Pit-1 binding sites mediate transcriptional responses to cyclic adenosine 3',5'-monophosphate through a mechanism that does not require inducible phosphorylation of Pit-1.
The ability of Pit-1 to mediate transcriptional responses to cAMP has been explored. To test the ability of Pit-1 to mediate transcriptional responses to cAMP, an expression vector was prepared for a mutant Pit-1 in which the major sites of phosphorylation by the cAMP-dependent protein kinase were eliminated. Before using the mutant Pit-1 to study transcriptional regulation, we first examined the ability of the protein to be phosphorylated in vivo in response to cAMP. Transfection and in vivo labeling experiments confirmed that the mutant Pit-1 did not support cAMP-inducible phosphorylation. The ability of the wild type or mutant Pit-1 to mediate transcriptional responses to cAMP was assessed in cotransfection experiments using reporter genes containing either the proximal region of the rat PRL gene or seven copies of a Pit-1 binding site placed upstream of a minimal promoter. Surprisingly, the wild type and mutant Pit-1 expression vectors supported similar responses to cAMP. To further assess the ability of Pit-1 to mediate responses to cAMP, a GAL4-Pit-1 fusion gene was prepared. Although a GAL4-cAMP response element binding protein fusion gene was found to permit transcriptional responses to cAMP, the GAL4-Pit-1 gene was unresponsive. These findings demonstrate that although Pit-1 can facilitate the ability of the PRL promoter to respond to cAMP, phosphorylation of Pit-1 is not required for this response. It seems likely that additional factors that interact with Pit-1 binding sites are important for mediating transcriptional responses to cAMP.
Okimura
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Y
Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 97201.
Howard
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PW
Maurer
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RA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D010903:Pituitary Gland, Anterior; D017354:Point Mutation / Q000235:genetics; D011388:Prolactin / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C494186:Pou1f1 protein, rat; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D011388:Prolactin; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases