Developmental expression of insulin receptor substrate-2 during dimethylsulfoxide-induced differentiation of human
D H Schacher1, R W VanHoy, Q Liu
1Laboratory of Immunophysiology, Department of Animal Sciences, College of Medicine, University of Illinois, Urbana 61801, USA.
Abstract:
Insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine by a number of cytokine receptors and is implicated in the activation of phosphatidylinositol 3'-kinase (PI3-kinase). Here, we demonstrate that induction of granulocytic differentiation of human promyeloid HL-60 cells leads to an increase in the amount of IRS-2 that is phosphorylated in response to insulin-like growth factor (IGF)-I. Although PI3-kinase is often activated following interaction with IRS-1, we could not detect IRS-1 protein, IRS-1 mRNA, or IRS-1-precipitable PI3-kinase enzymatic activity. However, PI3-kinase activity that was coimmunoprecipitated with either anti-phosphotyrosine or anti-IRS-2 following IGF-I stimulation was increased 100-fold. Heightened tyrosine phosphorylation of IRS-2 during granulocytic differentiation was not caused by an increase in expression of the tyrosine kinase IGF-I receptor, as measured by the amount of both the alpha- and beta-subunits. Instead, immunoblotting experiments with an Ab to IRS-2 revealed that induction of granulocytic differentiation caused a large increase in IRS-2, and this occurred in the absence of detectable IRS-1 protein. These IRS-2-positive cells could not differentiate into more mature myeloid cells in serum-free medium unless IGF-I was added. These data are consistent with a model of granulocytic differentiation that requires at least two signals, the first of which leads to an increase in the cytoplasmic pool of IRS-2 protein and a second molecule that acts to tyrosine phosphorylate IRS-2 and enhance granulocytic differentiation.
Insights
Granulocytic differentiation increases insulin receptor substrate-2 (IRS-2) phosphorylation, crucial for phosphatidylinositol 3-kinase (PI3-kinase) activation. This process enhances myeloid cell differentiation, independent of IRS-1.
Area of Science:
- Cellular signaling pathways
- Molecular mechanisms of differentiation
- Hematopoiesis research
Background:
- Insulin receptor substrate-2 (IRS-2) plays a role in cytokine receptor signaling and phosphatidylinositol 3-kinase (PI3-kinase) activation.
- Granulocytic differentiation is a critical process in myeloid cell development.
- The role of IRS-2 in IGF-I-mediated signaling during differentiation requires further elucidation.
Purpose of the Study:
- To investigate the role of IRS-2 phosphorylation in IGF-I-induced granulocytic differentiation of HL-60 cells.
- To determine the contribution of IRS-2 to PI3-kinase activation during differentiation.
- To explore the interplay between IRS-2 and IRS-1 in this cellular process.
Main Methods:
- Induction of granulocytic differentiation in human promyeloid HL-60 cells.
- Stimulation with insulin-like growth factor (IGF)-I.
- Immunoprecipitation and immunoblotting techniques to assess protein levels and phosphorylation status of IRS-2 and IGF-I receptor.
- Measurement of PI3-kinase activity.
Main Results:
- Granulocytic differentiation significantly increased IRS-2 tyrosine phosphorylation in response to IGF-I.
- A 100-fold increase in PI3-kinase activity was observed, coimmunoprecipitated with IRS-2.
- IRS-1 protein, mRNA, and activity were undetectable, suggesting IRS-2 is the primary mediator.
- Increased IRS-2 protein levels were observed during differentiation, independent of IGF-I receptor expression.
- IGF-I was required for the differentiation of IRS-2-positive cells into mature myeloid cells.
Conclusions:
- Granulocytic differentiation involves increased IRS-2 protein and its tyrosine phosphorylation, mediated by IGF-I signaling.
- IRS-2, not IRS-1, is the key substrate for PI3-kinase activation during this differentiation process.
- A two-signal model for granulocytic differentiation is proposed, involving IRS-2 accumulation and subsequent phosphorylation.
