The forkhead rhabdomyosarcoma transcription factor (FKHR) is a promising candidate to be the transcription factor that binds to the insulin response element of the insulin-like growth factor-binding protein-1 (IGF-BP-1) promoter and mediates insulin inhibition of IGF-BP-1 promoter activity. Cotransfection of mouse FKHR increased IGFBP-1 promoter activity 2-3-fold in H4IIE rat hepatoma cells; insulin inhibited FKHR-stimulated promoter activity ϳ70%. A C-terminal fragment of mouse FKHR (residues

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... 08 -652) that contains the transcription activation domain fused to a Gal4 DNA binding domain potently stimulated Gal4 promoter activity. Insulin inhibited FKHR fragment-stimulated promoter activity by ϳ70%. Inhibition was abolished by coincubation with the phosphatidylinositol-3 kinase inhibitor, LY294002. The FKHR 208 -652 fragment contains two consensus sites for phosphorylation by protein kinase B (PKB)/Akt, Ser-253 and Ser-316. Neither site is required for insulin inhibition of promoter activity stimulated by the FKHR fragment, and overexpression of Akt does not inhibit FKHR fragment-stimulated Gal4 promoter activity. These results suggest that insulin-and phosphatidylinositol-3 kinase-dependent phosphorylation of another site in the fragment by a kinase different from PKB/Akt inhibits transcription activation by the fragment. Phosphorylation of this site also may be involved in insulin inhibition of transcription activation by full-length FKHR, but only after phosphorylation of Ser-253 by PKB/Akt.