Prolactin-induced Jak2 phosphorylation of RUSH: a key element in Jak/RUSH signaling.
Jak2/Stat-mediated prolactin signaling culminates in Stat5a-DNA-binding. However, not all Jak2-dependent genes have Stat5 sites. Western analysis with inhibitors showed Jak2 is a proximal intermediate in prolactin-induced RUSH phosphorylation. Transfection assays with HRE-H9 cells showed the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcription of the RUSH gene. Jak2 inhibitors or targeted RUSH-site mutation blocked the prolactin effect. RUSH co-immunoprecipitated with phospho-Jak2 from nuclear extracts. Jak2 inhibitors abolished the nuclear pool of phospho-RUSH not the nuclear content of RUSH in HRE-H9 cells. Nucleolar-affiliated partners, e.g. nucleolin, were identified by microLC/MS/MS analysis of nuclear proteins that co-immunoprecipitated with RUSH/GST-RING. RUSH did not exclusively co-localize with fibrillarin to the nucleolus. MG-132 (proteasomal inhibitor) failed to block Tyrene CR4-mediated decrease in phospho-RUSH, and did not promote RUSH accumulation in the nucleolus. These studies authenticate prolactin-dependent Jak2 phosphorylation of RUSH, and provide functional implications on the RUSH network of nuclear interactions.
Helmer
Rebecca A
RA
Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, United States.
Panchoo
Marlyn
M
Dertien
Janet S
JS
Bhakta
Suhani M
SM
Hewetson
Aveline
A
Chilton
Beverly S
BS
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D053614:Janus Kinase 2 / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011388:Prolactin / Q000494:pharmacology*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects; D014018:Tissue Distribution; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000097796:Nucleolin
D004268:DNA-Binding Proteins; D053614:Janus Kinase 2; D010750:Phosphoproteins; D011388:Prolactin; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D000097796:Nucleolin; C104270:SMARCA3 protein, Oryctolagus cuniculus