Rapid changes in nuclear protein tyrosine phosphorylation after growth hormone treatment in vivo. Identification of phosphorylated mitogen-activated protein kinase and STAT91.
Growth hormone (GH) plays a central role in regulating growth and intermediary metabolism in vertebrates, although the mechanisms by which GH initiates these actions are largely unknown. The GH receptor, a member of the cytokine receptor superfamily, does not demonstrate homology with any known tyrosine kinases. However, addition of GH to cells in vitro has been shown to stimulate tyrosine phosphorylation of various intracellular proteins including mitogen-activated protein kinases (MAP kinases) and the newly described Janus kinase, JAK2. Subsequent steps in GH-mediated signal transduction have not been delineated. In the present study, we have examined early events in GH action in vivo. Hypophysectomized juvenile male rats were treated with GH for 15, 30, or 60 min. Rat liver whole cell and nuclear extracts were prepared and analyzed via SDS-polyacrylamide gel electrophoresis and Western blotting techniques. GH rapidly stimulated the tyrosine phosphorylation of at least 8 nuclear proteins of 205, 91, 83, 80, 65, 53, 44, and 42 kDa, and caused the dephosphorylation of a single approximately 149-kDa protein. Using specific antibodies, we have identified three of these nuclear phosphoproteins as 42- and 44-kDa MAP kinases, and as STAT91, a 91-kDa component of the interferon-stimulated gene factor-3 protein complex. One consequence of the activation of STAT91 in the nucleus is the appearance of GH-stimulated DNA binding activity, as assessed by gel-mobility shift assay using an oligonucleotide containing a c-sis-inducible element from the c-fos promoter. These results show that nuclear protein tyrosine phosphorylation is a prominent early event in GH action in vivo and demonstrate a link between GH-stimulated signal transduction and target gene expression.
Gronowski
A M
AM
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri 63110.
Rotwein
P
P
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007016:Hypophysectomy; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D019000:Phosphotyrosine; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D012016:Reference Values; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C494081:Stat1 protein, rat; D015534:Trans-Activators; D019000:Phosphotyrosine; D014443:Tyrosine; D013006:Growth Hormone; D017871:Calcium-Calmodulin-Dependent Protein Kinases