Modifications of eukaryotic initiation factor 4F (elF4F) in adult cardiocytes by adenoviral gene transfer: differential effects on elF4F activity andtotal protein synthesis rates

In adult feline cardiocytes, increases in eukaryotic initiation factor 4F ( eIF4F) activity are correlated with accelerated rates of total protein synt hesis produced in response to increased load. Adenoviral gene transfer was employed to increase either eIF4F complex formation or the phosphorylation of eIF4E on Ser-209. To simulate load, cardiocytes were electrically stimul ated to contract (2 Hz, 5 ms pulses). Non-stimulated cardiocytes were used as controls. Adenovirus-mediated overexpression of wildtype eIF4E increased the total eIF4E pool by 120-140% above endogenous levels after 24h and pro duced a corresponding increase in eIF4F content. However, it did not accele rate total protein synthesis rates in-quiescent cardiocytes; neither did it potentiate the increase produced by contraction. To modify the affinity of eIF4F, cardiocytes were infected with a mutant (eIF4E/W56F) with a decreas ed binding affinity for the mRNA cap. Overexpression of eIF4E/W56F increase d the quantity of eIF4F but the rate of total protein synthesis was decreas ed in quiescent and contracting cardiocytes. Overexpression of a mutant tha t blocked eIF4E phosphorylation (eIF4E/S209A) increased the quantity of eIF 4F without any significant effect on total protein synthesis rates in quies cent or contracting cardiocytes. Overexpression of the eIF4E kinase Mnk-1 i ncreased eIF4E phosphorylation without a corresponding increase in eIF4F co mplex formation or in the rate of total protein synthesis. We conclude the following: (1) eIF4F assembly is increased by raising eIF4E levels via aden oviral gene transfer; (2) the cap binding affinity of eIF4F is a rate-limit ing determinant for total protein synthesis rates; and (3) increases in the quantity of eIF4F alone or in eIF4E phosphorylation are not sufficient to accelerate total protein synthesis rates.