Characterization of the nuclear proteins binding the CACCC element of a glucocorticoid-responsive enhancer in the tyrosine aminotransferase gene.

The nuclear proteins which act synergistically with the glucocorticoid receptor to induce transcription of the tyrosine aminotransferase gene include factors recognizing the CACCC element. We have purified and characterized the proteins from rat liver nuclei which bind to the CACCC motif in the glucocorticoid-inducible enhancer of the gene. Three protein-DNA complexes (C1, C2, and C3) were detected in band-shift assays. The protein component of complex C1 also binds a GC motif (a Sp1 binding site) and is recognized by anti-Sp1 antiserum. The proteins forming complexes C2 and C3 have been purified by DNA-affinity chromatography and their molecular masses (75-80 kDa and 35-40 kDa, respectively) have been determined by ultraviolet cross-linking to radio-labelled DNA and SDS/PAGE. The DNA-affinity-purified C2 and C3 activities do not bind significantly to the GC motif and are not recognized by anti-Sp1 antiserum. Methylation interference analysis indicates that the nucleotides of the CACCC element bound by the C2 and C3 proteins correspond to those of the glucocorticoid-responsive enhancer which are contacted in vivo following glucocorticoid administration. Our data suggest that these proteins contribute to glucocorticoid-induced transcription of the tyrosine aminotransferase gene.

Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation.

A ribosomal protein S6 kinase copurifies with phosphatase-1 activating factor.

A highly purified preparation of phosphatase-activating kinase (Fa) from rabbit skeletal muscle phosphorylated ribosomal protein S6. The two activities copurified on DEAE-Sephadex, CM-Sephadex, and phosphocellulose chromatography and upon further chromatography on Sephacryl S-300 and FPLC Mono-S and Mono-Q columns. On the latter column, two separate peaks of Fa activity were observed when it was developed in Tris buffer as opposed to beta-glycerophosphate. S6 kinase activity was obtained only with the Fa which adhered to the resin. The Mr of the Fa and S6 activities was determined to be 83,200 by gel permeation on a Sephacryl S-300 column. The Fa preparation phosphorylated serine residues on S6; two tryptic phosphopeptides, A and C, were identified by two-dimensional phosphopeptide analysis. The enzyme also showed good activity toward initiation factor eIF-4B. Based on specificity toward ribosomal proteins and initiation factors, the Fa and a mitogen-stimulated S6 kinase purified from insulin-stimulated 3T3-L1 cells were similar. These results suggest that a form of Fa and an insulin-stimulated S6 kinase may be related or closely associated.