Related Experiment Videos
Growth hormone, interferon-gamma, and leukemia inhibitory factor promoted tyrosyl phosphorylation of insulin receptor
L S Argetsinger1, G W Hsu, M G Myers
1Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622, USA.
Abstract:
The identification of JAK2 as a growth hormone (GH) receptor-associated, GH-activated tyrosine kinase has established tyrosyl phosphorylation as a signaling mechanism for GH. In the present study, GH is shown to stimulate tyrosyl phosphorylation of insulin receptor substrate 1 (IRS-1), the principle substrate of the insulin receptor. Tyrosyl phosphorylation of IRS-1 is a critical step in insulin signaling and provides binding sites for proteins with the appropriate Src homology 2 domains, including the 85-kDa regulatory subunit of phosphatidylinositol (PI) 3'-kinase. In 3T3-F442A fibroblasts, GH-dependent tyrosyl phosphorylation of IRS-1 was detected by 1 min and at GH concentrations as low as 5 ng/ml (0.23 nM). Tyrosyl phosphorylation of IRS-1 was transient, with maximal stimulation detected at 30 min and diminished signal detected at 60 min. The ability of GH receptor (GHR) to transduce the signal for IRS-1 tyrosyl phosphorylation is mediated by the intracellular region of GHR between amino acids 295 and 380 by a mechanism not involving the two tyrosines in this region. This region of GHR is required for GH-dependent JAK2 association and activation (VanderKuur, J. A., Wang, X., Zhang, L., Campbell, G. S., Allevato, G., Billestrup, N., Norstedt, G., and Carter-Su, C. (1994) J. Biol. Chem. 269, 21709-21717). When other cytokines that activate JAK2 were tested for the ability to stimulate the tyrosyl phosphorylation of IRS-1, stimulation was detected with interferon-gamma and leukemia inhibitory factor. The correlation between JAK2 tyrosyl phosphorylation and IRS-1 tyrosyl phosphorylation in response to GH, interferon-gamma, and leukemia inhibitory factor and in cells expressing different GHR mutants, provides evidence that IRS-1 may interact with JAK2 or an auxiliary molecule that binds to JAK2. GH is also shown to stimulate binding of IRS-1 to the 85-kDa regulatory subunit of PI 3'-kinase. The ability of GH to stimulate tyrosyl phosphorylation of IRS-1 and its association with PI 3'-kinase provides a biochemical basis for responses shared by insulin and GH including the well characterized insulin-like metabolic effects of GH observed in a variety of cell types.
Insights
Growth hormone (GH) activates tyrosyl phosphorylation of insulin receptor substrate 1 (IRS-1), a key step in GH signaling. This process involves JAK2 and leads to IRS-1 binding with phosphatidylinositol 3-kinase, explaining shared metabolic effects of GH and insulin.
Area of Science:
- Molecular Endocrinology
- Cellular Signaling Pathways
Background:
- Janus kinase 2 (JAK2) is a growth hormone (GH) receptor-associated tyrosine kinase, indicating tyrosyl phosphorylation as a GH signaling mechanism.
- Insulin receptor substrate 1 (IRS-1) is a primary substrate of the insulin receptor, crucial for insulin signaling and mediating protein interactions via Src homology 2 domains.
Purpose of the Study:
- To investigate the role of GH in stimulating IRS-1 tyrosyl phosphorylation.
- To elucidate the involvement of JAK2 and the GH receptor (GHR) intracellular domain in this signaling pathway.
- To explore the functional consequences of GH-induced IRS-1 phosphorylation, specifically its association with phosphatidylinositol 3'-kinase (PI 3'-kinase).
Main Methods:
- Stimulation of 3T3-F442A fibroblasts with GH and measurement of IRS-1 tyrosyl phosphorylation over time.
- Analysis of GH receptor (GHR) mutants to identify critical regions for signal transduction.
- Assessment of IRS-1 tyrosyl phosphorylation in response to other JAK2-activating cytokines (interferon-gamma, leukemia inhibitory factor).
- Evaluation of GH-stimulated binding between IRS-1 and the p85 regulatory subunit of PI 3'-kinase.
Main Results:
- GH rapidly stimulates IRS-1 tyrosyl phosphorylation in a dose- and time-dependent manner (detectable within 1 min, peaking at 30 min).
- The intracellular region of GHR (amino acids 295-380), independent of its tyrosines, mediates GH-induced IRS-1 tyrosyl phosphorylation and JAK2 activation.
- Interferon-gamma and leukemia inhibitory factor also induce IRS-1 tyrosyl phosphorylation, suggesting a common pathway involving JAK2.
- GH stimulates the association of IRS-1 with PI 3'-kinase, a critical step in insulin signaling.
Conclusions:
- GH activates IRS-1 tyrosyl phosphorylation via JAK2, mediated by a specific region of the GHR intracellular domain.
- The findings suggest IRS-1 may interact with JAK2 or an associated protein.
- GH-induced IRS-1 phosphorylation and PI 3'-kinase association provide a molecular basis for GH's insulin-like metabolic effects.