁷GTP-sepharose was pulled down and western blotted with antibodies against eIF4G and 4EBP1 revealed the relative amounts of eIF4E-associated proteins. The anti-4EBP1 antibody recognizes both the endogenous 4EBP1 and the 4EBP1^M proteins.

(D) Protein synthesis rates in thymocytes derived from AKT^T, AKT^T;4EBP1^M, and AKT^T;rpS6^P−/− mice were measured by [³⁵S]-methionine incorporation and TCA precipitation represented as percentage of increase over WT levels; *p = 0.0006; **p = 0.001, n > 6/genotype.

(E) Cell size analysis of thymocytes from the indicated genotypes; **p < 0.00001, n = 8; *p < 0.0007, n = 6. Data are presented as the average ± SEM. See also Figure S2.