title: Growth hormone-induced phosphorylation of epidermal growth factor (EGF) receptor in 3T3-F442A cells. Modulation of EGF-induced trafficking and signaling.
abstract: Growth hormone (GH) promotes signaling by causing activation of the non-receptor tyrosine kinase, JAK2, which associates with the GH receptor. GH causes phosphorylation of epidermal growth factor receptor (EGFR; ErbB-1) and its family member, ErbB-2. For EGFR, JAK2-mediated GH-induced tyrosine phosphorylation may allow EGFR to serve as a scaffold for GH signaling. For ErbB-2, GH induces serine/threonine phosphorylation that dampens basal and EGF-induced ErbB-2 kinase activation. We now further explore GH-induced EGFR phosphorylation in 3T3-F442A, a preadipocytic fibroblast cell line that expresses endogenous GH receptor, EGFR, and ErbB-2. Using a monoclonal antibody that recognizes ERK consensus site phosphorylation (PTP101), we found that GH caused PTP101-reactive phosphorylation of EGFR. This GH-induced EGFR phosphorylation was prevented by MEK1 inhibitors but not by a protein kinase C inhibitor. Although GH did not discernibly affect EGF-induced EGFR tyrosine phosphorylation, we observed by immunoblotting a substantial decrease of EGF-induced EGFR degradation in the presence of GH. Fluorescence microscopy studies indicated that EGF-induced intracellular redistribution of an EGFR-cyan fluorescent protein chimera was markedly reduced by GH cotreatment, in support of the immunoblotting results. Notably, protection from EGF-induced degradation and inhibition of EGF-induced intracellular redistribution afforded by GH were both prevented by a MEK1 inhibitor, suggesting a role for GH-induced ERK activation in regulating the trafficking itinerary of the EGF-stimulated EGFR. Finally, we observed augmentation of early aspects of EGF signaling (EGF-induced ERK2 activation and EGF-induced Cbl tyrosine phosphorylation) by GH cotreatment; the GH effect on EGF-induced Cbl tyrosine phosphorylation was also prevented by MEK1 inhibition. These data indicate that GH, by activating ERKs, can modulate EGF-induced EGFR trafficking and signaling and expand our understanding of mechanisms of cross-talk between the GH and EGF signaling systems.
authors.affiliation: Department of Medicine, Division of Endocrinology and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: Yao
authors.initials: Y
authors.lastname: Huang
authors.forename: Sung-Oh
authors.initials: SO
authors.lastname: Kim
authors.forename: Jing
authors.initials: J
authors.lastname: Jiang
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Frank
mesh_terms: D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002352:Carrier Proteins / Q000378:metabolism; D050822:Cytokine Receptor gp130; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D019382:Human Growth Hormone / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D053614:Janus Kinase 2; D048369:MAP Kinase Kinase 1; D008301:Maleimides / Q000494:pharmacology; D008562:Membrane Glycoproteins; D051379:Mice; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D050721:Proto-Oncogene Proteins c-cbl; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D014162:Transfection; D044767:Ubiquitin-Protein Ligases
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002352:Carrier Proteins; D050822:Cytokine Receptor gp130; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D005419:Flavonoids; D019382:Human Growth Hormone; D007211:Indoles; D053614:Janus Kinase 2; D048369:MAP Kinase Kinase 1; D008301:Maleimides; D008562:Membrane Glycoproteins; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010750:Phosphoproteins; D010768:Phosphoserine; D010769:Phosphothreonine; D019000:Phosphotyrosine; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D050721:Proto-Oncogene Proteins c-cbl; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D044767:Ubiquitin-Protein Ligases; C118387:Cblb protein, mouse; C494199:IL6ST protein, human; C494200:Il6st protein, mouse; C492211:CBLB protein, human; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; C070515:bisindolylmaleimide I; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one