Hydrophobic residues Phe751 and Leu753 are essential for STAT5 transcriptional activity.
One facet of cytokine signaling is relayed to the nucleus by the activation, through tyrosine phosphorylation, of latent cytoplasmic signal transducers and activators of transcription (STAT) family members. It has been demonstrated that the C termini of STATs contain the transactivation domain and are essential for the transactivation of target genes. To better understand the function of the STAT C terminus, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding. Using GAL4 chimerae with the C terminus of STAT5, we have defined a 12-amino acid region essential for STAT5 transactivation. Surprisingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein. Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain. Using site-directed mutagenesis, we show that within this 12-amino acid region the acidic residues were non-essential for transactivation. In contrast, the non-acidic residues were crucial for transactivation. Mutating either Phe(751) or Leu(753) to alanine abolished transactivation suggesting that these residues were essential for connecting STAT5 to the basal transcriptional machinery.
Callus
B A
BA
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. Bernard_Callus@dfci.harvard.edu
Mathey-Prevot
B
B
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics; D007930:Leucine / Q000378:metabolism*; D051379:Mice; D008894:Milk Proteins; D010649:Phenylalanine / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D008894:Milk Proteins; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D029701:Saccharomyces cerevisiae Proteins; C494093:Stat5a protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D014443:Tyrosine; D010649:Phenylalanine; D004247:DNA; D007930:Leucine