Prolactin signals through RUSH/SMARCA3 in the absence of a physical association with Stat5a.
Jak/Stat-mediated prolactin signal transduction culminates in the sequence-selective binding of Stat5a. However, in the absence of Stat-binding sites, a RUSH-binding element mediates the prolactin signal in the rabbit uteroglobin promoter. Speculation about the existence of a Jak/RUSH pathway prompted this series of experiments to examine potential interactions between RUSH and Stat5a. Profiles of Jak/Stat pathway-specific genes by RT-PCR showed that mRNA for Jak2 and Stat5a is expressed in the endometrium of estrous, progesterone-treated, and 5-day pseudopregnant rabbits. Interspecies microarrays showed that transcripts for Stat5a were present at equal concentrations in the endometrium regardless of hormone treatment. The absence of a physical interaction between RUSH and individual Stat proteins bound to enhancer sites was demonstrated with transcription factor interaction arrays. These studies confirm that transmission of the prolactin signal through RUSH occurs in the absence of a physical association with Stat5a. Although a strong physical interaction between RUSH and Egr-1 was identified with the same arrays, no Egr-1 consensus sites were found in the region of the uteroglobin promoter (-175/-80) that contains the authentic RUSH site. Because the major transducer molecules (Jak2, Stat5a) are activated by tyrosine phosphorylation, Western analysis of immunoprecipitated samples, and gel shift assays were used to show that tyrosine phosphorylation is required for RUSH-DNA binding. The precise role for Jak2 in this process remains undefined. By comparison, serine-threonine-specific protein phosphorylation had no effect on RUSH-DNA binding.
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Aveline
A
Hewetson
Shelli L
SL
Moore
Beverly S
BS
Chilton
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D004717:Endometrium / Q000378:metabolism; D004971:Estrus / Q000378:metabolism; D005260:Female; D017874:Immediate-Early Proteins / Q000378:metabolism; D053614:Janus Kinase 2; D008894:Milk Proteins / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D011374:Progesterone / Q000494:pharmacology; D011388:Prolactin / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011555:Pseudopregnancy / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C104270:SMARCA3 protein, Oryctolagus cuniculus; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D014443:Tyrosine; D011374:Progesterone; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; D053614:Janus Kinase 2