Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.
Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.
Wang
Y
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Department of Cell Biology, University of Alabama at Birmingham 35294.
Fuller
G M
GM
D000818:Animals; D015703:Antigens, CD; D001483:Base Sequence; D001693:Biological Transport, Active; D050822:Cytokine Receptor gp130; D004789:Enzyme Activation / Q000187:drug effects; D015850:Interleukin-6 / Q000494:pharmacology*; D053614:Janus Kinase 2; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
D015703:Antigens, CD; C494200:Il6st protein, mouse; C494201:Il6st protein, rat; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D050822:Cytokine Receptor gp130; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; C507927:Jak2 protein, rat; D053614:Janus Kinase 2